Note: Descriptions are shown in the official language in which they were submitted.
2~
RAN 4093/48
The use of human chorionic gonadotropin (hereinafter
HCG) in the preparation of latex reagents usef`ul for the
detection of pregnancy is well known in the art. For
example, U.S. Patent No. 3,857,931, issued December 31, 1974,
teaches the preparation and -utilization of such reagents.
Further, there are a number of comrnercial reagents for the
determination of pregnancy which are of the latex HCG type.
The commercially available latex HCG pregnancy tests
utilize, in the most instances, HCG in a partially purified
form coupled to a serologically inert latex polymeric
carrier. In use, a drop of urine from a female subject is
placed on a clean glass slide and mixed with a drop of anti-
HCG serum and a drop of the HCG coupled to the latex carrier
in aqueous suspension. The slide is then examined within a
few minutes for the presence of agglutination which indi-
cates a negative result. This is only a general statement of
methodology as there are variations both in technique and
materials among the various preparations recognized in the
art.
Because there are a number of HCG latex-based
pregnancy tests commercially availab].e, there is a constant
striving improve on such tests :in the competitive environ-
rnerlt in terms of, e.g. sensitivity, cost of materials and
preparation and ease of handling and use. In the search for
a more specific test, it has been recognized in the art that
Klt/16.1.80
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splitting HCG into its alpha and be~a components or subunits
and then purifying the beta subunit would produce a reagent
which is highly specific, Morgan and Canfield, Endocri-
nology, Vol. 88, p. 1045 (1971).
There are methods recognized in ~he art whereby
beta-chain HCG can be purified as suggested above; however,
the methods previously known for the purification of beta-
chain HCG are very costly and, therefore, even ~hough
increased specificity could be achieved with a pure beta-
chain reagent, the lncreased costs inherent in the prepa-
ration thereof preclude its commercialization as a latex-
based test. As a result of the costs involved in the
purification of` beta-chain HCG, the only latex-ba~sed
product on the market utilizing beta-chain material utilizes
beta-chain antiserum and, therefore, contains no purified
beta-chain HCG as antigen.
~ '
In accordance with the present invention, a method
has been found whereby crude HCG such as is commercially
available can be treated without the need for additional
Z0 expensive purification procedures to obtain beta-chain
material which, unexpectedly, is immunologically very re-
active. The beta-chain HCG material of the invention can be
readily bound to immunologically inert latex particles to
yield a reagent which is from two-to four-fold more sensi-
tive than similar reagents known in the art which utilize
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commercially available HCG. The highl.y sensitive reagent ofthe subject invention is markedly advantageous i.n that it
can be prepared from relati.vely impure HCG without the need
for expensive purification techniques heretofore thought
to be essential for the use of beta-chain HCG.
In accordance with the present invention, relatively
impure HCG such as is commercially available is treated with
a chaotropic agent as will be defined hereinafter to split
it into its alpha- and beta-chain subunits which are then
separated by conventional methods such as, for example, ion
exchange chromatography. The beta-chain HCG thus obtained
has been found to be highly sensiti.ve immunologically even
though it still retains a large amount of the proteins, e.g.
urinary proteins, associated with the impure starting
material.
I'he beta-chain HCG obtained in accordance with the
method of the present invention can be readily bound to
immunologically inert latex carrier particles by con-
ventional methodology. The resulting reagent has been found
to possess from two-to four-fo].d sensitivity :immunol.ogically
in cornparison with commerc:ial preparations comprising
commerc.i.ally available HCG bound to an appropriate latex.
L~
The method of the present invention utilized as a
s~arting rnaterial HCG in a relatively impure form such as
is usually commercially available. ~his material contains
HCG in the presence of proteinaceous material. The amount
of HCG in such commercially available preparations varies
usually between about 2000 and about 5000 International
Units/mg. The proteinaceous material presen~ in such
preparations, at least in the context of the present
invention, is not considered to be immunologically contaml-
nating and merely dilutes the HCG. In the context of thepresent invention, such HCG preparations will hereinafter
be referred to as partially purified HCG.
In contrast to the partially purified HCG described
above, purified ~ICG is recognized in the art as having a
potency of between about 10,000 and about 20,000 Inter-
national Units~mg. It has been found in accordance with
the present inventi~on that partially purified HGG, i.e.,
an HCG preparation containing from about 2,000 to about
5,000 Internatlonal Units/mg., may be treated with a
chaotropic agent and that the beta-chain subunits isolated
therefrom still containing in substantial measure the
proteinaceous material ~rorn the partially purified
preparation may be utilized to prepare a highly sensitive
latex pregnancy reagent. It is considered to be within
the purview of the present invention to utilize as a
starting material HCG preparations which are regarded as
.
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being partially purified or "crude" even though their po-
tency may be appreciably higher than 5,000 IU/mg. The ran-
ge of 2,000 IU/mg. to 5,000 IU/mg. is given as being indi-
cative of presently available commercial preparations desi-
gnated as "crude" or "partially purified" and is not inten-
ded to be limiting on the scope of the present invention.
Whether commercial preparations containing signi~icantly
higher concentrations of HCG can, as a practical matter,
:
be utilized in the practice of the present invention will
be dependenb on the cost of such preparations.
,~
By the term "ohaotropic agent" as utilized ln the
practice of th~e~present invention is meant an agent wh1oh;
is capable o~f~rupturing hydrogen bonds of a polypeptide
molecule. Chaotr~oplc~ agents characteristically wlll not
rupture covalent~bonds ln such molecules. ln the context
of the present invention, the chaotropic agent is believed
,
to act more~-or-less~on the surface of the HCG molecule to
break hydrogen bcnds~and to split the alpha-and beta-chaln ~ -
subunlts thereof.~
Although the~exact mechanism by which the chaotropic
; agents render partially puri~ied HCG significantly more
sensitive in accorda~nce with the method of the present
invention is~nob known, it is believed that the chaotropic
a~ent acts to reveal more antigenic sites on the surface
of the HCG moleoule.~This phenomenon renders the HCG more
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immunospecific and, therefore, more sensitive without the
need to resort to costly procedures to purify it.
~ Suitable chaotropic agents useful in the practice
; of the present invention include, for example, an aqueous
solution of urea of from about 7 to about 10 molar concen-
tration, an aqueous solution of lithium bromide at similar
concentration, an aqueous solution of guanidine hydrochlo-
ride at from about ~,5 to about 6 molar concentration and
an aqueous solution of ammonium thiocyanate at from about
2 to about 3 molar concentration. The urea solution is pre-
ferred. The present invention is in no way intended to be
limited to the specific- chaotropic agents named herein. It
is considered to be within the scope of the invention to
,~ ~ utilize any other substances~recognized as possessing simi-
!: :
lar properties whioh can be utilized without deleterious
effect to the desired properties of the HCG. Whether a spe-
cific substance possesses chaotropic capability with regard
to partially purified HCG as described herein, without
i
'~ harmful effect, can be determined easily and is considered
; 20 to be within the skill of the art.
~:
, ;
! It will be appreciated that ~he chaotropic agerlts
enumerated above could be utilized in concentrations grea-
ter than those given. It has been found, however, that con-
centrations appreciably higher than those given herein do
not improve the efficiency of the treatment. Further,
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greatly increasing the concentration of the chaotropic
agent may, in certain instances, have a negative effect on
the desired results. Generally, the chaotropic agents are
utilized to treat the partially purified HCG in accordance
with the present invention at a pH in the range of from
about 4.0 to about 5.5 preferably about 5Ø An inorganic
acid, preferably hydrochloric acid, is utilized to adjust
the pH of chaotropic agent to the preferred range. The
preferred pH may vary slightly with the individual chaotro- `
pic agent be;1ng~utilized. For example, the preferred pH
when using urea as the chaotropic agent is about 4.5.
Generally, the chaotropic agent will be allowed to
remain in CoDtaCt with the partially purified HCG in aque-
ous solution for f~rom about one-half to about two hours,
15 ~ preferably for~about one hour, after which the pH of the
reaction mixture is raised to slightly above neutral, for
example, to~about 7.5. Generally, a temperature of from
about 20 C. to~about~50 C., pref`erably about 37C. is
utilized. Care must be taken to avoid raising the pH to a~
level where the~ HCG~may become~denatured, i.e., a pH of
about 9.5 or above. Likewise, temperature substantlally in
excess f`o 60C. ~hould be avoided to prevent denaturation
of the urinary proteins present. A suitable inorganio base
such as, for example, an alkali metal hydroxide such as
sodium hydroxide or potassium hydroxide is preferably uti.li-
zed to adjust the pH of the HCG solution after disassocia-
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tion to sligtly above neutral.
To prevent recombination of the alpha~ and beta-
chain subunits of HCG after treatment with the chaotropic
; agent, the HCG/chaotropic agent mixture is subjected to
conventional treatment to separate them such as, for exam-
ple, ion exchange media, batch or column, electrophoresis
and/or methods recognized in the art as being capable of
separating molecules based on differences in elcctrostatic
charge and the like. Ion exchange chromotography is prefer-
; 10 red. Any conventional ion exchange material may be utilized
to effect separation of the alpha- and beta-chain subunits
of HCG in accordance with the invention. A preferred mate-
rial is a hydrophilic, water-insoluble, cross-linked dex-
tran polymer gel. This material and the method of its
manufacture are taught in British Patent No. 854,715. The
; preferred gel material, which is commercially available
~ from Pharmacia Fine Chemicals, Uppsala, Sweden, under the
,~ tradename Sephadex, comprises a three dimensional macrosco-
'~ pic network of dextran substances bonded or cross-linked
i 20 together which is capable of absorbing water with swelling.
,
; The ability of the gel to take up water is inversely pro-
portional to the degree of crosslinking of the dextran
9ubstances. The gel material is available in a variety of
different grades differing with respect to degree of poro-
sity. Other similar materials recognized in the art may
; likewise be utilized. By the use of such separation techni-
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3 !324
que one may obtain the beta~chain HCG substantially free of
contaminating alpha-chain subunit. It has been demonstrated
that this technique permits recovery of consistently higher
than 80~ of the HCG antigenic activity. The partially puri-
fied beta-chain HCG thus-prepared is thereafter bound to a
suitable, immunologically inert latex carrier.
As used in the context of the present invention,
"immunologically inert latex polymers" or "immunologicaliy
inert latex polymeric carrier particles" includes latex
polymers which are water-insoluble, have a particle size
in the range of ~rom about 0.01 microns to about 0.9 mi-
crons, a specific gravity near that of water so that, a~ter
being bound to the beta-chain HCG in accordance with the
invention, the speclfic gravity of the particles LS appro-
ximately that of water or slightly above, i.e., 1.01 toabout 1.1 enabling`them to remain in aqueous suspension.
The latex polymer particles must have sufficient surface
,
~` charge density so that, when bound to beta-chain HCG, their
repulsive charges are sufficient to prevent aggregation.
The latex particles must be inert with respect to~immunolo-
gical diagnost~ic~test and, preferably, would have actlve
groupa which are capable of forming a covalent linkage with
beta-chain HCG such as, for examp].e, carboxyl groups, amine
groups or groups convertible into them. Typioal suitable
groups on the latex particles in a preferred embodiment of
the invention are those containing an active hydrogen suoh
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as, for example, -C~OH, -CON~2, or a primary amine group.
Typical suitable latex carrier particles are those
supplied commercially in the form of aqueous latex suspen-
sions, usually in concentrations of from about 50% to about
60% solids. Many types of latex polymers are suitable for
the practice of the present invention provided, however,
that they meet the criteria set forth above. The present
invention comprehends the use of all such suitable latex
polymers.
.~
The beta-chain HCG may in accordance with the pre-
sent invention be bound physically andtor chemically to the
immunologically inert latex polymer particles. In a prefer-
red embodiment, the beta-chain ~ICG is covalently bound to
the latex polymer particles with a water-soluble carbo-
diimide coupling~agen: through an amide linkage.
Examples of suitable latex polymers usable in the
practice of the lnvention include carboxyl-containing
polymers and copolymers such as polystyrene, polyvinyl
pyridine, styrene~butadiene copolymers, vinyl acetate/acry-
late copolymers, vinyl chloride-acrylate copolymers and the
like. The carboxyl groups may be incorporated into the la-
texes by conventional emulsion polymerization techniques or
by further reaction of a latex polymer or copolymer af'ter
preparation as is recognized in the art. For example, a
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monomer such as acrylic acid, methacrylic acid, itaconic
acid, maleic acid or the like can be added to the monomer
mixture. Alternatively, carboxyl groups can be formed on
the surface of the latex by conventional techniques such
as, for example, oxidation of hydroxyl groups, hydrolysis
of surface acrylate or methacrylate groups and the like.
The latexes may be prepared by a wide ~ariety of conven-
tional emulsion polymerization techniques such as, for
example, batch emulsion polymerization, seeded emulsion
polymerization, semi-continuous or continuous emulsion
polymerization. Preferred for the incorporation of carbo-
:
xyl groups is seeded emulsion polymerization wherein a seedlatex is added to the polymerization reactor before the
reaction is initiated to control the number of particles.
In this instance, a carboxy-containing monomer is polymeri-
zed on the surface of the seed latex particles. Commercial-
ly available latexes suitable for the practice of the pre-
sent invention include, for example, carboxylated styrene-
;~ butadiene copolymers marketed under the trademarks Dow
, 20 816 and Dow 421 by Dow Chemical Company and the like.
'-
In accordanoe with a preferred embodiment of the
present invention, beta~chain partially purified HCG is
covalently coupled to the aforementi.oned polymeric latexes
through an amide linkage utilizing, as a condensing agent,
a water-soluble oarbodiimide selected from those represen-
; ted by the ~ormula
`
12
R N=C=N R
wherein R is cycloalkyl havlng from 5 to 6 carbon atoms inthe ring; a straight- or branched-chain a]kyl of from 2 to
12 carbon atoms such as, f`or example, ethyl, n-propyl,
isopropyl, n-butyl, sec.-butyl, tert. butyl, amyl, hexyl,
octyl, nonyl, undecyl, dodecyl and the like; monoarylsubs-
titued lower alkyl radicals, for example, benzyl, a-or ~-
phenethyl and the like; monoaryl radicals such as, for
example, phenyl; morpholino; piperi~yl; morpholinyl substi-
.~
tuted lower alky~l radicals, e.g. morpholino ethyl;di-lower alkylamino-lower alkyl radicals; pyridyl substitu-
ted lower alkyl~radicals, e.g., ~,~, and y methyl or ethyl ~i
pyridyl; acid addi~tion salts thereof and quaternary amine
; ~ salts thereof.
Preferred coupllng agents in accordance with the in-
~; vention are l-cyolohexyl-3-(2-morpholinoethyl) carbodiimide
metho-p-toluenesul~fonate and l-ethyl-3-(3-di~ethyl-amino-
:
propyl)-carbodiimide hydrochloride. The reaction of teta-
chain partially purified HCG and the suitable latex carrier
is preferably carried out in the presence of the carbo-
diimide coupling agent in aqueous medium, preferably at
room temperature, i.e., about 20~ to about 25 C. Tempera-
tures of from about 5~C. to about 40C. are also suitable
for the reactlon.;In order to insure that efficient cou-
pling of beta-chain HCG to the latex carrier is realized,
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13
an amount of the water~soluble carbodiimide is utilized
which is equal to from about 0.05~0 by weight to about
2.0~ by weight based on the weight of the carrier parti-
cles. More preferably, about 1% by weight of the coupling
agent, based on the weight of the carrier particles is uti-
lized. The amount of the carbodiimide coupling agent utili-
; zed can vary within rather wide limits, however, depending,
for example, on the density of the carboxyl groups and the
presence of additional negatively charged groups, e.g.,
sulfonate groups, on the surface of the latex particles.
The pH of the coupling reaction is irnportant andshould be between about 5 and 8, preferably between about
6and 7. It is preferable to preadjust the pH of the latex
suspension to the desired range, i.e., between about 6 and
7, before adding it to the reaction mixture.
The reaction is completed in from about 5 minutes to
about 24 hours. Generally, from about 1 1/2 to about 2 1/2
hours is sufficient. The resulting product is a water-
insoluble material suspended in an aqueous medium having a
pH of from about 7 to about 8.5, preferably about 8Ø The
specific ~rav.ity of the material is about the same as water
with the result that the suspension of the product is stable.
The product can be isolated, e.g., by centrifugation, and
is a white, somewhat thixotropic, viscous, clay-like
material. Chemically, the product comprises a
2~
14
monolayer of beta-chain partially purified HCG bound physi-
cally and/or chemically to discrete particles of the immu-
nologically inert carrier by means of an amide linkage. Any
contaminating proteins present, as previously stated, do
not interfere serologically with the specificity of the
beta-chain HCG.
Physical binding of the proteins to a polymer latex,
as opposed to chemical binding, can be brought about by hy-
drophobic bonding of the reactants. For such a bond, it is
preferable to use a latex with a comparatively low order
` of reactive hydrophilic group, such as, for example, carbo-
xylic acid groups. Simple polystyrene polymers would, for
example, be suitable. The formation of such a hydrophobic
bond is further facilitated by binding in a high pH buffer
;15 such as a bicarbonate buffer at pH 10 to 11 and/or by trea-
ting the protein to remove acidic groups, such as, for
example, acidic sugar or neuraiminic acid groups ~rom the
beta-chain to render the molecule less hydrophilic. This
can be accomplished by heating the protein to about 80 C.
or by treatment with an enzyme, such as neuraiminidose or a
proteolytic enzyme which splits peptides that contain aci-
dic sugar groups. Examples of such enzymes include trypsin,
papain and bromelin.
The reagent prepared :in accordance with the present
invention is packaged for commercial purposes as an aqueous
suspenslon containing from about 0.5% by weight to about
3.5~ by weight beta-chain HCC linked to the immunologically
inert carrier material. More preferably, such suspensions
contain from about 1.0% by weight to about 2.5% by weight
5 of such particles. In a pref-erred embodiment, beta-chain
HCG is linked through an amide linkage to a carboxylated
butadiene-styrene copolymer containing a monomer ratio of
about 45% butadiene and 55% styrene and having a density of
carboxyl groups of about 1% by weight to about 5% by weight,
10 usually about 3% by weight.
The aqueous suspension of beta-chain specific
partially purified HCG coupled to the immunologically inert
carrier material prepared in accordance with the present
invention is conveniently packaged in a reagent kit which
15 contains, in a second container, the appropriate antiserum,
i.e. anti-HCG serum. Such anti-serum is derived by means
well known in the art. Thus, rabbits or goats are immunized
with highly purified HCG or the purified beta-chain peptide
of HCG and the anti-serum is collected, tested for titer,
20 then stored for use.
The following examples further illustrate the in-
vention.
16
EXAMPLE_l
A total of 100 mg. of commercially available crude
HCG containing approximately 2700 IU/mg. was dissociated
according to the method described by Morgan and Canfield
in Endocrinology, Vol. 8~, p. 1045 (1971). In this method,
the crude HCG is dissolved in 15 ml. of a 10 M aqueous
solution of urea and the pH of the resulting solution
adjusted to pH 4.5 with hydrochloric acid. The solution
is allowed to incubate at 37C. for one hour after which
3 ml. of a 0.03 M aqueous solution of glycine was added and
the pH adjusted to pH 7.5 with sodium hydroxide.
A solution containing dissociated alpha- and beta-
chain subunits of HCG prepared as above was subjected to
ion exchange chromatography to physically separate and
prevent the recombination of the subunits. The chromato-
graphy was carried out in a 2.5 X 40 cm chromatography
column packed to a helght of 20 cm with Sephadex A-50
(Pharmacia). This product has a particle size (dry) of from
40-120 microns, a bed volume/ml./g. dry gel of 25-33 and a
capacityof approximately 3 g. hemoglobin/g. of gel. The
column was equilibrated with an aqueous soluti.on of glycine
(0.03 M) and urea (~ M), pH 7.5.The down-flow rate was
adjusted to 60 ml/hr. and the HCG subunit solution applied
to the column. The columrl was thereaf'ter flushed with 500 ml
of the glycine/urea so].ution and the eluate, which contained
the alpha subunit of HCG, collected as one batchand discarded.
*Tra~e Mark - 16 -
t
17
The column was flushed a second time with 500 ml. ofan aqueous solution of glycine (0.2 M), sodium chloride
(1.0 M) and urea (8 M), pH 7.5. The eluate was collected
as one batch, diluted with an equal volume of distilled
water and then eoncentrated to 20 ml. by ultrafiltration
in an Amieon 402 stirred eell equipped with a PM-10
membrane (Amieon Corp.).The beta-chain containing HCG
fraetion was then equilibrated with 0.1 M sodium borate
buffer, pH ~.0 which eontained 0.02 pereent by weight sodium
a~ide,
Analytie disc electrophoresis performed on samples
of the first and the second eluates indieated that the
seeond eluate contained the beta-chain subunit of HCG free
of the alpha-chain subunit but containing urinary proteins
whereas the first eluate eontained alpha-chain HCG conta-
minated with some beta-chain subunit and urinary proteins.
Latex agglutination indicated that better than 80% of the
HCG aetivity in the starting erude material was reeovered.
EXAMPL,E 2
Beta-ehain HCG prepared from erude, eommereially
available HCG in aeeordanee with the proeedure of Example 1
was eovalently bound to immunologieally inert latex earrier
partieles by the following proeedure.
*Trade M.ark
- 17 -
'
18
A total of 5.0 ml. of a carboxylated styrene buta-
diene copolymer (Dow No. 816), washed, milled and adjusted
to between 78 and 82 mg./ml. was placed in a suitable
vessel equipped with a stirrer. To the stirred latex was
added 1.0 ml. of the beta-ch-ai.n HCG prepared in accordance
with Example 1 and assayed at 3000 IU HCC/ml.
~ hen the beta-chain HCG had become well dispersed in
the latex, 3.0 ml. of a 1% by weight solution of l-cyclo-
hexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene-
sulfonate in distilled water was added dropwise withstirring. After the addition of the coupling agent was
complete, stirring of the mixture was continued for an
additional two hours.
The coupled latex was then contrifuged at 15,000 rpm
at 5 to 10C. for 30 minutes during which the latex com-
pletely separated from the suspending medium. The super-
natant was analyzed for HCG activity and discarded. The
residue was washed with 10 ml. distilled water, centrifuged
as before and the supernatant analy~ed for HCG activity
and discarded. The residue was washed with 10 ml. of a
buffer cornprising 0.1 M Tris-HCl and 0.01% merthiolate-pH
8.0-8.2. The latex was then centrifuged as before, the
supernatant checked for HCG activity and discarded. The
residue was again washed in the above-mentioned buffer
and resuspended in 10 ml. of the same buff`er. The sequence
*Trade Mark
- 18 -
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.~
A 19
of centrifuging checking of the supernatant, washing and
resuspending was repeated until two consecutive buffer
wash supernatants were free of HCG activity. The coupled,
washed latex then ready for packaging into a diagnostic
reagent kit and was resuspended in 20 ml. of the above-
mentioned buffer.
A typical reagent kit including the beta-chain HCG
latex prepared according to the above-described procedure
would also contain as a separate reagent anti-HCG serum.
Such antiserum was raised in goats by conventional
procedure, i.e., by innoculating the animals with highly
purified HCG or the purified beta-chain peptide, collecting
the antiserum, and testing for titer. Subtractive a~finity
chromatography was utilized to eliminate cross-reactivity
of the anti-HCG serum with human urinary proteins.
For this procedure, 150 ml. of washed Sepharose 4 B
(Pharmacia) was activated according to the method described
by Primus et al., J. of` Immuno., Vol 188, p. 55 (1977).
Urinary protein, 0.25 gm % in 200 ml. of 0.1 M sodium
borate, p~l 8.0, was mixed with the activated Sepharose
4 ~ at 4C. f`or 18 hours, thus coupling the prote.in thereto.
The product was sequential:Ly washed with 1. llter of 0.1 M
sodium borate, pH 8.0, to remove unbound protein, 500 ml.
of a 1.0 M aqueous solution of 2-aminoethanol, pH 8.0,
to reduce activated groups on the Sepharose 4 ~ gel and
. *Trade Mark
- 19 -
re-equilibrated with 1.0 sodium borate, pH 8Ø The
resulting urinary protein adsorbent was packed into a 5 cm
diameter chromatographic column. To this column was added
in sequence 10 ml. of normal goat serum (1:2 in 0.1 M
sodium borate buffer, pH 8.o), 300 ml. of 0.1 M sodium
borate buffer, pH 8.0 and 100 ml. of 3.0 M ammonium thio-
cyanate in 0.1 M sodium phosphate, pH 7Ø The column was
then re-equilibrated with 500 ml. of 0.1 M sodium borate
buffer.
The anti-HCG serum collected above was diluted with
an equal volume of 0.1 M sodium borate buffer, pH 8.o, and
passed through the column. A recycling automated affinity
- chrornatography apparatus was utilized to control the ad-
sorption process. The diluted antiserum was automatically
cycled through the apparatus until all had been chromato-
~ graphed. A sample volume of 15 ml. was chromatographed at a
; downward flow rate of 60 ml./hr. The column eluate was
continually monitored by adsorbance and, before the
appearance of protein in the eluate, the flow was stopped
for one hour to allow the sample to react with the ad-
sorbent. The unadsorbed protein, specific antiserum to HCG,
was eluted first and thereafter the adsorbed protein con-
; taining antibodies to urinary proteins was eluted with 100 ml.
of 3.0 M ammonium thiocyanate. The unadsorbed fract~ns were
pooled and concentrated by ultrafiltration to the originalvolume of the sample in an Amicon 402 unit equipped with a
.~ *Trade Mark - 20 -
21
XM-50 membrane (Amicon Corp.).
Latex agglutination tests showed a recovery of anti-
body activity to be better than 90% after adsorption.
Specificity of the antisera was determined by immunodif-
fusion and confirmed by immunoelectrophoresis.
EXAMPLE 3
The sensitivity of the beta-chain HCG prepared in
accordance with Example 1 was demonstrated as follows. An
antiserum to HCG, i.e., goat serum adsorbed with urinary
protein was titrated in a physiologic saline buffered medium.
In the presence of a constant amount of the latex conjugates
tested, the anit-HCG was diluted to determine the last
dilution demonstrating agglutination or flocculation after
incubation for 120 min. The last dilution is considered the
end point and titer of that antiserum and its corresponding
antigen. The results are set forth in the Table I. The latex
conjugates compared are the beta-chain latex prepared in
Example 2, a similarly prepared alpha-chain latex and a
commercial pregnancy test comprising whole HCG conjugated
to a latex.
Table I
Dilution of La~ex Conjugate
Anti-HCG HCG ALPHA BETA
1:2,000 3-4 3-4 2-3
5 1:4,000 3-4 3 3_4
1:8,000 3-4 neg. 3-4
1:16,000 neg. neg. 3-4
1:32,000 neg. neg. 2
1:64,000 neg. neg. trace
-10 A rating of 3-4 is a strong reaction, 3 is medium
strong, 2 is medium to weak, trace is very weak and neg. is
no reactlon.
The higher the dilution of antiserum, the corres-
pondingly less HCG required to neutralize it. The results
in the above table for whole HCG approximately correlate to
a sensitivity of1.0-1.25 international units of HCG per ml.
of urine. The sensitivity, i.e., the least amount detectable,
of the beta-chain latex is about 0.25 I.U./ml. of urine.
This correlation may be demonstrated as follows. A
sample of antiserum was calibrated in terms of the amount
of ~ICG in International Units which would react therewith.
A series of dilutions of this sample in the aforementioned
buffer were added to samples of urine to be tested. In each
instance, 2 ml. of buffered antiserum were combined with
; 25 1 rnl. of urine in a test tube. One hundred microliters of the
23
latex to be tested were added to each sample and the whole
mixed by inversion. The lower end of the test tube was heated
to 37C for 2 hours. Agglutination or flocculation would be
visible after this time unless sufficient HCG in the urine
inhibits the reaction. The comparison was carried out between
the whole HCG pregnancy test reagent re~erred to above and
the beta-chain product of Example 2. The results are given
in Table II. The notations have the same meanings as those
in Table I.
Table II
Concentration of Latex Con~jugate
HCG in
Sample (IU/ml.) HCG BETA
0.0 3-4 3-4
0.1 _ 2
0.2 -- l/2
0.25 3-4 neg.
0.3 -- neg.
~~ neg.
0.5 3-4 neg.
0.75 2-3
l.0 2
1.25 neg.
This data demonstrates the improved sensitivity of
beta-chain latex of Example 2 over the whole HCG pregnancy
test reagent tested.
