Note: Descriptions are shown in the official language in which they were submitted.
3ti53
STABLE IM~NOCHE.MICAL ASSAY REAGENT FOR
THE DETERMINATION OF HAPTENS, AND ASSAY
METHOD USING SAID REAGENT
Thls invention relates to stable immunochemical
assay reagents Eor haptens and to an assay method using
these reagents.
Methods have long been known for determining
traces of biologically active substances existing in
body fluids such as blood or urine by immunochemical
techniques. For example, there is a method which
comprises sensitizing erythrocytes or red cells with
an antigen or antibody, and reacting the sensitized
erythrocytes with an antibody or antigen existing
in a body fluid of a given subject, thereby inducing
immunochemical agglutination or an agglutination
inhibiting reaction. In another known method, non-
biological particles such as a synthetic resin latex,
bentonite, collodion, cholesterol crystals and quartz
are used as a solid carrier in immunochemical reactions
instead of the erythrocytes.
In connection with such immunochemical assay
methods, it is known to use as a kind of hapten antigen
a hapten-carrier linked material obtained by chemically
binding a steroid, a kind of hapten, with a lysine
residue of a protein through an amide linkage (Journal
of Biological Chemistry, Vol. 22~, pages 713-727, 1957).
A method is also known to determine a
steroid in an assay sample immunochemically by using
X - 1 - ~
~3~:;53
both an antibody obtained by immunizing a mammal with an antigen protein having
a free amino group to which a steroid, a kind of hapten, is linked, and a car-
rier sensitized with a steroid protein complex obtained by linking a protein
other than the aforesaid antigen protein to the aforesaid steroid (see Japanese
Laid-Open Patent Publication No. 123819/75).
Recently, there was suggested an immunochemical assay method for the
determination of haptens which involves the use of both an antibody-sensitized
carrier obtained by sensitizing a carrier with a hapten antibody, and a hapten-
carrier linked material obtained by linking a hapten capable of reacting with
the hapten antibody to an antigenic carrier or a carrier sensitized with this
linked material (see Japanese Laid-Open Patent Publication No. 41420/78).
Since a hapten cannot produce an antibody by itself when it is used
singly to immunize an animal, it is the usual practice to render the hapten cap-
able of producing an antibody by linking an antigenic substance such as proteins
or polysaccharides to the hapten and then immunizing an animal with the linked
product under proper condition. Hence, a reagent resulting from the linking of
a protein or polysaccharide to the same hapten is used as an antigen. In such a
case, however, the stability of the reagent is reduced because the protein or the
like linked to the hapten is denatured, decomposed, or putrefied. It is neces-
sary therefore to store the reagent at low temperatures or under ice cooling,and various difficulties are encountered particularly during long-term storage.
It is an object of this invention therefore to provide a method which
can rapidly determine traces of haptens existing in blood, urine and other body
fluids with high sensitivity by an immunochemically reproducible and stable op-
eration. We have now found that the above object can be achieved very effectively
.
.
i53
by using an immunochemical assay reagent comprising a carboxyl-containing
water-soluble mono-olefinic polymeric compound combined with a hapten or its
chemically modified product, or a latex (having a particle diameter from
about 0.01 to about 2 microns) linked to the carboxyl containing water-soluble
mono-olefinic polymeric compo~md combined with a hapten thapten-holder).
To the best of the knowledge. of the present inventors, it has not
been known h0retofore to use such a reagent in the immunochemical assay of
haptens.
Accordingly, one aspect of the invention provides an immunochemical
assay reagent selected from the group consisting of:
~1) a combination of a carboxyl-containing water-soluble monoolefinic
polymeric compound chemically combined with a hapten or its chemically
modified product (component A-l) with a hapten antibody-supported carrier
comprising a carrier sensitized or chemically bound with the hapten antibody
(component B-2),
(2) a combination of a hapten-supported latex tcomponent A-2) obtained
by chemically combining with a polymeric latex having a particle diameter of
about 0.01 to about 2 microns, a carboxyl containing water-soluble mono-
olefinic polymeric compound chemically combined with a hapten or its chemically
modified product (component A-l), with a hapten antibody (component B-l), and
(3) a combination of a hapten-supported latex (component A-2) obtained
by chemically combining with a polymeric latex having a particle diameter of
about 0.01 to about 2 microns, a carboxyl containing water-soluble mono-
olefinic polymeric compound chemically combined with a hapten or its
chemically modified product (component A-l), with a hapten antibody-supported
carrier (component B-2) comprising a carrier sensitized or chemically bound
with the hapten antibody.
` ,~ 1
.
~3~5~
Another aspect of the invention provides a method for
immunochemically determining a hapten which comprises applying to an assay
sample containing a hapten an immunochemical assay reagent assaying an
agglutination inhibiting reaction of the reagent by the hapten present in the
assay sample said immunochemical assay reagent being selected from the group
consisting of:
(1) a combination of a carboxyl-containing water-soluble mono-olefinic
polymeric compound chemically combined with a hapten or its chemically
modified product (component A-l) with a hapten antibody-supported carrier
comprising a carrier sensitized or chemically bound with the hapten antibody
(component B-2);
(2) a combination of a hapten-supported latex (component A-2) obtained
by chemically combining with a polymeric latex having a particle diameter of
about 0.01 to about 2 microns, a carboxyl-containing water-soluble mono-
olefinic polymeric compound chemically combined with a hapten or its
chemically modified product (component A-l), with a hapten antibody (component
B-l); and
(3) a combination of a hapten-supported latex ~component A-2) obtained
by chemically combining with a polymeric latex having a particle diameter of
about 0.01 to about 2 microns, a carboxyl containing water-soluble mono-
olefinic polymeric compound chemically combined with a hapten or its
chemically modified product ~component A-l), with a hapten antibody-supported
carrier (component B-2) comprising a carrier sensitized or chemically bound
with the hapten antibody.
In the immunochemical determination of haptens present in body
fluids using the reagent of this invention, it is possible to use both this
reagent and any antibody capable of reacting with the aforesaid reagent or a
- 3a -
!
3~ 3
carrier sensitized or chemically combined with the antibody, and to determine
an agglutination inhibiting reaction between the two.
We have found however that haptens can be determined advantageously
with higher sensitivity within shorter periods of time by a stable operation by
using
~ A) a hapten-holder, or a hapten-holder linked latex resulting from the
chemical linking of a carboxyl containing water-soluble mono-olefinic polymeric
compound combined with a hapten or its chemically modified product to a latex
having a particle diameter of about 0.01 to about 2 microns, and
~b) a hapten antibody, or a hapten antibody supported latex or a hapten
antibody-supported carrier comprising a latex having a particle diameter of
about 0.01 to about 2 microns sensitized or chemically combined with the
hapten antibody or a carrier sensitized or chemically combined with the hapten
antibody, and by determining the agglutination inhibiting action of the two
reagents (A) and tB) due to the hapten in a body fluid o a given subject.
The present invention is described in greater detail below.
53
- 4 -
Yaptens
The ~'haptensi', as used in the present inven-
tion, denote lo~ nolecular compoundg which ~re no-t
antigenic by themselv~s, but which show antig~nici-ty when
bound to antigenic subs-tAnces. for examplc antigenic high-
molecu]a~ compounds s~lch as proteins and polysaccharides,
and have reactivity lJith antibodies formed by i~ uniz-
in~ animals t!ith such antigenic substances. Components
existing in anirnals includin~ physiologically active
substances and metabolites thereof and drugs admin-
istered and ~etabolites thereof` are especially
important haptens to ~rhich the prese~nt invention is
applicable. Specif`ic exatnples of the haptens are
given below.
(I) Steroidal haptens
(i) Estrogens such as estrone9 estradiolg
estriol, estetrol, equilin and equilemin.
(ii) ~atural gestagens which are produced in
animals or exist ?S ~etabolites, such
as progesterone, pregnanediol and
pregnanetriol, and synthetic gestagens
such as 19-norethisterone 9 and
chlormadinone acetate.
(iii) Androgens such ?S testosterone, dehydro-
epiandrosterone, 'aihydrotestosterone 9
androsterone and etiocholanolone.
(iv) Corticoids such as cortisol, cortisone,
deOXyCOrtiCOSterOlle 9 aldosteroneg
tetrahydroaldosterone an(l tetrahydro-
cortisol.
(v) Other steroids such ?S vitamin D
derivatives 9 bile acids SllCh as cholic
acidg desoxycholic acid aild chenocholic
acidg cardenolic'es~ saponins9 ard
sapogenins.
(II) Haptens of` biologically active amines
(i~ Catechol amines and their metabolites,
~436S3
^- 5 -
such as epine~hrine 9 norepinephrine,
dipamine and ephedrine.
B roloc~ Ic~l ly
: (ii) Bi~ e~ active allcaloids such as
rlorphine, codeine, heroin, morphine
glucuronide, cocaine1 mc-~scaline,
papaverine, narcotine 7 yohinbine,
reserpine 7 ergotamine ancl strychnine.
(iii) Amine drugs such as l,SD, amphetamine,
meprobamate and metarnphetamirle.
(III) Other haptens
Low molecular peptides which do not act
as anti~ens~ such as T~H ancl LH-RH 9
thyroid hormones such as diiodothyronine,
triiodothyronine and thyroxine 9
prostaglandin derivatives such as
prostaglandin E2 9 prostaglandin E3, and
prosta~landin Fl~;
vitamins such as vitarllin A, vitamin B
derivatives (for example vitamin Bl?
B2~ El6, and Bl2, vitamin ~ and vitamin K;
antibiotics such as penicillin
derivatives9 actinomycin~ chloromycetin
ancl tetracycline.
The haptens contemplated by this invention,
however, are not limited to chese specific examples.
Chernical modification of haPtens
In the present invention 9 the haptens are
chemically combined with carbox~l-containing water-solu-
~le mono-olefinie polym~ric com~ounds (to be referred to
as "holder" for convenience) either directly or after
they have been chemically modlfied.
Various methods have been knol~n to m,odify
haptens chemically. In ~he present invention, any
modifying methods can be employed which chemically
bind the haptens to the functiona], groups ~e.~
carboxyl or hydroxyl) of the holders. Especially
preferred methods for chemically modifying haptens
1 ~3~S3
are those which introduce a carboxyl group, a primary amino group, a secondary
amino group, or a hydroxyl group, above all the carboxyl group or the primary
amino group, into haptens. Some examples are described below.
A carboxyl group can be introduced into a hapten having a carbonyl
group by converting it to the corresponding carboxymethyl oxime (for example,
Journal of Biological Chemistry, Vol. 234, pages 1090-1094, 1959). Or the hap-
ten may be brominated, and then reacted with thioglycolic acid to introduce a
carboxyl group into it (for example, Steriods, Vol. 19, pages 357-375, 1972).
It is kno~n that reduction of an oxime of a carbonyl compound yields a
primary amino compound. This product can also be utilized as a chemically mod-
ified product of hapten.
A hapten having a hydroxyl group can be modified by reacting it with
monochloroacetic acid to form the corresponding carboxymethyl ether (for ex-
ample, Science, Vol. 168, pages 1347-1348, 1970), or reacting it with succinic
anhydride to form a hemisuccinate (for example, Journal of Clinical Endocrinol-
ogy, Vol. 33, pages 775-782, 1971).
A carboxyl group may be introduced into a hapten having a phenolic hy-
droxyl group by coupling a para-carboxybenzene diazonium salt with the ortho- or
para-position to the hydroxyl group of the hapten (for example, Steroids, Vol.
20 18, pages 555-563, 1971).
With regard to glucuronides which are metabolites of steroids, their
carboxyl groups can be utilized (for example, Journal of S~eroid Biochemistry,
Vol. 3, pages 275-288, 1972). If these carboxyl groups cannot be directly uti-
lized, they may be converted to amino compounds by reaction with suitable diamine
derivatives.
Haptens having a secondary amino group can be converted to primary
amine derivatives by, for example,
~3~S3
alkylating the amino group thereof with N-protected aminoalkylhalogen compounds,
and then splitting off the protective group (for example, FEBS Letters, Vol. 36,
pages 339-342, 1973). Or a carboxyl group can be introduced into such haptens
by reaction with bromoacetic esters followed by hydrolysis (for example, Chemical
and Pharamaceutical Bulletin, Vol. 25, pages 838-8~0, 1977).
Haptens which do not have suitable functional groups may first be sub-
jected to such means as hydroxylation by microorganisms, and then to the afore-
said methods to introduce the desired functional groups.
Holders
The holders, used in this invention may be any carboxyl-containing
water-soluble mono-olefinic polymeric compounds. The term "water-soluble", used
herein, means that when at least 1 part by weight of such a polymeric compound
is dissolved in 1000 parts by weight of distilled water, a clear solution forms.
So long as the solubility of the polymeric compound is above the lower limit
specified, it may be as high as is desired. The polymeric compound may have a
weight average molecular weight of about 103 to 107 or higher. Usually, those
having a weight average molecular weight of some ten thousand to several million
can be suitably used. These polymeric compounds may have a hydroxyl group as a
functional group in addition to the carboxyl group. These functional groups are
involved in chemical binding to the functional groups of haptens or their chem-
ically modified products or to the functional groups of latices to be described
hereinbelow, and also serve to impart water solubility to these polymeric com-
pounds as holders.
The polymeric compounds used as holders in this invention are consid-
ered to be biologically inactive substances, and generally do not have antigen-
icity.
~ 7 ~
~3~3
Examples of these polymeric compounds include homopolymers or copoly-
mers of acrylic acid or methacrylic acid, a copolymer of maleic acid and vinyl
acetate or a saponification product thereof, copolymers of maleic acid with
vinyl alcohol, lower alkylvinyl ethers, acrylic acid, lower alkyl acrylates,
methacrylic acid, or lower alkyl methacrylates, and hydrolysis products of these
copolymers. These polymeric compounds may also be a copolymer of acrylic acid
or methacrylic acid with beta-hydroxyethyl acrylate or acrylamide, or a terpoly-
mer containing the aforesaid monomers as structural units.
Chemical binding of haptens or their
-
modified products to holders
Preferably, the chemical binding of haptens or their chemically modi-
fied products to holders is carried out through an amide or ester linkage, espe-
cially through an amide linkage. This binding can be effected by using a known
amide linkage reaction or ester linkage reaction as described below.
For example, when a hapten or its chemically modified product (to be
inclusively referred to as "hapten") contains an amino group and the holder con-
tains a carboxyl group, or vice versa, the hapten can be chemically bound to the
holder through an amide linkage by any of the following methods.
(Methods for binding haptens to holders through an
amide linkage)
(1) Carbodiimide method
This method comprises forming an amide linkage between the amino group
and the carboxyl group by dehydrocondensation. This can be achieved by adding
an amo~mt of a carbodiimide compound which is required for the desired binding
or is slightly in excess of it to a solution of the two compounds, and reacting
them at room temperature or under ice cooling. The carbodiimide compound itself
changes to a urea derivative.
- 8 -
-NH2 + -COOH ~ R-N=C=N-R
~ -NH-CO- + R-NHCONH-R
Any reaction solvent can be used which does not have a functional
group that will participate in the reaction. For example, there can be used
ethyl acetate, tetrahydrofuran, dioxane, dimethylformamide, and chloroform as
the reaction solvent. The reaction solvent may contain any desired proportion
of water, and the reaction may be performed in aqueous solution. When the re-
action is to be carried out in an organic solvent, dicyclohexylcarbodiimide is
most frequently used as the carbodiimide compound. In a reaction in a water-
containing solvent, a water-soluble carbodiimide such as 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride may be used.
(2) Carbonyl diimidazole method
This method, like the carbodiimide method, comprises inducing dehydro-
condensation between the amino group and the carboxyl group. It can be per-
formed by adding a required amount of carbonyl diimidazole to a solution of the
two components. Since this reagent is sensitive to moisture and immediately de-
composes upon contact with water, it is preferred to use a solvent which does
not contain water.
~3) Mixed acid anhydride method
The carboxyl group forms a mixed acid anhydride with, for example, a
chloroformic acid ester in the presence of an organic base. The mixed acid an-
hydride readily reacts with the amino group to form an amide linkage.
-COOH + Cl-COOR ~ -COO-COOR
~¦,-NH2
-CONH-
Examples of the chloroformate are lower alkyl esters (e.g., ethyl,
isopropyl or isobutyl esters) Gf chloroformic acid, and isobutyl chloroformate
is most frequently used. Tertiary amines such as
_ g _
~3~53
triethylamine or N-methylmorpholine are used as the organic base.
The same reaction solvents as described hereinabove with regard to the
carbodiimide method can be used. Since the mixed acid anhydride is unstable in
water, care must be taken not to use hydrous materials in the formation of the
mixed acld anhydride. The reaction temperature during the formation of the
mixed acid anhydride is about -10C to -20C, and after adding the amino com-
pound, the reaction is continued at room temperature. Water or a water-contain-
ing solvent may be used when the amino compound is added.
(4) Active ester method
The theory of the active ester method is that when the carboxyl group
is converted to an ester of a compound having a great electron attracting prop-
erty, the electron density on the carbonyl group decreases and consequently the
carbonyl group attacks a functional group having a high basicity such as an
amino group to form an amide linkage. F.xamples of the active ester include
esters of phenol derivatives such as para-nitrophenol, 2,4-dinitrophenol, penta-
chlorophenol, thiophenolJ naphthol and 3-hydroxyquinoline, es~ers of N-hydroxy
compounds such as N-hydroxysuccinimide and N-hydroxypiperidine and alkyl esters
such as cyanomethyl. The same solvents as described above with regard to the
carbodiimide method can be used.
~5) Azide method
Conversion of a carboxyl compound to an ester and subsequent reaction
with hydrazine affords an ~cid hydrazide. By the action of nitrous acid, the
acid hydrazide is converted to an acid azide which reacts with the amino group
to form an amide linkage.
HN02
-coNHNH2 ~ > -CON3 -CONH-
-NH2
hydrazide
- 10 -
53
Specifically, the amide linkage can be formed by a classical method
whlch comprises reacting hydrazide with sodium nitrite in dilute hydrochloric
acid, isolating the resulting acid azide, and then reacting it with an amine
compound in a suitable organic solvent; or a method which comprises treating
hydrazide with an a]kyl nitrite such as t-butyl nitrite or i-amyl nitrite in
the presence of hydrogen chloride in an organic solvent (which may contain
water) to form an acid azide, and without isolating the acid azide, reacting
the resulting product with an amine compound.
(6) Acid chloride method
The most common method comprises converting a carboxyl compound into
an acid chloride, and reacting it with an amino compound to form an amide link-
age. Conversion to the acid chloride may be performed by a direct method which
comprises reacting the carboxyl compound with phosphorus pentachloride, thionyl
chloride, phosphorus oxychloride, etc., or an exchange reaction method which
comprises reacting the carboxyl compound with oxalyl chloride. The reaction of
the acid chloride with the amino compound can be performed by the "Schotten-
Baumann method" which comprises performing the reaction in water while adding an
alkali, or a method which comprises performing the reaction in an inert solvent
such as benzene in the presence of an organic base such as pyridine or triethyl-
amine.
(7) DPPA method
There can also be employed a method which comprises adding diphenyl-
phosphoryl azide (DPPA) to a solution of a carboxyl compound and an amino com-
pound, and then adding an organic base such as triethylamine or N-methylmorphol-
ine to form an imide linkage. In this method, dimethylformamide can be conve-
niently used as a solvent. The reaction temperature may be
- 1 1 -
~ ~,
36~3
from that attained under ice cooling to room temperature.
The chemical bonding can be effected by any of the above methods (1)
to (7). }lowever, any method which requires severe conditions should be avoided
in the present invention because haptens may be unstable depending upon their
other substituents. The carbodiimide method (1) and the DPPA method (7) can be
used most suitably. To a solution of the holder and a suitable amount of a hap-
ten is added an equimolar amount or slightly excessive amount, to the hapten, of
a carbodiimide or DPPA, and they are reacted (in the case of DPPA, an organic
base is added further prior to the reaction). After the reaction, the entire
reaction mixture is dialyzed against water in a cellophane tube whereupon the
unreacted hapten and reagent and by-products are driven to the external solu-
tion. Concentration or lyophilizing of the dialyzate affords the desired hapten
(or modified product thereof)- holder bound product.
(Method for binding haptens to holders through an
ester linkage)
When the hapten or its chemically modified product has a suitable re-
active hydroxyl group and the holder has a carboxyl group~ or vice versa, the
hapten can be chemically bound to the holder through an ester linkage.
When the hapten or its chemically modified product has a hydroxyl
group and the holder has a carboxyl group, the carboxyl group is reacted with,
- for example, thionyl chloride to form an acid chloride and then reacted with the
hapten to form a hapten-holder bound product through an ester linkage. If the
holder is a copolymer containing maleic anhydride, it is directly reacted with
the hapten to form a hapten-holder bound product through an ester linkage.
Typical examples of the hapten-holder bound
- 12 -
~3~53
product are given below.
Polyacrylic acid combined with 17-amino-1,3,5(10)-estratrien-3-ol,
methyl vinyl ether/maleic anhydride copolymer combined with 17-amino-
1,3,5~10)-estratrien-3-ol,
polyacrylic acid combined with estriol 16-glucuronide,
methyl vinyl ether/maleic anhydride copolymer combined with estriol
16-glucuronide,
polyacrylic acid combined with estriol 16, 17-dihemisuccinate,
polyacrylic acid combined with preganediol 3-glucuronide,
methyl vinyl ether/maleic anhydride copolymer combined with pregane-
diol 3-glucuronide,
polyacrylic acid combined with preganetriol 3-glucuronide,
methyl vinyl ether/maleic anhydride copolymer combined with pregane-
triol 3-glucoronide,
polyacrylic acid combined with 3~ ,17a,21 tetrahydroxypregnan-20-
one 3-glucuronide,
methyl vinyl ether/maleic anhydride copolymer combined with 3a,11~,
17a,21-tetrahydroxypregnan-20-one glucuronide,
polyacrylic acid combined with carboxymethylmorphine,
methyl vinyl ether/maleic anhydride copolymer combined with carboxy-
methylmorphine,
polyacrylic acid combined with etiocholanolone hemisuccinate,
methyl vinyl ether/maleic anhydride copolymer combined with thyroxine,
polyaerylic acid combined with thyroxine,
polyacrylic acid combined with metanephrine,
methyl vinyl ether/maleic anhydride copolymer combined with metane-
phrine,
- 13 -
~ `~
~1 ~3~53
Reactive polymeric latex
If desired, the holder may further be chemically combined with a re-
active polymeric latex. The polymeric latex llas an average particle diameter of
about 0.01 to about 2 microns and contains a functional group capable of react-
ing with the holder. Those having an average particle diameter of about 0.05 to
about 1.5 microns are especially suitable.
As such reactive polymeric latices, various polymeric latices composed
of a substrate polymer such as polystyrene, a styrene/butadiene copolymer, a
styrene/divinylbenzene copolymer, a styrene/divinylbenzene copolymer, polyvinyl
toluene and a vinyltoluene/tertiary butyl styrene copolymer and having a func-
tional group such as a carboxyl group, a primary amino group or a carbamide
group ~-CONH2) are commercially supplied under various tradenames. All of these
polymeric latices can be used in this invention. The substrate of the polymeric
latices, however, is not limited to these exemplified polymers or copolymers.
When the polymeric latex contains a primary amino group as a function-
al group, it is directly reacted with the carboxyl group of the holder to bind
the holder chem1cally to the polymeric latex through an amide linkage. Forma-
tion of such an amide linkage can be effected by using the reaction procedures
already described hereinabove with regard to the reaction between the hapten (or
its modified product) and the holder.
When both of the polymeric latex and the holder contain a carboxyl
group as a functional group, it is possible to modi-fy the carboxyl group of the
latex chemically by the following method to introduce a primary amino group and
then bind the latex to the holder through an amide linkage. For example, this
can be achieved by a method which comprises reacting
f~!, - 14 -
53
the carboxyl-containing latex with a polymethylenediamine such as heptamethyl-
enediamine in the presence of a water-soluble carbodiimide to introduce a prim-
ary amino group ~for example, Journal of Cell Biology, Vol. 64, pages 75-88,
1975).
Alternatively, an alkylenediamine derivative in which one amino group
is protected, such as N-~-tert.butoxycarbonyl lysine methyl ester of N-phthaloyl-
N'-methyltrimethylenediamine may be reacted with a latex having a carboxyl
group. The reaction can be carried out by using a method selected from those
employed for the formation of an amide linkage to bind the hapten to the holder.
It is necessary, however, that the reaction be carried out so as not to change
the physical properties, such as the particle diameter, of the latex. For this
purpose, it is desirablc to carry out the reaction in a system containing water.
A carbodiimide method to be effected in water using a water-soluble carbodiimide
is especially-preferred. Elimination of the amino protecting group after the
reaction gives a reactive polymeric latex having an am-ino group as a reactive
group. By reacting the resulting reactive polymeric latex with the holder com-
bined with the hapten or its modified product containing a carboxyl group in ac-
cordance with the method of forming an amide linkage as described hereinabove,
the assay reagent of this invention can be obtained. When the use of N-~-tert-
butoxycarbonyl lysine methyl ester is taken up as an example, the aforesaid re-
action is expressed by the following scheme.
~'
- 15 -
NHCOO-C-CH3
Llatex¦ -COOH + (CH2)4 CH3
H2N-CHCOOCH3
1st step
C~13
NNCOO-C-CH3
(CH2)4 CH3
~ CONH-CH-COOCH3
2nd step
N~H2
~,CH2)4
~ 1- CONH-cH-cOOCH3
3rd step HOOC- ~ - ¦hapten~
NHCO - ~ ~ - ¦hapten~
/ (CH2)4
- CONH-CH-COOCH
The reaction in the third step can be carried out in quite the same
way as in the reaction in the first step.
A similar assay reagent can be obtained also by reversing the se-
quence of reactions as shown below.
- 16 -
,C~13
NHcoo-c-cH3
¦Hapten¦ - ¦Holde~ - COOH + ~CH2)4 CH3
H2N-CI-I-COOCH3
C~13
NI-ICOO-C-CH3
(,CH2)4 C~13
- ¦IIolder¦ - CONH-CH-COOCH3
NH2
(CH2)4
- ¦HcIder¦ - CONH-CH-COOCH
HOOC - ¦latex¦
NHCO - ~latëx~
(C~I2)4
. . ~ .
IHapten - IHolder - CONH-CH-COOCH
., 3
Thus, by combining the hapten-holder bound product with the reactive
latex by a chemical reaction, a hapten-holder-latex bound product can be produced.
Specific examples of the hapten-holder-latex bound product are shown
below.
Latex linked to (polyacrylic acid combined with estriol 16-glucuronide),
latex linked to (polyacrylic acid combined with estriol 16,17-dihemi-
succinate),
latex linked to (polyacrylic acid combined with 17-amino-1,3,5(10)-
estratrien-3-ol),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with 17-amino-1,3,5(10)-estratrien-3-ol),
latex linked to (methyl vinyl ether/malelc anhydride:copolymer co~bined
~,~ - 17 -
3~3
with estriol 16-glucllronide)~
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with estriol 16, 17-dihemisuccinate),
Iatex linked to (polyacrylic acid combined with pregnanediol 3-glucur-
onide),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with pregnanediol 3-glucuronide),
latex linked to (polyacrylic acid combined with 3~ ,17~,21-tetra-
hydroxypregnan-20-one 3-glucuronide),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with 3~,113,17~,21-tetrahydroxypregnan-20-one 3-glucuronide),
latex linked to (polyacrylic acid combined with carboxymethylmorphine),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with carboxymethylmorphine),
latex linked to (polyacrylic acid combined with etiocholanolone succin-
ate),
latex linked to (polyacrylic acid combined with thyroxine),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with thyroxine,
latex linked to (polyacrylic acid combined with metanephrine),
latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with metanephrine).
Preparation of antibodies
The hapten antibody used in this invention can be produced by known
methods.
Since the hapten itself does not have antigenicity, it is combined with
a substance having antigenicity. The bound product is used as an antigen and an
animal is immunized with it to form an antiserum.
- 18 -
~3~3
The hapten used for this purpose may be any of those haptens or chemi-
cally modifled products thereof described hereinabove. The hapten is combined
with an antigenic substance such as bovine serum albumin, rabbit serum albumin,
human serum albumin, bovine gamma-globulin, rabbit gamma-globulin, human gamma-
globulin, tetanus toxin, and pneumonococcus polysaccharide, and for example, a
hapten-protein bound product may be applied to an animal for immunization.
Examples of the hapten-protein bound product used at this time are
steroid-protein bound products such as estriol-16,17-dihemisuccinate-BSA (bovine
serum albumin), estriol 16-glucuronide-BSA, 6-oxo-estriol 6-(o-carboxymethyl)-
oxime-BSA, dehydroepiandrosterone glucuronide-BSA, androsterone hemisuccinate-
BSA, and cortisol 21-hemisuccinate-BSA, and products obtained by binding haptens
other than the steroids to antigenic substances such as proteins. In immunizing
an animal with such a bound product, the joint use of adjuvants such as complete
Freund's adjuvant permits more effective production of an antiserum.
Animals that can be used for this purpose are mammals such as rabbits,
goats, sheep, and guinea pigs. The resulting antiserum is caused to be absorbed
by the substance having antigenicity used for bonding to the hapten, and gamma-
globulin is separated by such means as alcohol precipitation or salting out.
Thus, a hapten antibody can be obtained. Hapten antibody-supported carriers.
The carrier, as referred to in this invention, denotes any carrier
which can be used conventionally in immunochemical assay reagents, and includes,
for example, erythrocytes of humans, sheep, rabbits, etc., latices, bentonite,
collodion, cholesterol crystals, silica, and kaolin. Any materials which can
carry the hapten antibody may be used in the present invention.
Sensitization of the carrier with the hapten antibody, as referred to
in the present invention, means the adsorption of the antibody to the carrier,
- 19 -
~3653
and does not mean the chemical bonding of the antibody to the carrier. Accord-
ingly, when the antibody is combined chemically with a synthetic resin, etc.,
the product is referred to as a polymeric latex having the antibody chemically
combined therewith. These two types of carriers are inclusively referred to in
this invention as hapten antibody-supported carriers.
~y~ atices sensitized or
chemically combined with antibodies
In the present invention, the antibody obtained in the aforesaid man-
ner is chemically combined with a polymeric latex, etc. or the latex is sensit-
ized with the antibody. Such a latex may or may not contain functional groupsof the types exemplified hereinabove. However, when the antibody is to be chem-
ically combined with the latex, it is necessary to use polymeric latices which
have functional groups capable of reacting with such antibody. Such polymeric
latices have an average particle diameter of about 0.01 to about 2 microns, and
contain functional groups capable of reacting with the antibodies. Those having
an average particle diameter of about 0.05 to about 1.5 microns are especially
suitable.
Polymeric latices composed of a substrate such as polystyrene, a
styrene/butadiene copolymer, a styrene/divinylbenzene copolymer, polyvinyltolu-
ene, and a vinyltoluene/tert.-butylstyrene and having such a functional group as
a carboxyl group, a primary amino group or a carbamide group (-CONH2) are com-
mercially supplied under various tradenames as such reactive latices. The sub-
strate of the latices is not limited to these exemplified polymers or copolymers.
When the polymeric latex contains a carboxyl group or a primary amino
group as a functional group, it may be directly reacted with the amino group or
carboxyl group of the antibody to bind it chemically
f~
- 20 -
53
to the antibody through an amide linkage. Such an amide linkage can be formed
by any of the a-foresaid methods for chemically binding the hapten or its chem-
ically modified product to the holder.
When both of the latex and the antibody have a carboxyl group as a
functional group, it is possible to modify the carboxyl group of either one of
them chemically by the aforesaid method to introduce a primary amino group, and
to bind the latex to the antibody through an amide linkage. ~or example, there
is available a method which comprises reacting the latex containing a carboxyl
group with a polymethylenediamine such as heptamethylenediamine in the presence
of a water-soluble carbodiimide to introduce a primary amino group (for example,
Journal of Cell Biology, Vol. 64, pages 75-88, 1975).
However, since antibodies have both a carboxyl group and a primary
amino group in many cases, it is usually possible to chemically bind them to
0ach other by such a means as the carbodiimide method without the need for per-
forming the aforesaid chemical modification. Specifically, for example, by bind-
ing an anti-esteriol 16-glucuronide antibody to a carboxyl-containing latex by
the carbodiimide method, an anti-estriol 16-glucuronide antibody-linked latex
can be obtained.
Examples of the hapten antibody-linked latex are given below.
Latex combined with anti-estriol 16-glucuronide antibody,
latex combined with anti-estriol 16,17-dihemisuccinate antibody,
latex combined with anti-17-amino-1,3,5(10~estratrien-3-ol antibody,
latex combined with anti-pregnanediol 3-glucuronide antibody,
~r
~ 21 -
~3~53
latex combined with anti-pregnanetriol 3-glucuronide antibody,
latex combined with anti-3, 11~ 17, 21-tetrahydroxypregnan-20-one 3-
glucuronide antibody,
latex combined with anti-carboxymethylmorphine,
latex combined with anti-etiocholanolone hemisuccinate antibody,
latex combined with anti-thyroxine antibody,
latex combined with anti-me~anephrine antibody.
The latex combined with the anti-hapten antibody in accordance with
this invention, however, is not limited to the above specific examples.
As stated hereinabove, sensitization of the carrier with the hapten
antibody means the adsorption of the antibody to the carrier. An antibody-sen-
sitized carrier can be obtained by merely mixing a solution of the antibody with
a suspension of the carrier with stirring.
For example, erythrocytes as a carrier can be sensitized with the hap-
ten antibody by an ordinary method of producing antibody-sensitized erythro-
cytes. For example, the red cells are fixed with a suitable material such as
formalin, glutaraldehyde or pyruvaldehyde, and then sensitized with the antibody
optionally using tannic acid or other condensing agents to form antibody-sensi-
tized cells. These are used as a suspension, or if required, they are lyophil-
0 ized to form antibody-sensitized erythrocytes ~antibody-sensitized carrier).
A polymeric latex as a carrier may be sensitized with the antibody in
the same way as in the conventional method to produce an antibody-sensitized
latex. ~or example, by adding a suspension of such a carrier as a polystyrene
latex to a solution of the antibody in a suitable concentration and mixing them,
an antibody-sensitized latex can be obtained.
f - 22 -
.
,
~3~5~
Examples of the hapten antibody-sensitized carrier are given below.
Erythrocytes sensitized with anti-estriol-16-glucuronide antibody,
erythrocytes sensitized with anti-estriol 16,17-dihemisuccinate)
erythrocytes sensitized with anti-17-amino-1,3,5(10)-estratrien-3-ol
antibody,
erythrocytes sensitized with anti-pregnanediol 3-glucuronide antibody,
erythrocytes sensitized with anti-pregnanetriol 3-glucuronide antibody,
erythrocytes sensitized with anti-3~ ,17~,21-tetrahydroxypregnan-
20-one 3-glucuronide antibody,
erythrocytes sensitized with anticarboxymethylmorphine,
erythrocytes sensitized with antietiocholanolone hemisuccinate anti-
body, erythrocytes sensitized with anti-throxine antibody,
erythrocytes sensitized with anti-metanephrine antibody,
latex sensitized with anti-estriol 16-glucuronide antibody~,
latex sensitized with anti-estriol 16,17-dihemisuccinate antibody,
latex sensitized with anti-17-amino-1,3,5(10)-estratrien-3-ol antibody,
latex sensitized with anti-pregnanediol 3-glucuronide antibody,
latex sensitized with anti-pregnanetriol 3-glucuronide antibody,
latex sensitized with anti-3~ ,17~,21-tetrahydroxypregnan-20-one 3-
0 glucuronide antibody,
latex sensitized with anti-carboxymethylmorphine antibody,
latex sensitized with anti-etiocholanolone hemisuccinate antibody,
- 23 -
~',
3~3
latex sensitized with anti-thyroxine antibody, and
latex sensitized with anti-metanephrine antibody.
The hapten antibody-sensitized carrier in accordance with this inven-
tion, however, are not limited to these speciEic examples.
The present invention made up o:E the above described constituent ele-
ments thus provides a novel immlmochemical assay reagent comprising a combina-
tion of
(1) a carboxyl-containing water-soluble monoolefinic polymeric com-
pound combined with a hapten or its chemically modified product, or a latex
linked to the carboxyl-containing water-soluble mono-olefinic polymeric compound
combined with hapten, and
(2) a hapten antibody (or in dilution), or a hapten antibody-support-
ed carrier such as a carrier sensitized with the hapten antibody by adsorption
of a hapten antibody to a carrier, or a carrier chemically combined with the
hapten antibody such as a latex chemically combined with the hapten antibody,
and an immunochemical assay method characterized by using the aforesaid reagent.
Specific examples of the novel immunochemical assay reagent of this
invention are given below.
1. Immunochemical assay reagents for the determination of estrogen consisting
of A and B:-
(a) A: latex linked to (polyacrylic acid combined with estriol 16-glucuronide)
and
B: latex combined with anti-estriol 16-glucuronide antibody
(b) A: latex linked to (polyacrylic acid combined with estriol 16J17-dihemisuc-
cinate) and
B: latex combined with anti-estriol 16,17-dihemisuccinate antibody
(c) A: latex linked to (methyl vinyl ether/maleic anhydride copolymer combined
with estriol 16-glucuronide) and
B: latex combined with anti-estriol antibody
- 24 -
.~
53
(d) A: latex linked to ~methyl vinyl ether/maleic anhydride copolymer combined
with estriol 16,17-dihemisuccinate) and
B: latex combined with anti-estriol antibody
(e) A: latex linked to (polyacrylic acid combined with estriol 16-glucuronide)
and
B: latex sensitized with anti-estriol 16-gluc~uronide antibody
(~) A: latex linked to (polyacrylic acid combined with estriol 16,17-dihemisuc-
cinate) and
B: latex sensitized with anti-estriol 16,17-disuccinate antibody combined
0 (g) A: latex linked to (polyacrylic acid combined with estriol 16-glucuronide)
and
B: anti-estriol 16-glucuronide antibody
(h) A: la~ex linked to (polyacrylic acid combined with 17-amino-1,3,5(10)-
estratrien-3-ol) and
B: latex combined with anti-estriol 16-glucuronide antibody
(i) A: polyacrylic acid combined with estriol 16-glucuronide and
B: latex combined with anti-estriol 16-glucuronide antibody
(j) A: polyacrylic acid combined with estriol 16,17-dihemisuccinate and
B: latex combined with anti-estriol 16,17-dihemisuccinate antibody
0 ~k) A: methyl vinyl ether/maleic anhydride copolymer combined with estriol 16-
glucuronide and
B: latex combined with anti-estriol antibody
(1) A: methyl vinyl ether/maleic anhydride copolymer combined with estriol 16,
17-dihemisuccinate and
B: latex combined with anti-estriol antibody
(m) A: polyacrylic acid combined with estriol 16-glucuronide and
- 25 -
~3~53
26 -
B: l~tex ~ensiti,.ed with anti~e~trio' 16
glucuro~ide an'c,ibody or erythrocytes sensitizecl
~ith anti-estriol 16-glucuro~ide antibody
(n) A: polyacrylic acid combine~ with estriol
16,1';'-diher~lisuccinaSe ancl
B: latex combined with anti estriol 16,17-
dihe~isuccinate antibody or erythrocytes
sensitlze(l with anti-estriol ].6,17
dihenli.3uccinate antibocly~ (o~ A~ polyacryli.c acid combined with 17-amino-
1,3,5(10) estratrien 3 ol and
B: latex combined wlth anti~estriol 16
glucuronide antibody
2. Irnmunochemical assay reagents for the
deterraination of preDnandiol consist:ing of A and B:-
(a) A: latex linl~ecl to ~polyacryli.c acid combined
with pregnandiol 3~glucuronide) ancl
B: latex combined with anti-pregnandlol 3-^
glucuronide ant:i.body
(b) A: latex linked to (methyl vinyl ether~n~laleic
anhydride copolymer combined with pregnanediol
3~glucuronicle) and
B: latex combined with ant:i~pre,gna1lediol 3
glucuronide antibody
(c) A: latex linked to (polyacrylic acld combined
wit;h pregnanediol 3-glucuronide) and
B: latex sensitized with anti~pregnanediol
3-glucuronide antibocly
(d) A: latex linked to (polyacrylic acid combined
witlh pregnanediol 3-glucurorlide) a,ncl
B. anti-pregnanecliol 3-glucuronide antibody
(e) A: latex linked to (methyl vinyl ether/maleic
ar.hydride copolymer combined with pregnanediol
3~glucuronide) an~i
B: anti-pregnanediol 3-glucllronicle antibody
(f) A: po].yacrylic acid combîned with pregnanlediol
3-glucuronide and
;
~;3653
27 -
~: latex combined with anci--pre.L^n~ e~iiol 3~
Lrlllcuronide antibody
(g) A: n-ethyl vinyl el;her/rQaleic anhydride copolymer
combined with pregnanediol 3-glucuronide and
5]3: latex combined t~ith ant-L~prsgnanecliol 3-
,rlucuronide antlbodY
(h) A: polyacrylic acid combined with pregnanediol
3~-&~1uc~ronide
B: latex serlsici%ect witl anti-prer.-!anediol
103 glucuroni.c1e antibody
(i) A: polyacrylic acid combined with pre~nanetriol
3-glucuronide and
B: latex combined with anti--pregnanetriol
3-~glucuronide antibody
(j) A: methyl vinyl ether/maleic anhydride copolyrler
combined with pregnanediol 3~glucuronide and
B: latex combined ~ith anti~pre~nar!ediol 3
glucuronide antibody
3. Imlnunochemical assay reagent.C~ for the
determination of 17~-0HCS consisting of A and B:~
(a) A: latex linkecl to ~polyacryli.c acid combined
with 3a,11~,17a,21-cecrahydroxypregnan-20 one
3-glucuronide) and
B. latex combined with anti--3a,11~,17~721-
tetrahydroxypregnan 2-one 3-~glucuronide
ancibocly
(b) A latex linked co (r.!ethyl vinyl ether/maleic
~hydride copolymer combined with 3~ ,17~ 9
21-tetrahyclroxypregnan-~20 one 3~glucuronide)
and
B: latex combined with anci~3c~711~,17a921-
tetrahyclroxypregnan-20-one 3r-glucuronide
arltibody
(c) A: latex lin~ed to (polyacrylic acid combined
with 3a,11~517~921~tetrahydr(jxypregnan~20
one 3-glucuronide and
,
~3~;~3
28 -
B: latex sensitized with anti~3~ ,17~,21-
tetrahydroxypregnan-20~one 3 glucuronide
antibody
(d) A: latex linked to (polyacrylic acid combined
with 3u,11~,17~,21-tetrahydroxypregnan-20-one
3-glucuronide~ and
~: anti~3~ ,17~,21-tetrahydroxypregnan~20-one
3-glucuronide antlbody
(e) A: latex linked to (methyl vinyl ether~rllaleic
anhydride copolymer combined with 3~ ,17~,21--
tetrahydroxypregnan~20~one 3-glucuronide) and
B: latex sensitized with anti-3~ ,17~,21-
tetrahydroxypregnan-20-one antibody
(f) A: latex lin~ed to (methyl vinyl ether/maleic
anhydride copolymer combined with 3~ ,17~,
21-tetrahydroxypregnan-20 one 3-glucuronide)
and
B: anti-3~ ,17~,21--tetrahydroxypregnan 20-one
3-glllcuronide antibody
(g) A: polyacrylic acid combined with 3a,1].~,1?~,21-
tetrahydroxypregnan 20~one 3-glucuronide and
B: latex combined with anki~3~ 91'7a, 21~
tetrahydroxypregnan20~one 3-glucuronide
antibody
(h) A: methyl vinyl ether/maleic anhydride copolymer
combined with 3~ ,17~,21 tetrahydroxypregnan-
20-one glucuronide and
B: latex combined with anti3a,11~,17~,21
tetrahydroxypregnan-20one 3-glucuronide
antibody
(i) A: polyacrylic acid combined with 3~ ,17a,21-
tetrahydroxypregnan~20~one 3-glucuronide and
B: latex sensitized with anti-3~ ,17~,21-
tetrahydroxypregnan-20~one 3-glucuronide
antibody
(j) A: polyacrylic acid combined with 3~ ,17~,21
tetrahydroxypregnan-20~one 3 glucuronide and
,' ~ ' '
.
3~3
- 2~ ~
B: erythrocytes sensitlzed wlth anti-3~ ,17~,
21-tetrahydroxypregnan~20--one 3-glucuronide
antibody
(k) A: met~Ayl vin-jl ether/maleic a~hydride copolymer
co~bined with 3~ ,17~ tetrahydroxy-
pregnan-20~-one 3-~lucuronide and
B: erythrocytes sensltized with anti-3~ ,17a,
21~-tetrahydroxypregnan-~20-one glucuronide
anl;ibody
4. Immunocheraical assay reagents for the determ.ination
of morphine consisting of A and B:-
(a) A: latex linked to (polyacrylic acid combinecl
with carboxymethylmorE~hine)
~: latex combined with anti~carboxyrrlethylmorphine
antibody
(b) A: latex linked to (methyl vinyl ether/maleic
anhydride copoiymer combined with carboxymethyl-
morphine) and
B: latex combined with anti-carboxyrlethylr,lorphine
antibody
(c) A: latex linked to (polyacrylic acid combined
with carboxymethylmorphine) and
B: latex sensitized with anti~carboxymethyl
morphine antibody5 (d) A: latex linked to (rnethyl vinyl ether/maleic
anhydride copolymer combined with carboxymethyl-
morphine) and
B: latex sensitized with anti-carboxymethyl
morphine antibody
(e) A: latex linked to (polyacrylic acid combined
with carboxymethylmorphine) and
B: anti-carboxymethylmorphine antlbody
(f) A: latex linked to (methyl vinyl ether/maleic
anhydride copolymer corîlbined with carboxymethyl-
morphine) and
B: anti-carboxymethylmorphine antibody
lg) A: polyacrylic acid combii1ed with carboxyraethyl-
rnorphine
S3
- 30 -
B: latex combined with anti~carboxyrnethylmorphine
antibody
(h) A: methyl vlnyl ether/rnaleic anhydride copolymer
combined with carboxymethylmorphine and
B: latex combined with anti~carboxymethylr,lorphine
antibody
(i) A: polyacrylic acid combinecl ~!ith carboxymethyl-
morphine and
B: erythrocytes sensitized w:ith anti-carboxymethyl-
morphine antibody
(j~ A~ methyl vinyl ether/maleic anhydride copolyrner
combined with carboxymethylmorphine and
B: erythrocytes sensitized with anti~carboxymethyl
morphine antibody
(k) A: polyacrylic acid combined with carboxymethyl-
morphine and
B: latex sensi.tized with anti~carboxymethyl-
morphine antlbody
(l) A: methyl vinyl ether/maleic anhydride copolymer
combined with carboxymethylmorphine and
B: latex sensitized with anti-carboxymethyl~
morphine antibody
5. Immunochemlcal assay reagents ~or the determination
o~ 17~KS consisting of A and B:
(a) A: latex linked to (polyacrylic acid combined
with etiocholanolone hemisuccinate) and
B: latex combined with anti-etiocholanolone
hemisuccinate antibody
(b~ A: latex linked to (methyl ~inyl ether/maleic
anhydride copolymer combined with
etiocholanolone he~isuccinate) and
B: latex coMbined with anti-etiocholanolone
hemisuccinate antibody
(c) A: latex linked to (polyacrylic acid combined with
etiocholanolone hemisuccinatej and
B: latex sensitized with anti~etiocholanolone
hemisuccinate antibody
~3~3
- 31 -
(d) A: latex linked to !.nethyl vinyl ether/,maleic
anhydride copolyr~er combined wit~
etiocholanolone he~isuccinate) and
B: latex sensitized with anti-etiocholanolo~e
hel,1isuccinate antibody
(e) A: latex linked to (polyacrylic acid combined
with etiocholanolone hemisuccinate) and
B: anti-etiocholanolone hemisuccll1ate antibody
(f) A: latex linl{ed to (methyl v:lnyl ethertmaleic
anhydrlde copolymer combined with etio-
cholanolone hemisuccinate) and
B: anti-etiocholanolone hernisuccinate antibody
(g) A: ~olyacrylic acid co~bined with etiocholanolone
hemisuccinate and
B: latex combined with antietiocholanolone
hemisuccinate antibody
(h) A: methyl vinyl ether/maleic anhyclride copolymer
combilled with etiocholanolone hemisuccinate
and
B: latex combined with anti etiocholanolone
hemisuccinate antibody
(i) A: polyacrylic acid combilled with etiocholanolone
hemisuccinate and
B: late~ sensitized with anti-etiocholanolone
hemisuccinate antibody
(j) A: methyl vinyl ethertmaleic anhydride copolymer
combined with etiocholanolone hemisuccinate
and
~: latex sensitized anti~etiocholanolone antibody
(k) A: acrylic acid corlbined with etiocholanolone
hemisuccinate and
B: erythrocytes sensitized with anti-etiocholanolone
hemisuccinate antibody
(1) A. methyl v,nyl ether/maleic anhydride copolymer
cor,lbined with etiochlanolone hemisuccinate
and
B: erythrocytes sensitized with anti etiocholanolone
antibody
S3
- 32 ~-
6. I~nrmunoc.!e;~ical assay rea~ents f'or 'che determination
of thyroxine ~T4) consistlng of A and B:~
(a) R: latex linked to (polyacrylic acid combined with
thyro~ine) and
5B: latex combined with anti~-thyroxine antibody
(b) A: latex llnked to (methyl vin,yl ether/maleic
anl1ydride copol.ynler combinecl with thyroxine)
and
B: latex combined with anti--thyroxine antibody
ln (c) ~: ].atex linked to (polyacrylic acid combined
with thyroxine) and
B: latex combined with anti~thyroxine antibody
(d) A: latex linked to (methyl vinyl ether and
rnaleic anhydride combinecl wi.th thyroxine) and
15B: latex sensitized with anti~thyroxine antibody
(e) A: latex linked to (polyacrylic acid combined
with thyroxine) and
B: anti-thyroxine antibody
(f) A: latex linked to (methyl vinyl ether/maleic
20anhydride copolymer combined with thyroxine)
and
B: anti~thyroxine antibody
(g) A: polyacrylic acid combined with thyroxine and
B: latex combined with anti~thyroxine antibody5 (h) A: methyl vinyl ether/maleic anhydride copolyIDer
cornbined with thyroxine and
B: latex combined with anti thyroxine antibody
(i) A: polyacrylic acid combined with thyro~ine and
B: latex sensitized with anti-thyroxine antibody
30 ( j) R: methyl vinyl ether/maleic anhydride copolymer
combined with thyroxine and
B: latex sensitized with anti thyroxine antibody
(k) A~ polyacrylic acid combined with thyroxine and
B- erythrocytes sensitized with anti thyroxine
35antibody
~1) R: methyl vinyl ether/maleic anhydride copolymer
combind with thyroxine
1~3~3
- 33 -
B: ery~hrocytes sensitized wlth anti-thyroxine
antibody
7. Irnmunochemical assay reagents Lor t'ne determlnation
of catecholamine consisting o~ A anl B:~-
(a) A: latex lin~ed to (polyacrylic acid combinedwith r!3etanephrine) and }3: latex combi.ned ~lith anti~metanephrine antibody
(b) A: latex linl~ecl to (methyl vi.ny]. ether/maleic
anhydricle copolymer com~bined with metanephrine)
and
B: latex cornbined wi-th anti~-metanephrine antibody
(c) A: latex linked to (polyacryllc acid combined
with metanephrine) and
B: latex combined with anti~metanephrine antibody5 (d) A: latex linked to (methyl vinyl ether/rr,aleic
anhydride copolyr;ler combined with metanephrine)
and
B: latex sensitized with anti-metanephrine
antibody
~Q (e~ A: latex linlced to (polyacrylic acid combined
with metanephrine) and
B: anti--metanephrine antibody
(f) A: latex linked to (methyl vinyl ether/maleic
anhydride copolymer combined with Metanephrine)
and
B: anti~metanephrine antibody
(g) A: polyacrylic acid combined with metanephrine and
B: latex combined with anti~rnetanephrine antibody
rh) A: methyl vi.nyl ether/maleic anhydride copolymer
combined with metanephrine and
B: latex combined with anti~metanephrine antibod~
(i) A: polyacrylic acid com.bined with metanephrine and
B- latex sensitized with anti metanephrine antibody
(j) A: methyl vinyl ether/maleic anhydride copolymer
combined with .netanephrine and
B: latex sensitized with anti~rnetanephrine antibody
(k) A: polyacrylic acid combined with metanephrine and
3~3
34 .
B. erythrocytes .sensitize~ ith anti~me~anephrine
antibody
(1) A: l~ethyl vinyl ether~aleic anhydride copolymer
combined with metanephrine and
B: erythrocytes sensitized with anti~metanephrine
antlbody
The reagents in accordance with this invention,
however, are not limited to these .speclflc exalnples.
In the examples 1 to 7 above, polyacrylic acid
is mainly glven as the holder of the hapten bound holder
in A. If' deslred9 it can be replaced, for example, by
a methyl vlnyl ether/rnaleic anhydrlcls copolymer, and
in these examples, antibody-sensitized erythrocytes can
be used instead of the antibody-sensiklzed latices.
]5 Iml.nunochemical assay usin~ the novel reagent
of this invention can be performed in the f`ollowing
manner.
Traces of` haptens existing in urine, blood or
other body fluids can be eas.i].y and rapidly determined
by using the reagents of thls invention. The theory of
determination is based on an agglvtination inhibiting
reaction in which a hapten to be determined inhibits
an agglutlnation reacti.on between the hapten bound
carboxyl-containing mono-olefinic polymeric compound or
the latex combined with the hapten-bound carboxyl-
containing mono-olefinic po].ymeric cornpound and the
hapten antibody or the hapten antlbody-~sensitized (or
chemically bound) carrier, especially a hapten antibody-
bound latex.
Specific procedures of determination areshown in Examples to be given hereinbelow. General
methods of determination are as follows:
(1) One drop of an'assay sample (optionally
diluted) is placed on a clean slide, and then one drop
of a suspension of ~he aforesaid hapten antibody or
the hapten antibody-sensitized (or chemically bound~
~ ~3~i3
-35 --
latex i.s put onto 'che assay sample. l'he two are fully
mixed, and then one drc,l;~ of a SOlUtiO'l of the hapten~bound
carboxyl-contair.ing rnono-olefinic ooly.,leric cor,!pound or
a suspension o.f 'Ghe latex corl!bined l^Jith the hapten-~bound
polymeric compound is p~lt into the ~nixture. The slide
is rocked for 2 !~inutei~-i, and observecl~Jith tl1e naked e~e.
'I`he presellce of` an a~glutinated pattern is ~judf,ed to be
i~n-3gatiVe~ 9 and the presence of` an ag.~:luti.natiol1 inhibited
pattern (non~a2glutinated pattern? 7 i~po~siti.Ve;i .
The hapten concen'cratj.()n in the assay sample
can be determined by performing the cest ai'ter optionally
diluting the assay sample, and mul'ciplyirlg the maxi~^ilum
ratio of dilution at which a positivc paktern appears by
the assa~ sensitivity,
The assay method in accordance with this in~
vention is not lir~ited to the above one, For example,
it is possible to dissolve a predeterm~ined amount of
the hapten--bound product in a liquid ror diluting 'che
assay sample~ and react the dilu.~ecl assay sample with the
~0 antibody-~sensitized (or cheI~ically houncl) latex.
(2) 0.1 ml of an assay sample (optionally
cliluted~ is put into a clean round-bottorned test tube 9
and 0.3 mll of hapten antibody--sensiti.%ed erythrocytes
are added, They are fully s~irrelg and then 0,1 ml of
a solution of the hap'cen--bouncl carboxyl~containing water
soluble r,nono~olefinic polymeric co~lpound is added.
After stirring 9 the test tu.be is a.llowed to stand for
2 hours on a stand equipped with a mirror, Tl1en 9 the
hapten is determined by a pattern forrned at the bottorn
of the test tube. At this time 9 the agglutination
inhibited pattern forms a sedir,1entation rirK; and the
agglutinated pattern looi~s like a rila'c,
The method of this invent.ion is not lilllited
to these eri~bodiments. E~or exarilple, i'c is also possible
.~ 35 to dissolve a predeterrnined arnount o~ --he af`oresaid
6~ hapten bound product in a liquid for ~ , the assay
sample, and react prede'cer~ined amollnt of the diluted
S3
- 36 -
assay sample with the hapten antibody sensitiY,ed
erythrocytes.
Characteristic fea ures ancl advantages of
the invention
The characteristic feature of the reagent of
this invention is a combillation of
~A) a carboxyl-containing water-soluble
rnono-olefinic polyl,leric compound combined wlth a hapten
or its cheD~ically -,loclified procluct (to be soMetimes
referred to hereinbelow as component A 1), or a hapten-
supported latex (to be sometim.es referred to hereinbelow
as component A~2! resulting from the chemically binding
of the component A-l to a polymeric latex having a particle
diameter of about 0.01 to about 2 microns9 an~l
(B) a hapten antibody ~to be sometimes referred
to hereinbelow as con~ponent B^l! 9 or a hapten ankibody-
supported carrier ~to be some'cimes referred to hereinbelow
as component ~2) comprising a carrier sensitized or
chemically bound with the hapten an-cibody.
~hen the component A~l is used as the component
(A), it must be used in combination with the component
B-2.
Thus, examples of the combination of component
A with component B in this invention are as follows:
(1) a combination of component A-l with co--nponent
~ 2,
(2) a combination of component A~2 with component
E-l,
~3) a combination of component A-2 with component
B 2.
Of these 9 the combinations (2) and (3) are
preferred, and the combination (3) is especially preferred. ~-
In the method of determining haptens by an
immunological latex agglutination inhihiting reactiont
i'c has been the usual practice to use an agglutination
reaction system involving a hapten antibody and an
antigen-sensitized latex obtained by sensitizing a
3 ~5
3'i
poly;nerlc latex with an antigen resultin~ froI~ the bind--
ing of a '~!apten to a stronglv an'cigenic subs'cance such
as bovine serunl albur~.in, hulan seru!l` alburlin, bovine
gam~a~globulin or 'cetanus toxin~ and an agglutination
reaction syste!!l involving a hapten anti.bocly-sensitized
latex and the aforesaid strongl~ ankigenic substance
having a hapten bound theret,o. It llas no~l been found by
the present invention that the various characteristics
ancl advantages described below call be obtained by using
t,he carboxyl~containing water-~-soluble !lono--olefinic
polyYileric cor~pouncl ~Jhich is different fro~ the
conventionally used natural proclucts having strong
antigenicity such as protelns or ..nalog,s thereof',
chemically binding a hapten to sllch a polyrîleri.c coa~pound
(to forn~ cor.1ponent A~ , or further bi.nding 'che cor,lponent
A 1 chernically to a polyl~eric latex (to form component
A~2).
The cor~ponents A---l and A~2 show, very high
stability ln the form of an aqueous solution or aqueous
suspension and can f'ully withstand storage at room
temperature.
In the conventional reagents of this type,
the aforesaid natural products or analogs thereof are
used for combination with haptens~ and therefore in long~
term storage, these substances combinecl ~Jith haptens are
not stable because of dena~uration or decol~position.
Hence, the hapten~bound products which have been usecl
so far are unstable in the fori,~ of solution 9 and cannot
be stored even for a shor~ period of tii,le and in a cold
place. For this reason, they have to be storecl only
after lyophilization, Also, if' the latex sensitized with
hapten-bound holder is ].yophilized~ the properties of
the latex itself` will. be changed and r.~ay cause a
nonspecific agglutination reaction. I'hus9 in practice~
it cannot be lyophilized, and has to rely on storage in
a cold place.
In contrast1 the components A-l and A~2 used
3~S3
in this invention are very stable because they are prepared from carboxyl-con-
taining water-soluble mono-olefinic polymeric compounds which are quite irrel-
evant to the constituents of animals.
When -the carboxyl-con-taining mono-olcfinic polymeric compound combined
with a hapten is linked to a polymeric latex (component A-2), the product (com-
ponent A-2) has very high stability because of strong chemical binding to the
polymeric latex different from the conventional sensitization.
The method of this invention can achieve the desired assay in a short-
er reaction time than the conventional assay methods, and has a higher assay
sensitivity. The conventional methods require a reaction time of 3 to 5 min-
utes, and when the amount of the reaction liquid is small or when the reaction
time for assay reaches nearly 5 minutes owing to the environmental conditions
(e.g., temperature, humidity, etc.) of a room where the determination is made,
the reagent is apt to be dried, and a nonspecific reaction pattern tends to form
from its periphery. According to the method of this invention, determination
can be easily made in 2 to 3 minutes in the agglutination reaction systems (1)
and (2) described above. In particular, in the agglutination reaction system
(3), determination can be made easily in 1 to 2 minutes.
By a later agglutination inhibiting reaction, haptens can be deter-
mined by the inhibited pattern, as stated hereinabove, but the intensity of theagglutination pattern constitutes a factor for determining the ease of viewing
and judgement. According to this invention, an agglutinated pattern appears far
earlier than in the conventional methods, and moreover, the agglutinated pattern
can be clearly distinguished from an agglutination inhibited pattern. This is
presumably because the reactivity of the reagent with an antibody is increased
by the use of the carboxyl-containing
- 38 -
~3~;53
- 39 -
water-soluble Mono~olefinic polyrneric compound as a
holder unlike the conventional methods in which natural
products or the analogs thereof are vsed. I~ith the
agglutination system (3) described nereinabove, the
aforesaid effect can be expected co lncrease because
the antibody is bound to a latex ancl the antibody-bound
latex directly participates in the aggl-ltination reaction.
In the conventional agglutination reaction, the antigen-
sensiti~ecl latex reacts with clilutecl antiserum, and the
antibody which has reacted with che antigen of the
antigen-sensitized latex further reacts with the
antigen~sensitized latex. Thus, the agglutination of
the antigen-sensitized latex gradually grows and finally
forms an agglutinated pattarn that can be viewed with
the naked eye. In contrast, in the agglutination system
(3) in accordance with this invention, the antibody is
also bound to the latex, and therefore an antigen-antibody
reaction between the hapten bound to the surface of the
latex through the holder and the antibody supported on
the latex directly participates in agglutination. The
agglutination of the hapten and antibocly through the
latex grows, and ar. agglutinated pactern forms earlier
and more strongly. Moreover 9 the agglutination inhibiting
reac~/~n
actio~ can be discerned clearly and rapidly.
The carboxyl-containing water-soluble mono
olefinic polymeric compound (holder), and the component
A 1 obtained by chemically binding a hapten or its
chemically modified product to the holder clo not sub-
stantially have antigenicity and are immunologically
inactive, as shown in Referential ~xamples given herein~
below. It is believed therefore that the cornponent
A~2 which is obtained by chemica]ly binding the hapten-
bound holder ~component A-l) to a polyrneric latex neither
has substan-tial ancigenicity, and is therefore
immunologically inactive
In the prior art, a procein or its analog
having antigenicity is used as a carrier and is
~436S3
sensiti~ed with a hapten. In contrast, in the present invention, by using a
product (component A-l) obtained by chemically binding a hapten or its chemical-
ly modified product to an imm~mologically inert holder substantially having no
antigenicity, or a product ~component A-2) obtained by chemically binding the
component A-l to a pnlymeric latex, it is possible to obtain an imm~mochemical
assay reagent having very high storage stability and to determine the agglutina-
tion reaction of the hapten-bound antibody and the agglutination inhibiting re-
action, rapidly, accurately, and with high sensitivity.
In the present invention, the assay sensitivity of the reagent can be
adjusted to a level suitable for a subject to be determined by adjusting the
amount of the hapten to be bound ~o the carboxyl-containing water-soluble mono-
olefinic polymeric compound, the amount of the hapten-bound carboxyl-containing
mono-olefinic polymeric compound to be bound to the latex, and/or the ratio of
dilution of the corresponding antiserum or the amount of the antibody to be
bound to the polymeric latex; or by changing the concentration of rabbit serum
albumin, goat serum albumin, bovine serum albumin, etc. to be added at the time
of preparing the final latex; or by using a suitable combination of the above
methods.
Table 1 balow shows a comparison of the stability of the reagent of
this invention with that of a conventional reagent in determining urinary estro-
gen.
~ A) A combination of a solution of polyacrylic acid combined with 17-
amino-1,3,5~10)-estratrien-3-ol and a latex sensitized with anti-estriol 16-
glucuronide antibody (the reagent of the invention).
(B) A combination of a solution of bovine serum albumine combined with
estriol 16-glucuronide and a latex sensitized with anti-estriol 16-glucuronide
antibody (conventional reagent).
To compare the stability of (A) with that of (B), the assay sensitiv-
ity of each reagent is adjusted 40
r
l~
~3~3
_- 41 _
to 0.1 llg/ml. The solution of polyacrylic acid combirled
with l,-ar~ino 1,3~5(10)-estratrien--3-ol in (A), and the
bo~ine serun albu~.ln-~*~ee` with estriol 16~glucuronide
in (~) are storeci at 4C 9 or at room terlperat~lre, anrl
the antibocly--sensit:izecl latex is stored at 4C. The
stabilities of these solutions ~A) and (B) are cornpared.
Tab~e 1
, . ..... ... ___~ _
Assay sensitivity
Storage tenper-~ ._ _ _ __ _ _
ature 1 month 3 months 6 ~on'chs 12 months 24 ~onths
~ ~ .. . ,. ~_~. ~
(A) 4C 0.1 0.1 0.1 0.1 0.1
(B) 4C 0.1 0.1 0.0~ 0.05
__ _ __._
l.~onth 3 months 6inonths 12 months 24 nonths
.. ._~ _ __ ~ . __~
(A) room temper~ 0.1 0.1 0.1 0.1 0.1
ature
(B) room temper-~ 0.1 0.05 .. .
ature I ¦
- . _._ ~ . ... _ ... . _ ~
As shown in Table 1, (A) is stable even after
storage for 24 ~lonths at room -telnperatura9 but with (P),
the sensitivity increased af'ter s-corage at 4C for 6 to
12 months and after 24 ~onths 9 no reaction ls noted.
This tendency is rlorc clistinct during storage at; room
temperature. The sensitivity is douhled after storage
for 3 months 9 ancl after 6 inonchs 9 no reaGtion is noted.
In other words7 in (B) 9 agglutination with the antibocly-
sensitized latex becoines weal~ as 'che ti~ne elapses 9 ancl
the apparent sensitivi~cy increases. After storage for
24 months at 4C and 6 months at rooin teïlperature 9 a
reaction wllich intrinsically should S'ilOW ag~i7,l-ltinatiOn
does not 'cake place. ~t is seen fro~ these clata that
far higher stability can be obtalnecl by bindinF a hapten
'co a carboxyl containing water-soluble mono~olefinic
poJymeric compound than by blnding it to a protein.
Table 2 sho~is a comparison of the assay
sensitivity and the reaction ti~-ne between the method of
this invention and the conventional method in cleter~llining
urinary estrogen.
.: .
.
- 42
Table 2
___ _ _ ,
ri,e'chod of deter~ination Assay Reactior. ti--;e
sensitivity (.~.inutes)
( ~ 1 )
?-l thod of the inventlon 1 10 2 1 - 2
Method of the :invention 5 x 10 2 2
_ .. ,.. ~. ~_ ~ _ _
Conventiona:L ~ethod 5 x 10 1
,_ _
The i~.ethod ~A) of t'~lS lnvention is performed
by using a lysine--la'cex lin!~ed to polyacrylic acid
combined witll l',--amino--l.93~5(10)-estratri.en-3~ol and
a latex sensitized with antl-estriol 16~glllcuronide
antibocly. The methocl (B) of this inven'cion is performed
by using a lysine~],atex linked t,o pol~racrylic acid
, corlbined with 17~amino lf3,5(103--estratrien~3-ol and
diluted anti~-estriol 16--glucuron,i.de antiserul;l, The
conventional method is perfor~ned by using a latGx
sensitized with estriol 16-gluc-lronide~3S~ ancl dilutecl
anti-estriol 16-glucllronide antiseru!:l. As shown in the
table, 'che methods of this invention has 20 times (in
the case of A) and 10 times (in tlle case of B) as high
a sensitivity as the conventional ~ekhod, and t'ne reaction
til,le can be shortened to 1~25 to 1/5 of that of the
conventional metho(l. When an atkel-flpt is rnade in the
conventional r.1e'chod to increase the sensitivity further,
the agglutinated pattern, whlch is a negative pat'cern,
becomes weak. It is presun~ed fro.il this that ~he high
sensitivity in 'che present invention is due to the
increased reactivity of the antigen with an an'cibody as
a result of using the carboxyl~con'Gaining water-soluble
mono-olefinic poly~eric compound. Furthermore 7 according
to 'che ~ethod Gf this inven'cion, 'cransition from an
agglutination inhibited pattern 'co an agglutina'ced
pattérn i.s .n1ore distinc'c than in the conventional method.
S3
- 43 -
Moreover~ the margir! of concentration in transitiGn is
narro~er and the nethocl of this inverltion ila3 a ~reater
ass~ying ability.
Urinary estrogen which reflects the function
of the pl~centa of a pregnant wo~lan is rlainly cstriol
16-glucllronide. Stan(ard solutiors of estriol 16-
glucurol1ide shown in Table 3 are ~repared using glycine-
b~lffered saline solution9 and the distinctness of`
transition from zn a~lutlnation inhibited pattern to
an agglutinated pattern is deterlnined both in the method
of this invention and the conventional lethod. The
results are shown in Table 3. The ~nethod (A) of this
invention is perforlned by using~ a lysine-latex linked
to polyacrylic acid combined ~ith estriol 16 ~lucuronide-
lysine and ~ latex combined with anti-estriol 16~
glucuronide l~ntibody. The rnethod (B) of the invention
is performed by using the aforesaid linl~ed lysine-latex
and diluted anti--estriol 16 glucllronide antiserum. The
conventional r;~ethod is performed by IlSirl~, a latex
sensiti~ed with estriol 16 glucuronide--RSA and diluted
anti-estriol 16~glucuronide antiSerUIn. In these methods,
the sensitivity is adjusted to 0.1 g/.nl prior to the
testing .
Table 3
_ _
_ Concentration (~/rl as estriol) of
f~ Method of an estr iol lE glucu ^onide soluti )n
determination 0.2 0.12 0.10 0.08 0.06 0.04 a
__ .
l~lethod (A) of _ ~ _ ~- ~ ~ +-~
the invention
__ ~ . . _. _
Method (B) of
the invention ~ _ . rr to ~-l-+ ++~
~ _ ~ __
Conventional ~ _ _ ~ ~
method ~ ~ ~ _ _ _
1~3~53
- 44 -
The scale of evaluation is as follows.
-: a complete aggl~ltination inhibited pattern
-~: an agglutinated pattern slightly observed
visually
~ can be di~ferentiated from an agglutinated
pattern with the na!Qd eye
;-: a cleclr agglutinated pattern
In the above table 7 the forlnation o~ an
agglutinate~ pattern is shown by - in order to cor,'pare
the intensities of the agglutinated patterns.
As shown in the table 7 in the conventional
method, the agglutinated pattern shows a wea'~ intensity
even when usin~g only a buffer solution not containing
estriol 16-glucuronide, and the margin of concentration
which permits transition from an inhibited pattern to
an agglutinated pattern is large. ~ccording to the~ r,~ethod (B) of this invention9 the margin of concentration
fr~ss~on
i for taneltie~ is clearly narrower than in the conventional
method. In the method (A) of this invention, transition
from an inhiblted pattern to an agg]utinatod pattern is
clearly noted with a concentration rdargin of 0.02 ~g/;~
This effect is also considered to be clue to the use of
the water-soluble carboxyl containing mono oleflnic
polymeric compound.
In the conventional determination of haptens
by a latex agglutination inhibiting reaction, an antibody
to a hapten can be obtained for the first time by binding
a strongly anti~enic protein, etc. to the hapten and
immunizing a mammal with the resulting bound product
because the hapten itself cloes not have antigenicity.
Thus, it has been considered to be essential to sensitize
a latex with a product obtained by binding a hapten to
a similar strongly antigenic protein9 etc. In the
present invention,binding to a stron~ly antigenic
substance is essential as in conventional methods when
preparing an antibody to a hapten. However9 for binding
to a latex a substantially immunologically inactive
~3~53
- 4~ ~-
carboxyl~-containin~ water solubl,e -~orlo-olefir,ic polynn.eric
compound is used irrespective of' '~,he degree Gf its
antigenicity. As a result t the aforesaid charactesristics
and advanta$es not seen in the prior art can be obtained.
Furtllerrnore, by usirlg the correspondin r antibofly ln the
form bonded to the latex, or sensitizing the latex with
the antibody 9 the utility of the ar!tibody is further en-
hanced .
The followin~ Referentlal Examples show that
the water-soluble carboxyl~containing !llono~olef:inic poly-
meric cornpounds and the products obtained by binding
haptens or their chemically modified products to these
polymeric co.~pounds as holders (component A l) are
substantially devoid of antigenicity, or are substantial-
ly inert im~unologically.
Referential Exarnple 1
Antigenicity of holders~
(a) Imrnunization
As exarnples of the holder 3 polyacrylic acid
(PAA for short) having a ~olecular weight of 29000,000 9
polyacrylic acid having a !lolecular weigh'c of 250,000
poly(sodiurn acrylate) (PAA.Na salt for short) having
a molecular wei~ht of about l,500Sn009 and BSA (as a
control) were each dissolved in an amount of 2 mg in l
ml of physiological saline, and emulsifiecl with the sarneamount of co~plete Freund 9 s adjuv.lnt. The eTnul~sion was
injected into the subcutaneous site and the paw of
~ature rabblts. The injection was perfor~ned eight times
at two week intervals on ~r,roups each consisting of two
rabbits. Blood lettin,~r was conducted on a trial basis
from the test anir~als after three injections on the
seventh day after each injection 9 ancl the sera were
separated.
(b) Searching of antibodies
Each of the sera obtained in (a) was tested to
deterrnine the presence of an antibody by a precipitation
reaction in agrar ~el in accordance with the Ouchterlony
3~S3
- 46 ~
method and counter im~.lunoelectrophoresis (CI~'. The
results are sho~ln in 'i'able 4. It is seen fro~ the results
that ~ith nSA as a control, a clear precipitation pattern
was seen to form in the sera obtaine~ in all runs both
by the Ouchterlony rlethod and the counter ir:lmunoelectro-
phoresis, thus showing the formation of anti-BSA antibody.
ith PAA, however, no precip:itati.on patterr ~Jas seen to
forlll in any of the sera testcd, and anti--~AA a.ntibody ~1as
not detectecl.
T.able~ 4
Holder ~loocl Blood _ _ _ Total
letting letting sera
1st 2nd 3rd 4th 5th
. _ _ _ _ .
Ouchterlony ~?~ .. _ .. _
PAA CIE _ . _. _ ~
(~1: 2,000,000) Ouchterlony _ ._ _ _ . __ ~ _
C'IE - _ ~ _ .
_ . . _ _, _ _
Ouchter].ony ~ ~ ~ ~ _ _
~_ __ _ _
PAA CIE ~ ~ _ _ _
(~W: 2509000) ~ - _ _ _ =
_ ~ __ _~
Ouchterlony _ . ~ ~ j _ .
PAA.Na salt ~ ~ _ _ _
(~^1: about CIE _ ~ ~. ~
,5009000) _ _ _ _ _ _.__ _ _~ _
Ouchter~ony - -- ~ - ~
~__ _ __
Ouchterlony _ _ _ ~ . ¦ -
~SA CIE * -:- * . ¦ ~ + ¦
(ErY Fatty ~ __ _ _ l _____~
CTr -----= =
A precipitation pattern not for)!ed.
*: A precipitation pattern f'or~ed.
3f~S3
The concentration of each of the holders and BSA in the Ouchterlony
method and CIE was 5 mg/ml (Veronal buffer pll 8).
Referential Example 2
Antigenicity of a hapten-holder bound product:-
(a) Immunization
E3G-polyacrylic acid (molecular weight 250,000) bound product produced
by the same way as in Example 1 (e-3) given hereinbelow was used as an example of
the hapten-holder bound product, and E3G-BSA produced by the same way as in Ex-
ample 1 (a) was used as a control.
Each of these products was dissolved in an amount of 2 mg in 1 ml of
physiological saline, and emulsified with complete Freund's adjuvant. Each emul-
sion was subcutaneously administered to rabbits at the back. The injection was
performed 10 times at two week intervals on groups each consisting of two rab-
bits. The blood was collected on a trial basis from the test animals after com-
pleting injection three times on the seventh day after each injection, and the
sera were separated.
~b) Search mg of anti-E3G antibody
Each of the sera obtained in ~a) above was tested to determine the
presence of anti-E3G antibody by radioimmunoassay using 3H-E3G. Each serum was
diluted with a borate buffer ~containing 0.06% BSA and 0.05% bovine gamma-globu-
lin) having a pH of 8.0 to 10 times, 50 times, lOO times, and subsequently to
dilute to 204,800 times by a multiple dilution method. The antibody titer was
determined by the following method using the diluted sera.
10,000 dpm of a methanol solution of 3H-E3G was taken into a test tube,
and evapo-rated to dryness in a stream of nitrogen. Then, 0.25 ml of each of the
diluted sera obtained as described above was put into the test tube, and after
shaking well, reacted at room temperature for 30 minutes. Then, 0.25 ml of sat-
urated ammonium sulfate was added and mixed well with the reaction
- 47 -
~3~53
- 4~ -
mixture. After s'canfling for 10 minutes, khe mixture was
centrifuged for 10 minutes at a spee(l of 3,000 rpr..
Then, 0.2 ml of the supernatant liquid was taken into a
vial J and 10 ml of a dioxane scintillator was put into
it. Radio activity was measured by a liquid scintillation
counter, and the ratio of 3H~E3G bonded was calculated.
In the groups given E3G~BSA, the ratio of
dilution of the serum which showed a bonding ratio of
~lore than 70% at the ti~e of blood letting was r~ore than
3205. But in the groups to which E3G-PAA was administered,
the bonding ratio was only about 10% even when the serum
was diluted to 10 times. This dilution ratio was about
the same as that which was obtained when normal rabbit
serum (I~RS) was diluted si~ilarly and reacted.
Figure 1 attached to this application shows the
titers of the antl--sera which were detern~irled after 10
injections.
Figure 1 attached to this application shows
the titers of the antisera obtained from the blood samples
which were collected after the 10th adrïlinistration of
R3G-BSA and E3G-PAA. The solid lines with circular
marks show the titers of antiE3G~BSA antibody 7 and the
broken lines show those of anti-E3G PAA antibody. The
solid line with triangular rnarks shows the titer of NRS.
It is seen fro,-n the graph that the two rabbits
to which E3G-BSA was administered ,showed a high titer in
both, but the two rabbits given E3G~PAA clid not show an
increase in titer as obtained in the first blood col-
lection, and the titer was much the same as that of NRS.
It is conçluded therefore that in the adMinistration of
E3G-PAA, antibody to E3G ( i.e., hapten) was not formed 9
and the carrier effec'c of PAA was not recognized.
Combination of co~ponents A~2 and B-2
r
Exarnple 1
Determination of es'crogen in a pregnant
woman urine (I):-
(a) Procluction of estriol 16 glucuronide-BSA
Estriol 16 glucuronide (40 mg) was dissolved
..
.53
-- 49 ~
in 1 ml of r~ l-di!,1ethy]formamide5 and at less than 4C,
20.6 Microliters of tri-n-butyla:line w29 added.
Furtherr1ore, 11.2 microliters of isobutyl chloroformate
was added, and the mlxture was stirred for 30 minutes.
To the m.ixture l~as addcd a solution which hacl been
prepared by adding 150 microliters of lM aqueous sodium
hydroxicle sol;ltion to a solution of 117 mg of BSA
(bovlne serum albumill) in 2.8 ml of water and then
adding 2.0 ml of dimethylformar~lide and had been
maintained ac 8C. The mixture was then stirred at
8C, ancl one hour later, 16.6 microliters of lM aqueous
sodium hydro~ide solution was added. The mixture was
further stirred for 3.5 hours. Then, on a coluriln of
B Sephadex G-25, the unreacted estriol 15-glucuronide and
low~molecular weight reagents such as tri n~butylamine
were separated. The residue was dialyzed against
purified water, and then lyophilizecl to afford estriol
16-glucuronide-BSA. r~hen this lyophilized powder of
antigen was examined by the r'ober 7 S reaction, it was
20 confirmed that 27 to 30 moles of estriol 16-glucuronide
were bound per mole of BS~.
(b) Production of_anti-estriol 16~glucuronide
antibody
Two milligram.s of the estriol 16~glucuronide-
BSA produced in (a) above was disso]ved in 1 ml of
physiological saline, and emulsified with the same amount
of complete Freund9s adjuvant. The emulsion was injected
to the paw and subcutaneous site of mature rabbits. The
injection was performed at one ,-month intarvals. After
confirming the increase of 'che an'cibody titer, the total
blood was collected and antiseru.n was obtaine~. The
antiserum was immobilized at 56C for 30 minutes and
caused to be absorbecl by BSA. Subsequent salting out
with ammonium sulfate afforded anti-estriol~16 glucuronide
antibody.
(c) Production of a latex sensitized with anti-
estr _1 16-glucuronide antibody
Four milligrams of the anti-estriol 16
~e ~ c
1~3~53
- 5o
glucuronide antlbody produced in (b) above was clissolved
in 5 ml of a glycine buffered sodium chloride solution,
and 1 ml of a 10% polys'cyrene latex was added and mixed.
The mixture was 'created at 56C for 30 minutes. The
treated mixture was then centrifuged. The precipitate
was centrifugally washed with a glycine buffered sodium
chlori~le solution (pH 9.6). The precipitate was sub-
pended in 15 ml o~ a glycine-buffered sodium chloride
solution containing 0.05% of RSA (rabbit serum albumin)
to prepare an anti-estriol 16-glucuronide antibody-
sensiti~ed poly~ttyrene latex.
(d) Production of a reactive latex
_
(d--1) Lysine-latex
A solution of 260 mg of ~--tert butoxycarbonyl
lysine methyl ester in 3 ml of dirlethylformamide was
added to 5 ml of a 10% suspension of` a carboxyl-modified
polystyrene latex. The mixture was cooled to 0C, and
with stirring, 243 mg of 1-ethyl~3 (3-dirnethylal,linopropyl)
carbodiimide hydrochloride (a water-soluble carbodiimide)
was addecl. The mixture was stirred at 0C for 1 hour,
and at room temperature for 3 hours. Then, the mixture
was allowed to stand overnight at room temperature and
then centrifuged. The supernatant liquid was discarded,
and the precipitate was washed wi'ch a 50% aqueous solution
of dimethylformamide and then with water.
Then, 5 ml of ice cooled conc. hydrochloric
acid was added, and the mixture was al:Lowecl to stand
at 0C for 15 minutes with occasional shaking. It was
then diluted wi'ch ice water to about 2 times and
centrifuged. The precipitate was washed until the wash
liquid became neutral. Then, 10 ml of a 10% aqueous
solution of triethylamine was added. The .nixture was
stirred at room temperature for 15 minutes~ centrifu~ed
and repeatedly washed with water until the wash liquid
became neutral. Finally, the concentration of the
resulting suspension was adjusted to 10% to f`orm the
desired lysine-latex. This product showed a positive
~3~S3
result in a ninhydrin reaction. The product had the following structure.
NH2
(,CH2)4
CONH-CH-COOCH3
(d-2) Diaminoheptane-latex
2.5 ml of a 10% suspension of carboxyl-modified polystyrene latex was
centrifugedg and 17.5 ml of a O.OlM aqueous solution of aminoheptane was added
to the precipitated latex to suspend it. The suspension was cooled to 4C, and
with stirring, 33.4 mg of 1-ethyl-3-~3-dimethylaminopropyl)carbodiimide hydro-
chloride was added. ~Vhile the reaction temperature was maintained at 4C, the
mixture was stirred continuously overnight.
After the reaction, the reaction mixture was washed three times by
centrifugation and floating in water. Finally, the concentration of the latex
was adjusted to 10%. The product showed a positive result in a ninhydrin reac-
tion. Its structural formula was as follows:
¦latex¦ - CONH~CH2)7-NH2
(e) Production of a carboxyl-containing water-
soluble_mono-olefinic polymeric compound
combined with a chemically modified estrogen
~e-l) Production of polyacrylic acid combined with
17-amino-1,3,5(10)-estratrien-3-ol
The above aminosteroid (10.5 mg) and 100 mg of polyacrylic acid (aver-
age molecular weight of about 2,000,000) were dissolved in 5 ml of dimethylfor-
mamide, and 9 mg of dicyclohexyl carbodiimide (DCC) was added. Tne mixture was
allowed to stand at room temperature for 30 hours.
The reaction mixture was transferred into a cellophane tube, and dial-
yzed against 2 liters of distilled wa~er for 80 hours. The dialyzate was filter-
ed, and the filtrate was concentrated to afford an aqueous
53
~ C
solution Or the above-captioned product.
I:lhen tne aqueous solution was ccncentrated
to 8.0 rnl, it contained ~.26 mg/ml of the a~inosteroid
when dcterr1ined with light navin~!; a wa~elengGh of 280 m~.
(e-2) Production of a vinylDIethyl ether/m.aleic
anhydride copolymer combined with 17-amino-
1,3,5(10)-estratrien-3-~ol
PV~IA (100 mg) was dissolved in 5 ml of
dimethylformamide at an elevated temperature, and then
30 mg of the above~captioned anlinosteroid was added.
The resulting solution was allowed to stand at room
temperature for 4 clays.
The reac~ion mixture was transferred into
a cellophane tube, and dialyzed agaiNst 2 liters of
distilled water for 80 hours. The dialyzate was
filtered. ~hen the amount of the filtrate was adjusted
to 50 ml, it contained 0.44 mg/rnl of the aminosteroid
when determined with light having a wavelength of 280 m~. -
(e-3) Production of polyacrylic acid co~lbined
with estriol-16~glucuronide
(i) Estriol~16-glucuronide~lysine derivative
Estriol~16-glucuroYicle (23~ mg) ancl 233 mg of
~-benzyloxycarbonyl lysine methyl ester toluenesulfonate
were dissolved in 15 ml of dimethylformamide, and with
stirring under ice cooling, 164 n1g of dipLlenyl phosphoryl
azide and then 0.14 ml of triethy].amine were added.
After stirring for 1 hour at 0C 7 the mixture was allowed
to stand at room temperature for 48 hours. The reaction
mixture was dried under reduced pressure at a tzmper-
ature of below 40C. The residue was subjected topreparative thin-layer chron1akography to af`ford 240 Dlg
(65% of theory) of the desired product having the follow-
ing formula.
- 53 -
OH 1`!1-~-COOC~!2-~
O~l HO ~ ~OH C~l2)4
,O ~ O~CO ~ HC~I-COOCH3
~[~
HO
This product was not easily crystallized. lt gave a
single spot at Rf-0.55 (chloroforrn~methanol=5 1) in
silica gel thin-layer chroma'cography, and colored violet
by sulfurlc acid and orange red by ninhydrin.
~ii) The estriol~l6~glucuronide lysine derivative
(37 .mg) obtained in (i) above was dissolved in 15 ml
of a mixture of tertiary butanol and water (9 1), and
10 mg of 10% palladium-on~carbon was addecl. The mixture
was stirred in a stream of hydrogen at roorn temper-
ature and atrnospheric pressure. l~,~hen the reaction was
traced by thin-layer chromatography, the starting
material disappeared in 1.5 hours. Accordingly 9 the
catalyst was separated by f`iltration, and the filtrate
was washed. The filtrate ancl the wash liquid ~rere
dried under reduced pressure. Th3 resulting product and
100 mg of` polyacrylic acid were clissolved in 7 ml of
dimethylformarnide1 and 12.4 mg of clicyclohexyl carbodi-
imide (DCC) was added. The ~nixture was allowed to stand
for 48 hours at roorn temperature~ and then treated in
accordance with section le-l) above to afford 10 ml of
an aqueous solution of` polyacrylic acid cornbined with
estriol-16~glucuronide in an amount of' 2.01 mg/ml.
(e 4) Production of polyacrylic acid combined with
estriol-16~glucuronide (II)
(i) Estriol~16-glucuronide~hexar,lethylenediamine
derivative
Estriol-16~glucuronide ~93 mg) and 25 mg of
N-hydroxysuccinimide t~ere dissolved in 1.5 rnl of dimethyl-
for,narnide, and with stirring under ice cooling" 41 mg
,
.
~i~3ti~3
~ 54 --
of DCC was added. After a lapse of 30 r,linutes, a solu~
tion of 55 mg of monobenzyloxycarbonyl hexamethylenedi-
amine hydrochloride and 0.03 rl of triethylar~ine in 1
Inl of dimethylformamide was addec!. The mixture was
stirred f`or 2 hours urlder ice coollng, and for 12 hours
at room temperature~ Then, the mixture was evaporated
to dryness under reducecl pressure. The residue was
subjected to preparative thin layer chrorlatography to
afford 82 mg (55% of theory) of 'che above captioned0 product having the fol]owing structure.
OH
OEi HO l~ ~ ~OH
,O ~ COMH(CH2)6NHCOOCH
HO ~
This product showed a slngle spot at Rf=0.42
(chloroforlr~rnethanol =5:1) in silica gel thin-layer
chromatography.
(ii) F`ifty milligrams of the estriol-16-glucuronide-
hexamethylenediamine derivative obtained in (i) above
was dissol~ed in 3 r;11 of met'nanol~ and 10 mg of palladium
black was added. The mixture was stirred in a stream
of hydrogen at room termperature and atrnospheric pressure.
The reaction ended in 2 hours. The catalyst was separated
by filtration1 and the filtrate was concentrated under
reduced pressure. Addition of diethyl ether to the
residue afforded 35 mg of the desirecl product with the
splitting off of the carbobenzyloxy groupO
Ten milligrarns of this produc'c and 100 mg of
polyacrylic acid were dissolved in 2 rr.l of dimethylform
amide 9 and 4 mg of DCC was added. The mixture was allowed
to stand at room temperature for 50 hours. The reaction
mixture was dialyzed 9 and the dialyzate was filtered and
lyophilized to afford 'y5 r,~g of polyacrylic acid cor!~bined
with estriol 16-glucuronide as a white powder.
~3tiS~
- 55
~f) Production of a latex linked ~o 2 carboxyl-
containing ~lono olefinic ~olymeric coMpound
col~bined with a che.~ically ~odified estrogen
(f-1) Production of a latex linked to polyacrylic
acid co!;lbined with estriol^l6-F,lucuronide
The lysine-latex (0.1 ~) procluced in (d-l)
above was suspendecl in 1 ml of distilled water, and 1 !~1
(correspondir.~ to 0.3 mg of estriol-16-~;lucuronide) of
polyacrylic acid conbined with estriol-16-~lucuronide
which was produced in (e-3) above t~ras added. Then,
10 mg of l~ethyl~3~(3-dir~ethylanlinc~propyl)carbodiirllide
hydrochloride was added. I~!ith stirrirlg, the reaction
was performed overnight. After the reaction, the
reactlon llixture was centrifuged 7 and the resulting
precipitate was washed three times with 10 ml of` a
glycine~buffered sodiurn chloride solution. T'ne
precipitate was suspended in 30 ~1 of a glycine-buffered
sodiur2 chloride solution containing 0.05% RSA (rabbit
seruri~ alburnin) to produce a latex linked to polyacrylic
acid cor,~bined with estriol~l6-glucuronide.
(f-2) Produc'cion of a latex lin~ed to polyacrylic
acid combined with 17 a~ninoestrogen
The captioned latex was produced by the same
way as described in ~f 1) using the lysine-latex produced
by the r~ethod described in (d~l) a~.lcl polyacrylic acid
co!,lbined with 17~aminoestrogen producecl by the method
described ~e~
(f 3) Production of a latex linked to PVMMA
combine~ with 17-aminoestrogen
The captioned latex was producecl in the same
was as describecl in (f-l) using the ]ysine latex produced
in (d-l) and the 17-arninoestrogen-bound PVPli~'~ produced
in (e-2).5 (f-4) Production of a latex lin~;ed to polyacrylic
acid having estriol 16 glucuronide bound
thereto
The captioned latex was Produced in the same
~9t365~
56
way as in (f l) usin~ the dia!ainolleptarIe la'cex produced
in (d-2) and the polyacrylic acid having astriol-16-
glucuronide bound thereto procIuced in (e-~4).
(g) Deter!aination of urinary estrogen
Five assay samples of urine taken fro~,. pregnant
wo~Ien were dilutecl to 50, 100, 200, and 400 times. One
drop (0.03 ml) of each dilu~ed urine ~sar,Iple was droped
onto a slide. Then, one cIrop of the latex sensitized
r~ with anti-estriol--16-glucuronide antibody produced in
(~) was added. One drop of the latex linl.ed to poly-
acrylic acid having estriol-16-glucuronide bound thereof
produced in (f-l) was further ac,'dec!. The three were
mixed uniformly and shaI~en for 2 rainutes. Then, the
agglutinated pattern and the agglucination lnhibited
pattern were examined with the naked eye.
In 'chis Example, the assay sensitivity of the
reagent was adjusted to 0.1 ~g/l,~l. Thus, the estrogen
concentration in each urine sa~.ple was as shown in
Table 5.
Table 5
.
Sample ~o.Ratio Or dilutionestrogen
100 200 400 (I~g/ml~
_ " ~
~L ~1~ ., _ 105
3 ~ 20
4 .~ 10
.L~ 20
___ _~
Standards of cIe'cermina'cion ~
~: a clear agglutinated pattern
~ a completely agglutinatlon inhibited
pattern
The same standards of determination will
apply to the following Examples.
When the products obtaine(l in {i-2), (f-3),
53
~ 57 -
and ~f-4) were used as the 12tex lin!~ed to a rnodlfied
estrogen-bound carboxyl containin~ ~-nono-olefinic polymer,
the sar~e results were obtained as in the case of using
(f-l).
Example 2
Determination of urinary pregnaled _]
(a) Production of pre~nanecliol~3 gluc~lronide-BSA
This product was prepared in the sa1le way as
in Example 1, (a) using pregnanecliol~3-~lucuronide and
BSA,
(b) Production of anti~pre,~nanediol-3-glucuronide
antibody
The captioned antibody was produced by using
the pregnanediol-3~glucuronide~BSA produced in (a)
above, immunizin~ a goat with it in the same way as in
Exan~ple 1, (b), and collecting antiserum.
(c) Production of a latex sensitized with anti-
pregnanediol~3-glucuronicle antibocly
The cap-tioned polystyrene latex was produced
in the same way as in Example 19 (C) using the anti~
pregnanediol-3~glucuronide antibody produced in (b)
above.
(d) Production of PV'I~IA con!bine(l with pregnanediol-
3-glucuronide
25 (i) Pregnanediol 3-glucuronide~lysine derivative
Pregnanediol-3 glucuronide (99 m~) and 140 mg
of ~-benzyloxycarbonyl lysine methyl ester toluene--
sulfonate were dissolved in 12 ml of din~ethylformamide,
and with stirring under ice cooling 9 65 ~Ig of diphenyl
phosphoryl azide and 0.056 ml of triethylamine were
added. The mixture was treated in the sarne way as in
Exa.nple 19 ( e-3) to aff'ord 100 mg (5S% of theory~ of
the desired lysine derivative having the following
structural formula.
,C113
~I-C-OH
1--~ N~ICOOC~1
'IIO ~ O~l (, 2)4
b~ O ~ CON~I-C~I-COOC~13
(ii) The preganediol-3-glucuronide-lysine derivative (38 mg) obtained in
(i) above was catalytically reduced by the same way as in Example 1, ~e-3). The
product and 100 mg of PVhlMA were dissolved in 6 ml of dimethylformamide at an
elevated temperature~ and the solution was allowed to stand at room temperature
for 4 days. The reaction mixture was treated in the same way as in Example 1,
(e-2) to afford 30 ml of a solution of PVMMA combined with pregnanediol-3-glucur-
onide which had a pregnanediol content of 0.46 mg/ml as determined by coloration
with sulfuric acid.
(e) Production of a latex linked to PVMMA
combined with pregnanedlol-3-glucuronide
The captioned latex was produced in the same way as in Example 1, (f)
using the lysine-latex produced in Example 1, (d-l) and the PVMMA combined with
- pregnanediol-3-glucuronide produced in (d) above.
(f) Determination of urinary pregnanediol
Five assay samples of urine taken from pregnant women were each dilut-
ed to 50, 100, 200, 400, and 800 times with a glycine-buffered sodium chloride
solution. The urinary pregnanediol was determined by the same procedure as in
Example 1, (g) using (c) and (e) above. Since in this Example~ the assay sensi-
tivity of the reagent was adjusted to 0.05 ~g/ml~ the concentration of pregnane-
d.iol of each urine sa~ple was as shown in Table 6.
"~ - 58 -
.
,
53
Table 6
Sample No. ~ G~ dilution Amount of
__ . _ _ __ pregnane-
100 200 400 800 diol ~g/ml~
2 +.~ `_ _ _ _ 10
3 ~ *~ ~ _ 20
4 ~ _ _ 5
_ ~ -- , , _~ . ...... I O
Example 3
Measurement of urinary pregnanetriol:-
(a) Production of pregnanetriol-3-glucuronide-BSA
Pregnanetriol-3-glucuronide-BSA was produced by the same way as in Ex-
ample 1, (a) using pregnanetriol-3-glucuronide and BSA.
(b) Production of anti-pregnanetriol-3-
glucuronide antibody
The captioned antibody was produced by using the pregnanetriol-3-
glucuronide-BSA produced in (a) above and immunizing a rabbit with it in the
same way as in Example 1, (b).
(c) Preparation of a latex sensitized with
antipregnanetriol-3-glucuronide antibody
The captioned polystyrene latex was produced in the same way as in Ex-
ample 1, (c) using the antipregnanetriol-3-glucuronide antibody produced in (b)
above.
(d) Production of polyacrylic acid combined with
pregnanetriol-3-glucuronide
(i) Pregnanetriol-3-glucuronide-hexamethylenediamine derivative
Pregnanetriol-3-glucuronide (102 mg) and 63 mg of monobenzyloxycar-
bonyl hexamethylenediamine hydrochloride were dissolved in 4 ml of dimethylfor-
- 59 -
,
,
3~53
mamide, and after cooling with ice, 55 mg of diphenyl phosphoryl azide and 0.06
ml of triethylamine were added with stirring. The mixture was stirred at 0C
for 2 hours, and then allowed to stand at room temperature for 72 hours. The
reaction mixture was evaporatecl to dryness under reduced pressure. The remain-
ing product was subjected to preparative thin-layer chromatography to afford 61
mg (~1% of theory) of the desired pregnanetriol-3-glucuronide-hexamethylenedi-
amine derivative.
(ii) The pregnanetriol-3-glucuronide-hexamethylenediamine derivative (56
mg) obtained in (i) above was catalytically reduced in the same ~ay as in Ex-
ample 1, (e-4). The product and 200 mg of polyacrylic acid were dissolved in
20 ml of dimethylformamide, and 16 mg of DCC was added. The mixture was allowed
to stand at room temperature for 5 days. The reaction mixture was treated in
the same way as in Example 1, (e-4) to afford 168 mg of polyacrylic acid com-
bined with pregnanetriol-3-glucuronide as a white powder.
(e) Production of a latex linked to polyacrylic
acid combined with pregnanetriol-3-glucuronide
The captioned latex was produced in the same way as in Example 1, (f)
using the lysine-latex produced in Example 1, (d-l) and the polyacrylic acid
with pregnanetriol-3-glucuronide produced in (d) above.
(f) Determination of urinary pregnanetriol
Absorbent tampons of a certain fixed size were applied to the urine of
neonates or infants or to diapers to get the urine adsorbed therein. These
tampons were dried, and then leached with a predetermined amount of distilled
water to prepare assay samples. The assay samples were diluted with physiolog-
ical saline to 25, 50, 100, 200, and 400 times calculated on the basis of the
original urine. The urinary pregnanetriol was determined by the same procedure
as in Example 1, (g) using the products (c) and (e) above. Since in this Exam-
ple, the assay sensitivi~y of the reagent was adjus~ed to 0.1 ~g/ml, the amount
- 60 -
53
of pregnanetTiol in each of the assay samples was as shown in Table 7.
Table 7
_ _ ,~ .... . ...... ____ _ _
Sample Ratio of dilution Amount of Evaluation
No. pregnane-
25 50 100 200 400 (~g/ml)
...... ,.. _........ _ _ . . _ _
A _ _ _ _ .. <2.5 Normal
~ ++ ++ + >:` ~ rarenal
_ hyperplasia
As shown in Table 7, this test permi~s -the detection of congenltal suprarenal
hyperplasia which a neonate has. Thus~ by treatment with an adrenal cortical
hormone at the early stage, the neonate suffering from it can grow normally.
If it is a girl, she can grow up into a woman who can be pregnant and deliver
normally.
Example 4
Determination of urinary 17-OHCS
(a) Production of 3a,11~,17a,21-tetrahydroxy-
pregnan-20-one(THF) 3-glucuronide-BSA
THF-3-glucuronide-BSA was produced by ~he same way as in Example 1,
(a) using THF 3-glucuronide and BSA.
(b) Production of anti-THF 3-glucuronide antibody
The captioned antibody was produced by using the THF 3-glucuronide-
BSA produced in (a) above, and immunizing a rabbit with it in the same way as
in Example 1, (b).
(c) Production of a latex sensitized with anti-
THF-3-glucuronide antibody
The captioned polystyrene latex was produced in the same way as in
- 6il --
.
~36S3
Example 1, (c) using the anti-TI-iF-3-glucuronide antibody produced in ~b~ above.
(d) Production of polyacrylic acid combined with
TIIF 3-glucuronide
~;) TIIF 3-glucuronide-lysine derivative
Tlle desired lysine derivative was produced in accordance with the
method described in Example 1, ~e-3) from 53 mg of THF 3-glucuronide, 59 mg of
c-benzyloxycarbonyl lysine methyl ester toluenesulfonate, 33 mg of diphenyl
phosphoryl azide and 0.027 ml of triethylamine. Yield 57 mg; (theoretical
yield 71%)
~ii) Forty milligrams of the lysine derivative obtained in (i) above was
catalytically reduced by the same way as in Example 1, (e-3), and then reacted
with 14 mg of DCC and 100 mg of polyacrylic acid. The reaction mixture was
after-treated to afford 15 ml (THF content 0.96 mg/ml) of a solution of poly-
acrylic acid combined with THF 3-glucuronide.
(e) Production of a latex linked to THF 3-
~lucuronide combined with polyacrylic acid
The captioned latex was produced by the same way as in Example 1, (f)using the diaminoheptane latex produced in Example 1, (d-2) and the polyacrylic
acid combined with THF 3-glucuronide produced in (d) above.
(f) Determination of urinary 17-OHCS
Urine of a normal healthy man and urine of a normal healthy woman were
each diluted with a glycine-buffered sodium chloride solution to 10, 15, 20, 25,
30, and 40 times respectively. Using the above products (c) and (e), urinary
17-OHCS was determined by the same procedure as in Example 1, (g). The results
are shown in Table 8.
Since in this Example, the assay sensitivity of the reagent was ad-
justed to 0.2 ~g/ml, it was found that the urine of the healthy man contained
5 ~g/ml of 17-OHCS, and the urine of the healthy woman contained 4 ~g/ml of
- 62 _
~3~S3
17-OHCS.
Table 8
Urine Ratio of dilution Concentration
_ _ _ _ _ _ of 17-OHCS in
30 40 urine (~g/ml)
. _
Healthy ~ ~ ~ ~ _ _ 5
man l _
Healthy ~ +-l ~ _ _ _ 4
womcm
. _ _
Example 5
Determination of morphine
(a) Production of carboxymethyl mor~phine-BSA
BSA (lOO mg) was dissolved in 25 ml of distilled water, and 80 mg of
carboxymethyl morphine was dissolved in the resulting solution. The solution
was adjusted to pH 5.5, and 80 mg of 1-ethyl-3-~3-dimethylaminopropyl)carbodi-
imide hydrochloride was dissolved in the solution. The solution was stirred a~
room temperature overnight to perform the reaction. The reaction mixture was
dialyzed against distilled water. The dialyzate was lyophilized to afford car-
boxylmethyl morphine-BSA.
(b) Production of anti-carboxylmethyl morphine
antibody
Anti-carboxymethyl morphine antibody was produced by using the car-
boxymethyl morphine-BSA produced in ~a) above, immunizing a rabbit with it by
the same way as in Example 1, (b), and collecting antiserum.
(c) Production of a latex sensitized with anti-
carboxymethyl morphine antibody
The captioned sensitized latex was produced in the same way as in Ex-
ample 1, (c) using the anti-carboxylmethyl morphine antibody produced in (b)
above.
X - 63 -
~ ; S 3
(d) Production of polyacrylic acid combined with
carboxymethyl morphine
(i) Carboxymethyl morphine-lysine derivative
In the same way as in Example 1 J (e-3), 66.7 mg t78% of theory) was
produced from 51.5 mg of carboxymethyl morpl-ine, 61 mg oE F-tert-butoxycarbonyl
lysine metllyl ester acetate, 10 ml of dimethylformamide, 49 mg of diphenyl
phosphoryl azide and 0.042 ml of triethylamine.
(ii.) Forty milligrams of the lysine derivative obtained in (i) was dis-
solved in 5 ml of 98% formic acid. The solution was allowed to stand at room
temperature for 2 hours, and then evaporated to dryness under reduced pressure
below 40C. The residue was maintained under reduced pressure on potassium hy-
droxide for 24 hours, and then dissolved in 5 ml of dimethylformamide. Tri-
ethylamine (0.02 ml) was added, and 100 mg of polyacrylic acid and 3 ml of di-
methylformamide were added. Further, 21 mg of DCC was added, and the reaction
was performed for 48 hours. The reaction mixture was treated by the same way
as in Example 1, (e-3) to afford lO ml (carboxymethyl morphine content 2.0 mg/
ml) of a solution of polyacrylic acid combined with carboxymethyl morphine.
(e) Production of a latex_linked to polyacrylic
acid combined with carboxymethyl morphine
A latex linked to polyacrylic acid combined with carboxyme$hyl mor-
phine was produced by the same way as in Example 1, (f) using the lysine-latex
produced in Example 1, (d-l) and the polyacrylic acid combined with carboxy-
methyl morphine produced in (d) above.
(f) Determination of morphine
Morphine was dissolved in physiological saline and morphine-free urine
to the concentrations shown in Table 9. Morphine was determined by the same
procedure as in Example 1, (g) using the products (c) and (e) above. The re-
sults are shown in Table 9. The assay sensitivity of the reagent in this
- 64 -
X
~3bi53
Example was 50 ng/ml. It was found that this sensitivity was not affected by
the urine components.
Table 9
. . . . .
Solvent Concentration o morphine
0 ml) 50 100 200
.... _ . _ ~ _ ~ .
Physiological saline _ _ ++ ++ ++
Morphine-free urine _ ++ ++ ++
of a male
. _ __ _
Example 6
Determination of thyroxine (T4)
(a) Production of Thyroxine-BSA
BSA (50 mg) was dissolved in 25 ml of distilled water, and a solution
of thyroxine in 5 ml of dimethylformamide was added to the solution. With
stirring, 30 mg of 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide metho-p-
toluenesulfonate was added. Th0 reaction was performed overnight at room tem-
perature. The reaction mixture obtained was dialyzed against distilled water,
and lyophilized to produce thyroxine-BSA.
(b) Production of anti-thyroxine an~ibody
Anti-thyroxine antibody was produced by using the thyroxine-BSA pro-
duced in (a) above, immunizing a rabbit with it, and collecting;antiserum.
(c) Production of a latex linked _o anti-thyroxine
antibody
Five milligrams of the anti-thyroxine antibody produced in ~b) above
was dissolved in 5 ml of distilled water, and mixed with 1 ml of a 10% suspen-
20 sion of a carboxyl-modified latex. Then, 10 mg of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride was added, and the reaction was performed
overnight with stirring. After the reaction, the reaction mixtuTe was centri-
fuged. The resulting precipitate was washed with a glycine-buffered sodium
- 65 -
.
iS3
chloride solution. The precipitate was suspended in 15 ml of a glycine-buf-
fered sodium chloride solution containing 0.08% of rabbit serum albumin to pro-
duce a latex linked to anti-thyroxine antibody.
(d) Production of vinyl methyl ether/maleic
anllyclricle copolymer (PVMMA) combined with
thyroxine
In the same way as in Example l, (e-2), the above product was prepared
from PVMMA and thyroxine. By lyophilizing the dialyzate, the product was ob-
tained as a white powder.
0 (e) Production of a latex linked to PVMMA combined
with thyroxine
The captioned latex was produced in the same way as in Example 1, (f)
using the PVMMA combined with thyroxine produced in (d) above and the lysine-
latex produced in Example 1, (d-l).
(f) Determination of T4
8-Anilino-l-naphthalenesulfonic acid was added to 4 assay samples of
urine taken from normal healthy males. Each of the mixtures was diluted with
physiological saline to 1.5, 2 and 3 times, and T4 was determined by the same
procedure as in Example 1, (g). Since the assay sensitivity of the reagent in
this Example was adjusted to 30 ng/ml, the measured T4 concentrations were as
shown in Table 10.
Table 10
.... ~ ~ ....
Sample No. Ratio of dilution Concentration
= of T4 (ng/ml)
, _ ,_ , ,
2 ++ _ _ _ 45
3 *+ ++ _ 45
4 ++ ++ _ _ 45
- 66 -
~1~3~5;~
Example 7
Determination of catecholamine
~a) Production of metanephrine-BSA
Metanephrine-BSA was produced in the sams way as in Example 1, (a)
using metanephrine ~nd BSA.
(b) Production of anti-metanephrine antibody
Anti-metanephrine antibody was produced by using the metanephrine-BSA
produced in (a) above, immunizing a rabbit with it in the same way as in Example
1, (b), and collecting antiserum.
(d) Production of a latex sensitized with anti-
metanephrine antibody
The captioned latex was produced in the same way as in Example 1, (c)
using the anti-metanephrine antibody produced in (b) above.
(d) Production of polyacrylic acid combined with
metanephrine
Polyacrylic acid combined with metanephrine was produced from poly-
acrylic acid and metanephrine in the same way as in Example 1, (e-l).
(e) Production of a latex linked to metanephrine-
bound polyacrylic acid
A latex linked to polyacrylic acid combined with metanephrine was
produced in the same way as in Example 1, (f) using the lysine-latex produced
in Example 1, (d-l) and the polyacrylic acid combined with metanephrine pro-
duced in (d) above.
(f) Determination of metanephrine
Three assay samples of urine from normal healthy males were each di-
luted with physiological saline to 1.5, 2, and 3 times, and urinary metanephrine
was determined by the same procedure as in Example 1, (g). Since in this Ex-
ample, the assay sensitivity of the reagent was adjusted to 20 ng/ml, the
- 67 -
53
measured concentrations were as shown in Table II.
Table 11
:
Sample No. Ratio of dilution Concentration of
~ metanephrine
0 1.5 2 3 (ng~ml)
_ .. _ -
1 ++ ++ 30
2 *+ ++ ++ _ ~l0
3 ++ +~ ++ _ 40
... .. _ .. . . .... _ __ _ .. _ .. .
Combination of component A-l with
component B-2
Example 8
Determination of urinary estrogen (I)
Five assay samples of urine taken from pregnant women were each di-
luted with a glycine-buffered sodium chloride solution to 50, 100, 200, and 400
times respectively. One drop ~0.03 ml) of each diluted urine was added to a
reaction slide. One drop of the latex sensitized with anti-estriol 16-glucur-
onide antibody produced in Example 1, ~c) was added ~o it. After mixing, one
drop of a solution of the polyacrylic acid combined with 17-amino-1,3,5~10)-
estra~rien-3-ol produced in Example l,(e-l) having an estrogen concentration of
1.2 ~g/ml was added dropwise. The three were uniformly mixed, and shaken for 2
minutes. The agglutinated pattern and the agglutination-inhibited pattern were
observed with the naked eye. Since in this Example, the assay sensitivity of
the reagent was adjusted to 0.1 ~g/ml, the concentration of estrogen in each
urine sample was as shown in Table 12.
Tabl~e 12
~ : _
Sample No. Ratio of dilution Amount of
.__ estrogen
100 200 400 (~g/ml)
. _ _ ... _ .. , .....
2 ++ _ _ 10
3 ++ ++ ++ _ 20
. ... . .
X - 68 -
~L3~53
Table 12 Cont'd
.... .
Sample No. Ratio of dilution Amount of
_ _ _ _ estrogen
lO0 200 400 (~g/ml)
~ . __
4 +~ ~ lO
_ _ ~ ~ 20
When the products obtained in Example 1, ~e-2), (e-3) and (e-4) were
used as the carboxyl-containing mono-olefinic polymeric compound combined with
modified estrogen in the above example, similar results to those in the case of
using (e-l) were obtained.
Example 9
Det rmination of urinary estrogen (II)
(a) Production of erythrocytes sensltized with
anti-estriol 16-glucuronide antibody
To a 4% suspension of formalin-fixed goat erythrocytes (phosphoric
acid-buffered sodium chloride solution, pH 6.4) was added an equal volume of a
0.01% tannic acid solution, and they were reacted at 56C for 30 minutes.
Then, the erythrocytes were washed with a phosphoric acid-buffered sodium chlor-
ide solution to form an 8% suspension. Then, an equal volume of a 0.05% solu-
tion of the anti-estriol-16-glucuronide antibody produced in Example 1, (b) was
added, and reacted at 56C for 2 hours. After the reaction, the red cells were
centrifugally washed with a phosphoric acid-buffered sodium chloride solution,
and then diluted to a concentration of 1.5% (cell concentration) with a phos-
phoric acid-buffered sodium chloride solution containing 0.2% NRS (normal rab-
bit serum) and 5% of lactose. The dilution was poured in an amount of 0.1 ml
in each of ampoules, and then lyophilized to afford erythrocytes sensitized with
anti-estriol 16-glucuronide antibody.
(b) Determination of urinary estrogen (II)
Urine samples taken from healthy females at various stages of the
- 69 -
S3
menstrual cycle were each diluted with a phosphate-buffered sodium chloride
solution to 5, 10, 20, 40 and 80 times. Each of the urine dilutions was poured
in an amount of 0.1 ml into each of round-bottomed small test tubes. Then, one
ampoule of the erythrocytes sensitized with anti-estriol 16-glucuronide anti-
body produced in (a) above was suspended in 0.3 ml of a phosphate bufferized
sodium chloride solution. Tlle suspension was added to each of the urine dilu-
tion in each test tube, and mixed with stirring. Then, 0.1 ml of the solution
of polyacrylic acid combined with 17-amino-1,3,5(10)-estratrien~3-ol produced in
Example 1, (e-l) was added. The mixture was well stirred, and allowed to stand
for 2 hours on a stand equipped with a mirror. The presence of estrogen was de-
termined by the pattern formed on the bottom of the test tube. Since in this
Example, the assay sensitivity of the reagent was adjusted to 2 ng/ml, the
urinary estrogen concentration of each urine sample was as shown in Table 13.
Table 13
. . ___ . ,
Menstrual Concentra-
cycle Ratio of dilution of urine tion of
_ estrogen
in the
20 40 80 urine
(ng/ml)
.. ~
Follicular ++ _ _ 10
stage
.. ~ ._ ,
Ovulating .~+ ++ ++ ++ _ 80
stage
~ _
Luteal ++ .~ _ _ 20
stage
_ .___
Example 10
Determination of urinary pregnanediol
Five assay samples of urine taken from pregnant women were each di-
luted with a glycine-buffered sodium chloride solution to 50, 100, 200, 400 and
800 times, and the ur~nary pregnanediol was determined by the same procedure as
in Example 8 using the products obtained in Example 2, (c) and (d). Since in
~ 70 -
3~j3
this Example, the assay sensitivity of the reagent was adjusted to 0.05 ~g/ml,
the pregnanediol concentrations of the assay samples were shown in Table 14.
Table 14
. . _ ,.
Sample No. Ratio of dilution Amount of
~ pregnane-
100 200 400 800diol
_ _ _ _ _ ~g/ml)
2 ++ _ _ 10
3 ++ ++ ++ ++ _ 20
4 ++ ++ _ _ 5
_ _++ ++ - _ 10
Example 11
Determination of thyroxine (T4)
Four samples of sera of normal healthy males were diluted with physio-
logical saline to 1.5, 2 and 3 after adding 8-anilino-1-naphthalenesulfonic
acid. Using the products obtained in Examples 6, ~c) and ~d), thyroxine ~T4)
was determined by the same procedure as in Example 8.
Since, in this Example, the assay sensitivity of the reagen~ was ad-
justed to 30 ng/ml, ~he measured concentrations of T4 in the sera were as shown
in Table 15.
Table 15
Sample No.~ Ratio of dilution Concentration of
0 1.5 2 3 T4 ~ng/ml)
~ . . .. _ . . . , . . _ __ ~
1 ++ ~+ _ _ 45
2 ++ _ 30
3 ++ ++ _ _ 45
4 ++ ++ _ 45
- 71 -
. ~
53
Example 12
Determination of urinary pregnanetriol
Absorbent tampons of a predetermined size were applied to the urine of
neonates or infants or to diapers to get the urine adsorbed therein. They were
dried, and then leached with a predetermined amount of distilled water to pre-
pare as assay samples. The assay samples were each diluted with physiological
saline to 25, 50, 100, 200, and ~00 times calculated on the original urine.
Using the products obtained in Example 3, (c) and (d), the urinary pregnanetriol
was determined by the same procedure as in Example 8. Since in this Example,
the sensitivity of the reagent was adjusted to 0.1 ~g/ml, the amounts of preg-
nanetriol in the samples were as shown in Table 16.
Table 16
Sample Ratio of dilution Amount of Evaluation
_ _ pregnane-
100 200 400 triol
(~g/ml)
.... _ __ .... _ . _ _
A _ _ _ _ _ 2.5 Normal
C _ _ _ _ 2 5 "
D _~ _ _ _ _ 2.5 "
E ++ ++ ++ ++ ++ ~0 Suprarenal
hyperplasia
_ _
As shown in Table 16, this test permits the detection of congenital
suprarenal hyperplasia which a neonate has. Thus, by treatment with an adrenal
cortex hormone at the early stage, the neonate suffering from it can grow up
normally. If it is a girl, she can grow up into a woman who can be pregnant and
deliver normally.
X - 72 -
~ ~ ~3~53
Example 13
Determination of 17-KS
~a) Production of etiocholanolone hemisuccinate-BSA
Etiocholanolone hemisuccinate BSA was produced in the same way as in
Example 1, ~a) rom ethiochloanolone hemisuccinate.
~b) Production of anti-etiocholanolone
hemisuccinate antibody
The captioned antibody was produced by using the etiocholanolone
hemisuccinate BSA produced in (a) above, and immunizing a rabbi~ with it in by
the same way as in Example 1, ~b).
~c) Production of a latex linked to anti-
etiocholanolone hemisuccinate antibody
The captioned latex was produced by the same way as in Example 6, (c)
using the anti-etiocholanolone hemisuccinate antibody produced in ~b) above.
~d) Production of etiocholanolone hemisuccinate-
b~9~L~
~i) Etiocholanolone hemisuccinate-lysine derivative
Etiocholanolone hemisuccinate ~78 mg), 64 mg of -tert-butoxycarbonyl-
lysine methyl ester acetic acid salt and 25 mg of N-hydroxysuccinimide were dis-
solved in 2 ml of methylformamide. Then, 0.08 ml of triethylamine was added,and the mixture was cooled with ice. With stirring, 41 mg of DCC was added.
The reaction was performed for 3 hours under ice cooling, and then for 10 hours
at room temperature. The reaction mixture was evaporated to dryness under re-
duced pressure. The residue was purified by preparative thin-layer chromatog-
raphy to afford 64 mg ~51% of theory) the desired product of the following
structure.
,~
- 73
~3653
~`~
COO 11
C~12 N}ICOOC4119
C~12 ~C}l2)4
CONH.C}1.COOCH3
This product gave a single spot at Rf=0.64 (chlorof~m-methanol=lO:l) in silica
gel thin-layer chromatography.
(ii) Thirty milligrams of the etiocholanolone hemisuccinate-lysine deriv-
ative obtained in (i) above was dissolved in 8 ml of 98% formic acid. The so-
lution was allowed to stand at room temperature for 8 hours, and then evapor-
ated to dryness under reduced pressure at less than 45C. The product and lOO
mg of polyacrylic acid were dissolved in 5 ml of dimethylformamide, and 0.03 ml
of triethylamine was added. Then, 20 mg of DCC was added~ and the mixture was
allowed to stand at room temperature for 50 hours. The reaction mixture was
treated in the same way as in the last half of Example l, (e-3), ~ii) to afford
lOO mg of polyacrylic acid combined with etiocholanolone hemisuccinate (white
powder).
(e) Determination of urinary l7-KS
Two assay samples of urine taken from normal healthy males were each
diluted with a phosphate-buffered sodium chloride solution, and urinary 17-KS
was determined in the same way as in Example 8 using the latex linked to anti-
etiocholanolone hemisuccinate antibody produced in (c) above and the polyacrylicacid combined with etiocholanolone hemisuccinate produced in ~d) above. The
assay sensitivlty was adjusted to 0.5 ~g/ml. It was found that the two samples
each contained 4.0 ~g/ml of 17-KS.
- 74 -
.
'
6~3
Example 14
Determinatlon of morphine
(a) Production of erythrocytes sensitized with
anti-carboxymethyl morphine antibody
Erythrocytes sensitized with anti-carboxymethyl morphine antibody
were produced in the same way as in Example 9, ~a) using the anti-carboxymethyl
morphine antibody produced in Example 5, ~b).
(b) Determination of morphine
Morphine was dissolved in physiological saline and morphine-free urine
to the concentrations shown in Table 17, and morphine was determined by the same
procedure as in Example 9, ~b) using the products obtained in ~a) above and Ex-
ample 5, (d). The results are shown in Table 17. The assay sensitivity of the
reagent in this Example was 50 ng/ml, and it was found that this sensitivity
was not affected by the urine components.
Table i7
Solvent Concentration of morphine
(ng/ml)
.~
0 2~ 50 100 200
_ . . . ~ ~
Physiological saline _ _ ++ ++ ~+
. ., . _ ____
Morphine-free urine _ _ ++ ++ +l
Also, with a reagent containing both a latex sensitized with the
anti-carboxymethylmorphine and PAA combined with carboxymethylmorphine which
has the same assay sensitivity as that of the reagent obtained by Example 14,
(a) and (b), similar results were obtained.
Example 15
Determination,of metanephrine
Three assay samples of urine taken from normal healthy ma~es were
each diluted to 1.5, 2 and 3 times with physiological saline, and urinary
metanephrine was determined by the same procedure as in Example 8 using the
- 75 -
~3~S~
products obtained in Example 7, ~c) and (d). Since the assay sensitivity of
the reagent in this Example was adjusted to 20 ng/ml, the measured concentra-
tions of metanephrine in the urine were as shown in Table 18.
Table 18
, .,
Sample No. Ratio of dilution Concentration
- of metanephrine
0 1.5 2 3 (ng/ml)
. . . _
1 ~+ ++ 30
. ~ . . _ . __~__
2 ~+ ++ ++ 40
.. ~
3 ++ ++ ++ 40
. __ _ ._ . _
Combination of component A 2 with
component B-l
Example 16
Determination of urinary 17-KS
~a) Production of a latex linked to polyacrylic
acid combined with etiocholanolone
hemisuccinate
A latex linked -to polyacrylic acid combined with eticholanolone hemi-
succinate was produced in the same way as in Example 1, (f) using the lysine-
latex produced in Example 1, (d-l) and the polyacrylic acid combined with etio-
cholanolone hemisuccinate produced in Example 13, (d).
(b) Determination of urinary 17-KS
The antibody produced in Example 13, (b) was diluted with a glycine-
buffered sodium chloride solution, and by using the latex obtained in (a) above,
urinary 17-KS was determined while the assay sensitivity of the reagent was ad-
justed to 0.5 ~g/ml. Two assay samples of urine taken from normal healthy males
were each diluted with a phosphate-buffered sodium chloride solution to 2, ~, 8
and 16 times. Urinary 17-KS was determined in the same way as in Example 1,
(g) using the aforesaid diluted antibody instead of the antibody-sensitized
- 76 -
X
i53
latex in Example 1, ~g). It was found that both of these assay samples con-
tained 4.0 ~g/ml of 17-KS.
~ - 77 -
