Note: Descriptions are shown in the official language in which they were submitted.
I~MUNOCHEMICAL ASSAY OF HUMAN CHORIONIC GONADOTROPIN
AND REAGENT THEREFOR
This inventio~ relates to an immunochemical assay
of hl~an chorionic gonadotropin (hereinafter sometimes
referred to as "hCG").
Heretofore, two methods have been proposed for
enzyme immunoassay (hereinafter sometimes referred to
briefly as "EIA") of hCG, as follows:
(1) Competitive method: The assay is accomplished by
allowing a reagent containing a definite amount of hCG
labeled with an enzyme and a sample containing an unknown
amount of hCG to become bound to a known amount of an
anti-hCG antibody competitively, assaying the enzymic
activity of that portion of enzyme which has been bound
to the antibody or remains unbound and comparing the assay
result with the result obtained in the same manner with a
known amount of hCG.
(2) Sandwich method: The assay is accomplished by
trapping a sample containing an unknown amount of hCG with
an anti-hCG antibody supported on a carrier, allowing an
enzyme-labeled antibody to be bcund to the resulting
2Q comp]ex and assaying the enzymic activity.
The present inventor has already found that the use
of a certain ~-D-galactosidase as labeling agent and a
certain antibody in the above-mentioned competitive method
(1) enables microassay of hCG with high sensitivity and
high specificity (cf. European Patent Application Publi-
cation No. 37,110). The present inventor has also found
3~
that, in the above-mentioned sandwich method (2), an EIA which comprises using
an antibody supported on the carrier and an antibody to which the labeling
agent is coupled, and they are different antibodies which are not overlapping
in antigen-determining site and one of these different antibodies is specifi-
cally reactive to human chorionic gonadotropin, is highly sensitive, highly
accurate and highly specific (cf. European Patent Publication No. 49,898).
~ owever, even by these methods, it is difficult to assay hCG at levels
of about 0.1 to 2 mIU or be]ow. Accordinglyg a still more highly sensitive
assay method has been required f or more decisive diagnosis of malignant tumor
and observation of the course of such tumor as well as study of the physiolo-
gical meaning of hCG in normal human subjects.
Under these circumstances, the present inventor conducted further
investigations and found that, in EIA by the sandwich method which uses the
above-mentioned specific antibody, the use of an antibody-enzyme conjugate
obtained by coupling an anti-hCG antibody to peroxidase enables a highly
sensitive, highly accurate microassay. Continued studies based on the above
finding have now led to completion of the present invention.
The present lnvention relates to (1) an immunochemical assay of human
chorionic gonadotropin, comprising the use of an antibody supported on a
carrier, an antigen and an antibody labeled with a labeling agent, wherein
the supported antibody and the labeled antibody are dissimilar antibodies
which are not overlapping witll each other in antigen-determinant position,
the antibody to be supported on a carrier is specifically reactive to human
chorionic gonadotropin, the labeling agent is a peroxidase, and the peroxidase
is coupled with the corresponding antibody employing a compound represented
by the formula:
-2-
,. -i ~ -
o o
~- (Cll2) ~ -R_CO-O-N~ (I)
wherein n is an integer of 0 to 5 and R is a cllemical bond or a divalent 6-
membered cyclic hydrocarbon resicluc. The present inventi.on also pro-vides an
immunocllemical assay reagent for use in an assay of human chorionic gonado-
tropin, which comprises as component (a) a reagent consisting of a conju~Satc
which is prepared by coupling peroxidase with an antibody employing a compound
of the formula:
O O
~N (C~l ) R CO-O-I~ I
wherein n is an integer of 0 to 5 and R is a chemical bond or a di.valent 6-
membered cyclic hydrocarbon residue, and as component (b) an antibody support-
ed on a carrier, the latter antibody being not overlapping in antigen-deter-
minant position with the antibody to be coupled with peroxidase and being
speci.fically reactive to human cnorionic gonadotropin
'~le present invention further provides an antibody conjugate useful
in an assay of human chorionic gonadotropin, which conjugate is prepared by
coupling peroxidase with an antibody of human chorionic gonodotropin employ-
ing a compound of the formula:
O O
¦I~,N- (C~l2) -R-CO-O-N 1
wherein n is an iDteger of 0 to S and R is 2 chemical bond or a divalent 6-
membered cyclic hydrocarbon residue, the antibody bcing non-overlapping in
antigen-determinant position with an antibody which is specifically reactive
to human chorionic gonodotropin.
The present inventiOn still further provides a process for preparing
'~,
.. .. -3-
33~
an antibody conjugate useful in an immunochemical assay of human chorionic
gonadotropin, which process comprises:
coup]ing peroxidase with an antibody of human chorionic gonadotropin
employing a coupling compound of the -Formula:
O O.
N-(CH2)n -R-C0-0 N~
O O i'
wherein n is an integer 0 to 5 and R is a chemical bond or a divalent 6-
membered cyclic hydrocarbon residue, the antibody being non-overlapping in
antigen-determinant position with an antibody which is specifically reactive
to human chorionic gonadotropin.
The present invention further provides a method of immunochemical
assay of human chorionic gonadotropin, which method comprises:
(1) contacting an antibody conjugate reagent and a sample containing
human chorionic gonadotropin with an antibody supported on a carrier, wherein
the antibody supported on a carrier is specifically reactive to human chorionic
gonadotropin and the antibody conjugate reagent is prepared by coupling peroxi-
dase with an antibody employing a compound of the formula:
O O~
i ,~(CH2)n -R-CO-O-N
wherein n is an integer of 0 to 5 and R is a chemical bond or a divalent 6-
membered cyclic hydrocarbon residue, and
the peroxidase-coupled antibody is non-overlapping in antigen-derterminant
position with the antibody supported on a carrier,
(2) adding a substrate for peroxidase to the reaction mixture of
step (1), and measuring the substance produced by the action of peroxidase.
The present invention still further provides an immunochemical assay
kit for assay of human chorionic gonadotropin, which kit comprises:
-3a-
vv
(1) an antibody supported on a carrier, wherein the antibody i5
specifically reactive to human chorionic gonadotropin,
(2) an antibody conjugate which is prepared by coupling peroxidase
with an antibody of human chorionic gonadotropin employing a compound of the
formula
O 0
~ ( 2)n ~J
~ o o ~
wherein n is an integer of 0 to 5 and R is a chemical bond or a divalent 6-
membered cyclic hydrocarbon residue,
the antibody coupled with peroxidase being non-overlapping in antigen-deter-
minant position with the antibody supported on a carrier,
(3) a standard human chorionic gonadotropin,
(4) a buffer solution for diluting the reagents and the test sample,
(5) a substrate for peroxidase.
The carrier used in the present invention includes, among others,
beads of gels ~for example, agarose gel Ce.g. Sepharose* 4B, Sepharose 6B
(Pharmacia Fine Chemicals, Sweden)~ , dextran gel ~e.g. Sephadex* G75,
Sephadex G100, Sephadex G200 (Pharmacia Fine Chemicals)~ , polyacrylamide gel
~e.g. Biogel* P 30, Biogel P60, Biogel P100 (Bio-Rad Laboratories, U.S.A.)~
particles of cellulose ~for example, Avicel* (Asahi Chemical Industry Co.,
Ltd. Japan), ion exchange cellulose (e.g~ diethylaminoethyl-cellulose, carbo-
xymethyl-cellulose)~ , physical adsorbents Lfor example, glass (e.g. glass
beads, glass rods, aminoalkyl-glass beads, aminoalkyl-glass rods), silicone
rubbers, styrene resin (e.g. polystyrene beads, polystyrene granules)~ , ion
exchange resins Cfor example, weakly acid cation exchange resins re.g. Amberlite*
IRC-50 (Rohm and Haas, U.S.A.), Zeocarb* 226 (Permutit, West Germany)~ , weakly
basic anion exchange resins ¦e.g. Amberlite IR-4B (Rohm and Haas), Dowex* 3
-3b-
* Trade Mark
3~
(Dow Chemical, U.S.A. )3 ~ and so on.
In accordance with this invention, the antibody supported on a carrier
and the antibody coupled with a
c-
~8~
-- 4
labeling agent are two dissimilar antibodies which are not
overlapping with each other in antigen-determinant posi-
tion, and the antibody to be supported on the carrier is
an antibody specifically reactive to hCG.
As examples of the antibody specifically reactive
to hCG, there is mentioned (1) an antibody described in
Endocrinology, vol. 104 (1979), P. 396. Thus, an hCG-
specific peptide at the C-terminal of hCG-3 sub-unit and
a carrier protein such as bovine albumin or bovine-
thyroglobulin are condensed in the presence of a water-
soluble carbodiimide reagent such as l-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide, and using the resulting
condensate and Freund's complete adjuvant or incomplete
adjuvant, a warm-blooded animal such as rabbit i5 immunized
a plurality of times to produce an antibody. This procedure
gives an antiserum which reacts specifically to hCG.
(2) An hCG-specific anti-hCG antibody described in
European Patent Application Publication No. 37,110 is
mentioned. The antibody is prepared as follows: The
peptide of the formula [VIII]:
~-~-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH [VIII]
wherein Rlis a partial peptide of 1 to 14 amino acid
residues including 14-Gly of the peptide Alal-Pro2-Pro3-
PrO4-ser5-Leu6-pro7-ser8-pro9-serlo-Argll-Leul2-prol3
Glyl4
as immobilized on a carrier, is contacted with a body fluid
containing an anti-hCG antibody, and the anti-hCG anti~ody
specifically absorbed is separated by elution.
(3) Furthermore, as an antibody which reacts specifically
with hCG, there may be mentioned an antibody prepared by
condensing a peptide of the formula [VIII] mentioned above
with a carrier protein in the presence of glutaraldehyde
(hereafter briefly, GLA), inoculating a warm-blooded
animal other than man with the resulting condensate to
produce an antibody and recovering the same (European
33~30
Patent Application Publication No. 49,898).
The carrier protein referred to above is a substance
which is used as coupled with a hapten (a substance of
low molecular weight~ for producing an antibody to the
hapten such as a peptide which, as it is alone, cannot
induce formation of an antibody, and includes such proteins
as bovine serum albumin, bovine gamma-globulin, bovine
thyrogl0bulin, tetanus toxoid, hemocyanin and polyamino
acid.
The coupling of the peptide of the formula [VIII]
with a carrier protein in the presence of GLA can be
conducted by the conventional method [e.g~ "Hormone and
Metabolic Research", vol 8 (1976) P. 241]. The relative
amount of peptide [VIII] and carrier protein is preferably
about 1:1 to 2:1, and the reaction pH of about 7.3 gives
satisfactory results in many cases. The reaction time
is somewhere between about 2 and 6 hours and a reaction
time of about 3 hours is usually appropriate. The
condensation product thus obtained is dialyzed against
water of about 4C, lyophilized and stored as usual.
The condensation product thus obtained is used to
inoculate a warm-blooded animal other than man.
~ he warm-blooded animal other than man, which is
used in the production of the above hCG-specific antibody,
includes, among others, mammalian warm-blooded animals
(e.g. rabbit, sheep, rat, mouse, guinea pig, cattle,
horse, pig) and avian species (e.g. chicken, pigeon, duck,
goose, quail).
To inoculate the condensation product into such a
warm-blooded animal other than man, the condensation
product is used in an amount sufficient to produce the
desired antibody. For example, 2 mg per dose of the
condensation product is emulsified with equal volumes
(1 ml) of physiological saline and Freund's complete
adjuvant and the emulsion is injected subcutaneously
into dorsal sites and rear foot pads of a rabbit for a
1~83~0
-- 6
total of 5 times at intervals of ~1 weeks. The above
procedure yields the desired ant:ibody in many instances.
The antibody thus produced in the body of the
warm-blooded animal can be harvested as follows. Thus,
in the case oE a rabbit, for instance, blood is withdrawn
from the ear vein and centrifuged to separate the serum
normally at a time hetween 7 to 12 days after the last
immunization.
The carrier for supporting the antibody used as
irnmobilized thereon in the assay of hCG may be any of the
carriers mentioned above.
The coupling of the carrier with the antibody
may be effected by the conventional method. For
example, the cyanogen bromide method as well as -the GLA
method described in "Taisha" ~Metabolism and Disease),
vol, 8 (1971), P~ 696 published by Nakayama Shoten Inc.,
Japan may be mentioned. As a more expedient procedure,
the antibody may be physically adsorbed on the surafce of
the carrier.
As examples of the peptide of the formula [VIII],
there is mentioned a C-terminal fragment peptide [IX] of
hCG-~ (123-145) represented by the formula: H-Ala-Pro-
Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-
Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH. The fragment is produced,
for example, by a method described in European Patent
~pplication Publication No. 37,110.
Throughout this specification, when abbreviations
are used to amino acids, peptides, etc., they are either
the abbreviations according to IUPAC-IIJB Commission on
Biochemical Nomenclature or those of common use in this
field of art. The following is a partial list of the
abbreviations. It is to be understood that when optical
isomers exist in regard to amino acids, etc., L-forms are
meant unless otherwise indicated.
Ala : alanine
Pro : proline
830(~
-- 7 --
Ser : serine
Leu : leucine
Arg : Arginine
Gly : glycine
Asp : aspartic acid
Thr : threonine
Ile : isoleucine
Gln : glutamine
The various peptides which are employed for the
production s~f the specii~ic antibody accordirlg to the
present invention can be produced by procedures known
~ . While both of the solid-phase and the liquid-
phase methods of synthesis may be employed, the latter
method is more often advantageous. Such methods for
peptide syn~hesis include those described in the literature,
e.g. Schroder and Lubke: The Peptides, vol. 1 (1966),
Academic Press~ New York, U.S.A. and Izumiya et al.:
'iPeptide Gosei" (Peptide Synthesis) (1975), Maruzen Co.,
Ltd., Japan, namely the azide method, chloride method t
acid anhydride method, mixed acid anhydride method, DCC
method, active ester method, Woodward Reagent K method,
carbodiimidazole method, reduction-oxidation method, DCC-
additive (e.g. HONB, HOBt, HOSu) method and so on.
The coupling of the carrier with the specific anti-
boty may be effected by the conventional method. For
example, the cyanogen bromide method as well as the GLA
method described in the aforementioned "Metabolism and
Disease", vol, 8 (1971), P. 696 published by Nakayama
Shoten Inc., Japan may be mentioned. As a more expedient
procedure, the antibody may be physically adsorbed on the
surface of the carrier.
The antigen in the assay according to the invention
is the assay target, namely hCG.
Referring to the antibody labeled with a labeling
agent to be used in the practice of the invention, the
antibody therein is nonspecifically reactive with hCG and
3300
has an antigenic determinant which does not overlap with
any of the antigen-determinant position of the above-
mentioned antibody supported on a carrier.
An exampLe of the antibody nonspecifically reactive
to hCG is the antibody which shows cross-reactivity with
other structurally analo~ous protein hormones such as
hLH (human luteinizing hormone), which antibody may be
prepared by inoculating a warm-blooded animal other than
a human being with a purified hCG derived from human urine
in the conventional manner to produce an anti-hCG antibody,
subjecting the same antibody to salting-out to recover
y~globulin fraction, subjecting the same fraction to
affinity chromatography on a column packed with a solid
phase coupled with the C-terminal peptide of hCG-~ to
recover an effluent and purifying the effluent further
by affinity chromatography on a column of a solid phase
with which hCG has been coupled.
The labeling agent, namely peroxidase, may be of
any of various origins~ For example, peroxidase species
of the horseradish, pineapple, fig, sweet potato, broad
bean and corn origins are usable. In particular,
horseradish peroxidase extracted from horseradish is
preferable.
In coupling peroxidase to the antibody, a compound
of the formula:
O O
~ ~CH2)n-R-CO-O- ~ [I3
O
wherein n and R are defined as above, is used. In the
above formula, the divalent 6-membered-cyclic hydrocarbon
residue represented by R may be either saturated or
unsaturated. Examples of the saturated divalent 6-
membered-cyclic hydrocarbon residue are 1,2-, 1,3- and
1,4-cyclohexylene. Examples of t:he unsaturated 6-membered-
123L8~
cyclic hydrocarbon residue are 1,2-, 1,3- and 1,4- phenylene.
In said compound [I], n is preferably an integer of
1 to 5, and more preferably the integer 1. As the divalent
6-membered-cyclic hydrocarbon residue R, 1,4-cyclohexylene
is particularly preferable.
The compound [I] to be used in the method of the
present invention can be produced, for example, by the
methods described in The Journal of Biochemistry, vol. 79,
page 233 (1976), European Journal of Biochemistry, vol.
101, page 395 (1979), Japanese Patent Application Laid-
open No. Sho 52(1977)-85,1~3, Japanese Patent Application
Laid-open No. Sho 52(1977)-85,164 and German Patent
Application Offenlegungsschrift 2656155 or modifications
thereof. Thus, for instance, it can be produced by
reacting a maleimide compound of the formula:
o
~ N-(CH2)n-R-COX [II]
O
wherein X is a hydroxyl group or a halogen atom and n and
R are as defined above, with a succinimide compound of
the formula:
o
~
~ N-O-Y [III]
o
wherein Y is a hydrogen atom or an alkali metal atom, in
the presence of a dehydrating agent or a deacidifying
agent. Referring to the above formulas, the halogen atom
is, for example, chlorine or bromine, and the alkali metal
atom is, for instance, sodium or potassium. Examples of
the dehydrating agent to be used in the reaction are
sulfuric acid and dicyclohexylcarbodiimide. Pyridine
and triethylamine are examples of the deacidifying agent.
-- 10 --
The above-mentioned compound [II~ can be produced for
example, by the method described in Japanese Patent
Application Laid-open No. 52(1977)-85,164 or a modifi-
cation thereof. Thus, for instance, it can be obtained
by subjecting a compound [IV] of the formula:
HC-CO-NH-(CH ) --R-COOH
HC-COOH 2 n [IV]
wherein n and R are as defined above, to dehydration under
ring closure. Said dehydration-ring closure reaction can
be ef~ected by gentle heating with a dehydrating agent
such as acetic anhydride or acetic anhydride plus sodium
acetate (anhydrous).
Alternatively, it can also be produced by the method
described in Helvetica Chimica Acta, vol. 58 (1975), page
531 or a modification thereof. Thus, for instance, an
N~alkoxycarbonylmaleimide [V] of the formula:
~-COOZ [V]
O
wherein Z is~an alkyl group, with an amino acid [VI] of the
formula:
NH2(CH2)nRCH [VI]
wherein n and R are as defined above, to give a maleimide
compound [VII] of the formula:
~/o
N ( 2)n [VII]
o
wherein n and R are as defined above. Then, a succinimide
compound [III] is added and the reaction is carried out
in the presence of a dehydrating or deacidifying agent
such as mentioned above, to give the compound [I].
Referring to the compound of the formula [V], the
alkyl represented by Z is, for example, methyl or ethyl.
The reaction of compound [I] with peroxidase is
~18~0
accomplished by contacting both with each other in a
buffer having a pH of about 6 to 8 at a temperature of
about lO~C to 50C for about 10 minu-tes to 24 hours. Said
buffer is, for example, 0~1 M phosphate buffer (pH 7.0)
or 0.05 ~ phosphate buffer (pH 6.3).
The thus-obtained maleimidated peroxidase can be
purified by gel chromatography, for instance. Packing
materials adequate for said gel chromatography are
Sephadex G-25 (Pharmacia Fine Chemical, Sweden) and
Biogel P-2 (Bio-Rad Laboratories/ U.S.A.), among others.
An exemplary method for reacting the maleimidated
peroxidase with an anti-hCG antibody is described below.
The anti-hCG antibody IgG or the F(ab')2 fraction obtained
by pepsin degradation is reduced in the presence of a
mercaptoethylamine and the unreacted material is removed
by gel filtration. The resultant anti-hCG antibody IgG
or Fab' is reacted with the maleimidated peroxidase.
Said reaction can be efrected by contacting
both with each other in a buffer at a temperature
of about 0C to 40C for about 1 to 48 hours. Said
buffer is, for example, 0.1 M phosphate buffer containing
5 mM sodium ethylenediamine-tetraacetate (pH 6.0).
The thus-produced peroxidase-labeled antibody can
be purified by gel chromatography, for instance. As the
packing material for use in said gel chromatography, there
may be mentioned, among others, Ultrogel AcA 44 (LKB,
Sweden) and Sephacryl S-200 (Pharmacia Fine Chemical,
Sweden).
Thus obtained peroxidase~labeled antibody is
presumed to have the following structure:
o
Ab-S ~N- (CH2 ) n-R-CO-NH-Per
wherein R and n are the same meaning as defined above,
~830Ç~
Ab means a residue of the antibody (Ab-SH), and Per is
a residue of peroxidase (Per-NH2).
The assay procedure according to the present
invention is described in more detail hereinbefore.
(1) First, an hCG-containing sample to be assayed is
added to the antibody supported on a carrier, whereupon
the antigen-antibody reaction takes place. Thereafter,
the peroxidase~coupled anti-hCG antibody IgG or Fab'
obtained in the above manner is added for reaction.
As the hCG-containing sample to be assayed by the
enzyme immunoassay method according to the present înven~
tion, there may be mentioned urine, serum, plasma,
cerebrospinal fluid and various internal organ extracts,
among others. Among them, urine, serum and plasma are
most frequently used.
(2) A substrate for peroxidase is added to the reaction
product obtained in (1), and the absorbance or fluorescence
intensity of the substance produced is measured. The
enzymic activity of the above-mentioned reaction product
is known in this manner.
(3) The above serial procedures (1) and (2) is followed
with standard solutions containing known amounts of hCG
and a standard curve indicating the relationship between
the hCG level and the absorbance or fluorescence intensity
is constructed in advance.
(4) The hCG content in the test sample is determined by
conversion of the absorbance or fluorescence intensity
obtained with the test sample containing an unknown amount
of hCG using the standard curve.
An assay kit suited for use in immunochemical
assay of hCG by the sandwich method according to the
present invention mainly comprises:
(1) the antibody supported on a carrier,
(2) the peroxidase-labeled antibody fragment (Fab')
obtained by the method of the present invention,
(3) a standard hCG,
1~L83~0
(4) a buffer for diluting the above reagents (2) and ~3)
and a test sample (e.g. phosphate or glycine buffer
having a pH of about 6 to 9 and containing about 10%
of sheep serum and about 1% of bovine serum albumin
(hereinafter sometimes referred to as "BSA") in each
of which serum and proteinicsubstances are coexisting),
and
(5) reagents necessary for determination of peroxidase
activity (e.g. p-hydroxyphenylacetic acid as substrate
and hydrogen peroxide for fluorescent assay; o-
phenylenediamine and hydrogen peroxide for colorimetry;
a buffer for dissolving the substrate, preferably
phosphate buffer; and an enzymic reaction terminator
solution).
The above kit can be used, for instance, in the
following manner:
About 10 to 200 ~1 of the standard hCG or test
liquid is diluted with the reagent (4). Then, an adequate
amount of the reagent (1) is added. After about 1 to 48
hours of reaction at about 0 to 40C, the carrier is
washed with water and, then, about 10 to 300 ~1 of the
reagent (2) is added. After about 1 to 48 hours of
reaction at 0 to 40C, the carrier is washed an* the
activity of peroxidase on the carrier is determined.
Thus, about 10 to 1,000 ~1 of a solution of a peroxidase
substrate is added and the enzymic reaction is allowed to
proceed at about 20C to 40C for about 0.2 to 24 hours
and then terminated, followed by determination of the
absorbance or fluorescence intensity of the liquid reaction
mixture.
The reagents for immunochemical assay in accordance
with the present invention enables a highly sensitive
- assay of hCG by a simple procedure in a usual clinical
laboratory.
When the reagents according to the invention is used,
hCG can be assayed with very high sensitivity and accuracy
lZ1~33~
- 14 -
without suffering interference by similar hormones (e.g.
hLH: luteinizing hormone~. Accordingly, the present
invention provides a means very useful also for diagnosis
and prognosis of chorionic tumor and other types of hCG-
producing tumor, among others.
Brief Description of the Drawings:
Fig. 1 shows the gel chromatographic elution patterns
of the reaction product of peroxidase with rabbit anti-
hCG antibody (Fab' fragment) in Example l; Fig. 2 shows
the standard curves of hCG and hLH in the EIA using the
peroxidase-labeled anti-hCG antibody (Fab' fragment) and
hCG-specific antibody-polystyrene bead in Example l; and
Fig. 3 shows the standard curve of hCG in the single-
incubation assay in Example 2.
The following Reference Examples and Examples are
given to illustrate the present invention in
further detail.
Reference ExamFle 1
Production of anti-hCG antibody (anti-hCG serum):
In 1 ml of physiological saline was dissolved 1 mg
of hCG (approx. 10,000 IU/mg) purified from the human
urine by the conventional method, and 1 ml of Freund's
complete adjuvant [Tachibana et al.: Men-eki-no-Seikagaku
(Biochemistry of Immunity), p. 26, Kyoritsu Shuppan Inc.
Japan (1967)] was added and stirred well to prepare an
emulsion. This emulsion was injected into the bilateral
femoral muscles and subcutaneously at several dorsal
sites of a rabbit. The above procedure was repeated at
intervals of 3 weeks for a total of 5 times and a blood
sample was taken one week after the last immunization
for a pilot assay. In this manner, there was obtained an
anti-serum N305B having an affinity also for the C-
terminal fragment peptide [IX] of hCG-~.
- Reference Example 2
Production of specific anti-hCG antibody:
Five (5) mg of peptide [IX] was dissolved in 8 ml of
vo
- 15 ~
~.l M NaHCO3 containing 0.5 M NaCl. To this solution was
added l y of BrCN-activated Sepharose 4B previously
washed with 1/1,000 N HC1. The mixture was stirred at
5C overnight. Then, the Sepharose was washed well w~th
the 0.1 M NaHCO3 solution containing 0.5 M NaCl as used
above, followed by addition of 10 ml of 0.5 M ethanolamine
adjusted to pH 8 with hydrochloric acid. The reaction
was conducted at room temperature for one hour, after which
time the Sepharose was w~shed with (1) 0.1 M acetate
buffer containing l M NaCl (pH 4.0) r (2) 0.1 M borate
buffer containing l M NaCl (pH 8.0) and (3~ 0.02 M borate
buffer containing 0.15 M NaCl (pH 8.0) in the order
mentioned. The Sepharose was then packed into a column.
Eight (8) ml of the anti-hCG serum N305B obtained
according to Reference Example l was subjected to
fractional precipitation with 1.5 g of anhydrous sodium
sulfate and the resultant ~-globulin fraction was passed
through the above column of peptide [IX]-Sepharose 4B
(column size: 0.9 x 4 cm).
The column was washed with 0.02 M borate buffer
containing 0.15 M NaC1 (pH 8.0) to remove the anti~hCG
antibodies showing cross-reactivity with hLH, hFSH (human
follicle-stimulating hormone) and hTSH (human thyroid-
stimulating hormone). Then, elution was carried out with
0.17 ~ glycine-HCl buffer (pH 2. 3) to recover the specific
anti-hCG antibody N305BS having a strong affinity for the
C-terminal fragment peptide [IX] of HCG-~ (Protein content
1.2 mg).
Reference Example 3
Production of anti-hCG-~ C-terminal fragment peptide [IX]
antibody:
In 4 ml of 0.2 M phosphate buffer (pH 7.3) were
dissolved 25 mg of the C-terminal peptide [IX] of hCG-~
and 50 mg of bovine thyroglobulin (briefly, BTG), followed
by addition of 4 ml of 5% aqueous G~A. The mixture was
stirred at room temperature for 3 hours, after which it
~2~8300
- 16 -
was dialyzed against water at 4C (2 Q of water x 4) and
lyophilized to obtain an immunogen. In 0.75 ml of
physiological saline was dissolved 1.5 mg of the above
hCG-~ C-terminal peptide [IX]-BTG conjugate, followed by
addition of 0.75 ml of Freund's complete adjuvant. The
mixture was stirred well to prepare an emulsion. The
emulsion was injected intramuscularly into bilateral
femoral muscles and subcutaneously several dorsal sites
of a rabbit~ The above procedure was repeated four times
at intervals of 4 weeks and the blood was collected a week
after the last immunization, centrifuged to separate the
antiserum. In the above manner, anti-hCG-~ C-terminal
peptide [IX] serum N313B was obtained.
This antiserum N313B was precipitated with ammonium
sulfate in the conventional manner and the resulting y-
globulin fraction was applied to a Sepharose 4B column
(0.9 cm dia. x 4 cm long) carrying 2 mg of hCG.
The column was washed with 0.02 M borate buffer
containing 0.15 M NaCl (pH 8.0) and elution was carried out
with 0.17 M glycine-HCl buffer (pH 2.3), whereby a
specific antibody N313BS having a high affinity for hCG
was obtained.
Reference Example 4
Production of nonspecific anti-hCG antibody:
Antiserum N305B obtained in Reference Example 1 was
salted out with ammonium sulfate and subjected to affinity
chromatography using a column (0.9 cm in diameter and
4 cm in length) of Sepharose 4B coupled with 5 mg of the
C-terminal peptide ~IX~ of hCG-~. An antibody fraction
was recovered as the effluent. This fraction was then
passed through a column (0.9 cm in diameter and 4 cm in
length) of Sepharose 4B coupled wi'h 2 mg of hCG. The
column was washed with 0.02 M borate buffer (pH 8.0)
containing 0.15 M NaC1 and then eluted with 5 M MgC12 to
give nonspecific antibody N305BG having high affinity to
hCG.
i2183C~
-- 17 --
Example_l
(1) Introduction of maleimide group:
In 1 ml of 0.1 M phosphate buffer (pH 7.0), there
was dissolved 6 mg of horseradish peroxidase (Boehrinyer
Mannheim, West Germany), followed by addition of 4.8 mg
of a coupling agent, MMC (the compound of the formula [I]
wherein n=l and R=cyclohexylene~, dissolved in 50 111 of
N,N-dimethylformamide. The mixture was stirred at 30C
for 60 minutes. After the reaction, the resultant pre-
cipitate was removed by centrifugation and the supernatant
was passed through a Sephadex G-25 column (1.0 x 45 cm),
followed by elution with 0.1 M phosphate buffer. A
protein-containing fraction was collected and concentrated
with a collodion membrane. The thus-prepared maleimidated
peroxidase contained 1.0 to 1.2 maleimide groups introduced
per one molecular of peroxidase (calculation was made on
the assumption that the molecular weight of peroxidase
was 40,000 and El% nm = 22.75).
(2) Production of maleimidated peroxidase-anti-hCG antibody
(Fab' fragment) complex:
To 5 mg of antibody N305BG obtained in Reference
Example 4 was added 0.1 mg of pepsin, followed by overnight
reaction at 30C. The reaction mixture was passed through
a Sephadex G-150 column (2.5 cm in diameter and 55 cm in
length) . The thus-purified antibody F(ab')2 fraction was
reduced with 2-mercaptoethylamine, followed by purification
by gel chromatography using a Sephadex G-25 column to
give rabbit anti-hCG antibody (Fab' fragment).
To a solution of 1.5 mg of the maleimidated peroxi-
dase prepared in (1) above in 0.15 ml of 0.1 M phosphate
buffer (pH 6.0), there was added 0.15 ml of 0.1 M
phosphate buffer (pH 6.0) containing 1.8 mg of the
previously obtained anti-hCG antibody (Fab' fragment) and
5 mM sodium ethylenediamine-tetraacetate. After 20 hours
of reaction at 4C, the reaction mixture was subjected to
gel chromatography using a column (1.5 x 45 cm) packed
301D
~ 18 -
with Ultrogel AcA 44. Elution was performed with 0.1 M
phosphate buffer ~pH 6.5). The absoxbance at 280 nm and
enzymic activity of the eluate were determined. The
formation of a peroxidase-rabbit anti-hCG antibody (Fab'
fragment) complex was confirmed by the following method.
First, the enæymic activity was determined by the
method of Gilbert et al. [Analytical Chemistry, vol. 40
(1968), page 12563. Thus, each eluate fraction was
diluted 1,800 times with 0.1 M phosphate buffer (pH 7.0)
containing 0.1% of bovine serum albumin. To 10 ~1 of
each dilution was added 0.25 ml of a 0.5% solution of p-
hydroxyphenylacetic acid in 0.05 M sodium acetate buffer
(pH 5.0) containing 0.1% of bovine serum albumin, followed
by incubation at 30C for 20 minutes. Thereafter, 0.05 ml
of 0.01% hydrogen peroxide was added, followed by
reaction at 30~C for 20 minutes. The enzymic reaction
was terminated by addition of 2.5 ml of 0 1 M glycine
buffer (pH 10.3) and the intensity of fluorescence was
measured at 405 nm (excitation light wavelength: 320 nm)
regarding the intensity of fluorescence emitted by 1 ~g~ml
of quinine as 100. The results obtained are shown in
Fig. 1. In Fig. 1, -o- indicates the absorbance at 280 nm
and -~- indicates the peroxidase activity (in terms of
fluorescence intensity). It was confirmed that the forma-
tion of the peroxidase-anti-hCG antibody (Fab' fragment)
complex was very well recognizable in fraction 38 and
neighboring fractions.
(3) Preparation of antibody-coupled solid phase:
To 1,500 polystyrene beads (4.8 mm in diameter,
Precision Plastics Ball Co., Chicago, U.S.A.) were added
100 ml of 15 ~g/ml solution of specific anti-hCG antibody
- N305BS or N313BS mentioned in Reference Example 2 or 3,
respectively, in 0.01 M NaCl-0.01 M phosphate buffer (pH
8.0). After overnight incubation at 5C, the beads were
washed with 0.05 M phosphate buffer (pH 7.0) containing
0.1% of BSA and stored inthe cool until use thereof.
- 19 -
(43 EIA:
The following ~IA reagents were used.
(1) The peroxidase-rabbit anti-hCG antibody (Fab'
fragment) complex according to Example 1 (2).
(2) The anti-hCG antibody-coupled polystyrene bead
according to Example 1 (3).
(3) Standard hCG and hLH solutions.
(4) Buffer B: a 0.02 M phosphate buffer (pH 7.0)
containing 10% normal sheep serum, 1~ bovine serum
albumin, 0.1% NaN3 and 0.15 M NaCl.
(5) The reagents for determination of peroxidase
activity: a 0.1 M citric acid-disodium phosphate buffer
(pH 4.8) containing 0.02% hydrogen peroxide and 0.15%
o-phenylenediamine and a reaction terminator (lN-HC1).
Assay
Either hCG or hLH was reacted with the rabbit anti-
hCG-coupled polystyrene bead in 200 ~1 of buffer B at
room temperature for 1 day. After the polystyrene bead
was washed with water, 300 ~1 of peroxidase-rabbit anti-
hCG antibody (~ab' fragment) complex was added and the
reaction was conducted at 4~C for 1 day. After washing
the polystyrene bead, 500 ~1 of a 0.1 M citric acid-
disodium phosphate buffer (pH 4.8) containing 0.02%
hydrogen peroxide and 0.15% o-phenylenediamine was added.
The reaction system was allowed to stand at room tempera-
ture for 40 minutes, at the end of which time 2 ml of lN
hydrochloric acid was added to thereby terminate the
reaction. The absorbance of the reaction mixture was
measured at 492 nm. The standard curves constructed in
the above manner are shown in Fig. 2. In Fig. 2, -~-
represents the standard curve of hCG for the N305BS-
polystyrene bead; -o- the standard curve of hLH for the
N305BS-polystyrene bead; -~- the standard curve of hCG
for the N313BS-polystyrene bead; and - - the standard
curve of hL~I for the N313BS-bound polystyrene bead.
Thus, the assay method of the present invention was
12~8300
- 20 -
highly specific and sensitive to hCG without the inter-
ference of cross-reaction with hLH.
Example 2
Expedient assay:
Standard hCG, the peroxidase-anti-hCG antibody
(Fab' fragment) complex according to Example 1 (2), and
one anti-hCG antibody-N305BS-polystyrene bead according
to Example 1 (3) were simultaneously mixed together in
300 ~1 of a 0.02 M phosphate buffer (pH 7.0) containing
25% normal sheep serum, 1% bovine serum albumin, 0.002%
merthiolate and 0.15 M NaCl and reacted at 4C for 1 day.
After the polystyrene bead was washed with water, peroxi~
dase activity was determined by the procedure described
in Example 1 (4). As shown in Fig. 3, a satisfactory
standard curve of hCG was obtained.
Example 3
m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) coupling
method:
In 0.1 M phosphate buffer (pH 7.0) was dissolved
6 mg o~ horseradish peroxidase [Boehlinger-Mannheim, West
Germany] followed by addition of 4.8 mg of the coupling
agent MBS (the compound of the formula [I~ wherein n=0,
R=phenylene) as dissolved in 50 ~1 of N,N'-dimethyl-
formamide. The reaction was conducted under stirring at
25C for 30 minutes and the reaction mixture was then
passed through a Sephadex G-25 column (l.O x 45 cm), and
eluted with 0.05 M acetate buffer (pH 5.0). The protein-
containing fraction was taken and concentrated through
a collodion membrane. The number of maleimide groups in
the maleimidated peroxidase per one mole of peroxidase was
0.68 to 0.78.
To 5 mg of the antibody N305BG according to Reference
Example 4 was added 0.1 mg of pepsin and the reaction was
conducted at 30C overnight. The reaction product was
then purified by passing through a Sephadex G-150 column
(~.5 cm dia. x 55 cm long). The resulting antibody F(ab')2
8.300
fraction was reduced with 2~mercaptoethylamine and, then,
purified by gel chromatography on a Sephadex G 25 column
to give a rabbit anti-hCG antibody (Fab' fragment). A
solution (0.15 ml) of 1.7 mg of anti-hCG antibody (Fab'
fragment) in a 0.1 M phosphate buffer (pH 6.0) containing
5 m~l sodium ethylenediaminetetracetate was added to 0.15 ml
of a solution containing 1.5 mg of the maleimidated
peroxidase pr~pared above in 0.1 M phosphate buffer (pH 6.0)
and the reaction was conducted at 4C for 20 hours. The
reaction mixture was then subjected to gel-chromatography
on an Ultrogel AcA 44 column (1.5 x 45 cm~ and elution
was carried out with 0.1 M phosphate buffer (pH 6.5) to
give a peroxidase-rabbit anti-hCG antibody (Fab' fragment)
complex.
Assays using this complex by the procedure described
in Example 1 (2) and ~4) showed that it was highly sensitive,
with a very low non-specific adsorption on the solid phase.
The EIA reagents used were as follows.
(1) The anti-hCG antibody N305BS-polystyrene bead
according to Example 1 (3).
(2) The peroxidase-rabbit anti-hCG antibody (Fab'
fragment) complex according to Example 3.
(3) Standard hCG.
(4) Buf~er B [cf. Example 1 (4)].
(5) Reagents for determination of peroxidase activity.
An acetate buffer (pH 5.0) containing 0.5% p-
hydroxyphenylacetic acid, a 0.01% hydrogen peroxide
solution, and a reaction terminator (0.1 M glycine
buffer, pH 10.3).
Example 4
A hCG specific immunochemical assay kit, and the assay of
hCG:
Using the hCG immunochemical assay kit and procedure
described below, plasma hCG concentration was determined
in healthy subjects, ovariectomized patients, women in
the ovulation phase, pregnant women, and patients who had
:12~L830C~
- 22 -
undergone carcinectomy for choriocarcinoma.
hCG immunochemical assay kit:
(l) The 4.8 mm (dia.) polystyrene bead coupled
with about l ~g/bead of hCG-specific antibody as
obtained according to ~xample l (3).
(2) The peroxidase-anti-hCG antibody complex
according to Example l (2).
(3) 0 to lO0 mIU of starldard hCG.
(4) Buffer B [cf. Example l (4)] for dilution of
the above reagents (l) and (3) and the test sample.
(5) o-Phenylenediamine.
(6) Buffer C for dilution of reagent (2):
a 0.1 M phosphate buffer (pH 7.5) containing 0.1%
bovine serum albumin and 0.002% merthiolate.
(7) Buffer D for dilution of reagent (5):
a 0.1 M citric acid buffer (pH 4.8) containing 0.02%
of hydrogen peroxide and 0.002% of merthiolate.
(8) Reaction terminator: l N hydrochloric acid.
~9) Control serwn (normal sheep serum).
Procedure:
To 50 ~l of the test sample were added 250 ~ of
reagent (4) and one bead (reagent l), and the reaction was
conducted at room temperature for l day. After washing
the polystyrene bead with water, 300 ~l of reagent (~) as
diluted with reagent (6) (about 30 ng as the complex) was
added and the reaction was conducted at 4C for l day.
After the polystyrene bead was washed with water, 500 ul
of a 0.15% solution of reagent (5) in reagent (7) was
added. The reaction was conducted at room temperature
for 40 minutes, at the end of which time 1.5 ml of l N
hydrochloric acid was added to thereby terminate the
reaction. The absorbance was then measured at 492 nm.
As to standard hCG solutions, the above procedure was
repeated using 200 ~1 of reagent (4), 50 ~l of control
serurn, and one bead of reagent (l) to construct a standard
12~8300
- 23 -
cureve.
The hCG concentrations in the sera of healthy
subjects, ovariectomized patients, women in the ovulation
phase, pregnant women and patients who had undergone
carcinectomy for choriocarcinoma were determined by the
procedure set forth hereinbefore. The results are shown
in Table 1.
Table 1
Test sample (mIU/ml)
.
Serum of healthy 1 0O20
woman 2 0.25
3 0.35
4 0.18
0.27
... . . . _ . . _ . _
Serum of 1 0.33
ovariectomized 2 0.86
woman
3 0'54
4 0.23
0.77
Serum of woman in 1 0.34
ovulation phase 2 0.19
3 0.25
Serum of pregnant 1 1,000
woman 2 34,000
3 18,000
- - ~ _ _ _
Serumofpatient who 1 190
had undergone 2 9 8
carcinectomy for
choriocarcinoma 3 5,3
It will be apparent from Table 1 that very small
amounts of hC~ in serum, inclusive of the sera of healthy
3(~(~
- 24 -
subjects, can be successfully assayed without the inter-
ference of cross-reaction with hLH and other analogous
hormones. Moreover, this assay method is very useful as
a test of cure in the prognostic management of patients
undergoing carcinectomy for choriocarcinoma.
