U.S. patent number 3,839,153 [Application Number 05/206,952] was granted by the patent office on 1974-10-01 for process for the detection and determination of specific binding proteins and their corresponding bindable substances.
This patent grant is currently assigned to Akzona Incorporated. Invention is credited to Antonius Hermanus Wilhelmus Maria Schuurs, Bauke Klaas VAN Weemen.
United States Patent |
3,839,153 |
Schuurs , et al. |
October 1, 1974 |
PROCESS FOR THE DETECTION AND DETERMINATION OF SPECIFIC BINDING
PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES
Abstract
The invention relates to a process for the detection and
determination of a component of the reaction between a specific
binding protein and the corresponding bindable substance contacting
a fluid containing one of these components with a conjugate of the
bindable substance with an enzyme and of antibodies against the
specific binding protein which latter component is brought in an
insoluble form, if necessary, in combination with the specific
binding protein, and determining the activity of the liquid or
solid phase of the resulting reaction mixture.
Inventors: |
Schuurs; Antonius Hermanus
Wilhelmus Maria (Oss, NL), VAN Weemen; Bauke
Klaas (Oss, NL) |
Assignee: |
Akzona Incorporated (Asheville,
NC)
|
Family
ID: |
19811894 |
Appl.
No.: |
05/206,952 |
Filed: |
December 10, 1971 |
Foreign Application Priority Data
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|
|
|
|
Dec 18, 1970 [NL] |
|
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7018838 |
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Current U.S.
Class: |
435/7.93;
435/188; 435/971; 436/518; 436/808; 436/818; 435/7.95; 435/964;
435/975; 436/530; 436/531; 436/817 |
Current CPC
Class: |
G01N
33/541 (20130101); G01N 33/743 (20130101); Y10S
436/818 (20130101); Y10S 435/975 (20130101); Y10S
435/964 (20130101); Y10S 436/817 (20130101); Y10S
435/971 (20130101); Y10S 436/808 (20130101) |
Current International
Class: |
G01N
33/541 (20060101); G01N 33/536 (20060101); G01N
33/74 (20060101); G01n 031/14 () |
Field of
Search: |
;195/13.5R ;424/12
;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Nidgley et al. "Karolinska Symposia on Research Methods in
Reproductive Endocrinology," 1st Symposium, Sept. 23-25, 1969 in
Acta Endocrinologica, Supplementum (Copenhagen), 142: 247-256
(1969). .
Miles et al. "Nature" 219: 186-189 (1968)..
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Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Weisberger; Hugo E.
Claims
1. Process for the detection and determination of a bindable
substance selected from the group consisting of an antigen and a
hapten, comprising the steps of:
a. providing a given quantity of a conjugate of said bindable
substance with an enzyme;
b. providing a corresponding given quantity of an antibody against
said bindable substance;
c. admixing a sample of a fluid containing the bindable substance
to be determined with the reactants of steps (a) and (b) to form a
reaction mixture and allowing the reaction to go to completion;
d. separating the resulting mixture into a liquid phase and a solid
phase by adding an insolubilized antibody against the antibody of
step (b); and
e. determining the quantity of the bindable substance from the
measure of
2. The process of claim 1 in which the bindable substance is
determined by first adding thereto the antibody, then the enzyme
conjugate, and finally
3. Process for the detection and determination of an antibody in a
fluid sample containing the same utilizing the reaction between
said antibody and a bindable substance selected from the group
consisting of an antigen and a hapten having an affinity therefor,
comprising the steps of:
a. providing a given quantity of a conjugate of said bindable
substance and an enzyme;
b. providing a given quantity of an insolubilized antibody against
the antibody to be determined;
c. admixing said given quantities of steps (a) and (b) with said
sample, and allowing the components to react;
d. separating the reaction mixture into a liquid phase and a solid
phase; and
e. determining the quantity of the antibody from the measure of
enzyme
4. A test pack for the detection and determination of a bindable
substance selected from the group consisting of an antigen and a
hapten in a fluid sample, comprising:
a. a known amount of a conjugate of said bindable substance with an
enzyme;
b. a corresponding known amount of an antibody against said antigen
or hapten;
c. a known amount of an insolubilized antibody against said
antibody;
d. a stabilizer; and
e. a substrate for the determination of the enzyme activity and
thus of the
5. A test pack for the detection and determination of an antibody
in a fluid sample, utilizing the reaction between said antibody and
an antigen or hapten to said antibody, comprising:
a. a known amount of a conjugate of the antigen or hapten with an
enzyme;
b. a corresponding known amount of an insolubilized antibody
against the antibody to be determined;
c. a stabilizer; and
d. a substrate for the determination of the enzyme activity and
thus of the
6. A test pack for the determination of pregnancy by the detection
and determination of human chorionic gonadotropin in a sample of
urine, utilizing the reaction between human chorionic gonadotropin
and an antibody therefor, comprising, in separate lyophilized
layers:
a. a predetermined amount of a conjugate of human chorionic
gonadotropin and an enzyme;
b. a predetermined amount of an antibody against human chorionic
gonadotropin;
c. a predetermined amount of an antibody against the human
chorionic gonadotropin antibody, in insolubilized form;
d. a stabilizer; and
e. a substrate for the determination of the enzyme activity and
thus of the quantity of human chorionic gonadotropin enzyme
conjugate and human chorionic gonadotropin to be determined.
Description
It is known that a component of the reaction between a specific
binding protein and the corresponding bindable substance can be
detected and determined by incubating one of the named components,
labelled by a marker, in a reaction mixture which at least contains
the other component, and then effecting a separation between the
marked component that is and that is not bound to its binding
partner, and finally determining the marker in at least one of the
two fractions obtained. The distribution of the marked component
over the two fractions is a measure of the amount of substance to
be determined and present in the sample. Three systems can be
described, in which the above method is applied.
A. antibodies as specific binding proteins, and the corresponding
antigens as bindable substances. Substances which can be determined
with this system are, inter alia, protein hormones and their
antibodies, or virus antigens and their antibodies;
B. antibodies as specific binding proteins, and haptens as bindable
substance. Here the haptens are defined as protein-free substances,
which can react with antibodies, without being able to induce them.
Substances which can be determined in such a system are, inter
alia, steroid hormones and vitamines;
C. proteins which in the body act as receptor or transport
molecules as specific binding proteins, and substances which are
bound by them as bindable substances. This system is e.g., suitable
for the determination of steroid hormones, but also of thyroxine
and triiodothyronine, vitamine B.sub.12, intrinsic factor, and
adrenocorticotropic hormone.
The most important points in the methods of determination described
are the application of a marker and the separation of the marked
component into a fraction which is, and a fraction which is not
bound to the corresponding component.
In the methods practically used up to now only radio-active atoms
have been applied as marker, e.g., 131.sub.I, 125.sub.I, 14.sub.C,
3.sub.H, 57.sub.Co. This method usually stands out by a high
sensitivity. However, the application of this method is limited to
institutes which have the required specialized apparatus
available.
The separation methods can be divided as follows:
A. methods depending on the difference in physical properties
between the non-bound, marked component and its complex with the
binding partner, such as gelfiltration, electrophoresis, salt
precipitation, and adsorption to dextran-coated charcoal;
B. the so-called solid phase methods, in which beforehand one
component has already been brought into an insoluble form by
cross-linking or by covalent binding or physical adsorption to a
solid carrier.
c. the so-called double antibody method in which the complex
formed, antigen (or hapten)-antibody, is precipitated with the help
of antibodies against the antibody in the complex, which method is
only known for systems in which antibodies are involved. Whilst the
methods mentioned under (a) and (c) are relatively complicated to
perform, those mentioned under (b) suffer from the disadvantage
that as a result of bringing a reaction component in an insoluble
form, its affinity for the reaction partner usually decreases. A
high affinity is, however, essential for realizing a sensitive
system of test.
Now a process was found for the detection and determination of a
component of the reaction between a specific binding protein and
the corresponding bindable substance, applying the known binding
affinity of such components for one another, characterized in that
use is made of a given amount of a coupling product of the
combinable substance with an enzyme, and of antibodies against the
specific combining protein, brought in an insoluble form, and that
after the reaction the enzyme activity is determined in the liquid
or solid phase of the reaction mixture, which activity is a measure
of the amount of the component to be determined.
A frequent and advantageous use of this method is made when
antibodies are made to act as specific binding protein and antigen
or hapten as corresponding bindable substance.
In the present description the term conjugate and enzyme conjugate
are used as synonyms for the coupling product of the bindable
substance and the enzyme.
The bindable substance can be detected and determined by bringing
together the unknown sample or a dilution series of same with a
known amount of a conjugate of the substance to be determined and
an enzyme, and with an amount of specific binding protein,
depending on the amount of enzyme conjugate that was added. Then an
amount, preferably an excess, of the insoluble-made antibodies
against the specific binding protein is added, so that all the
enzyme conjugate that has reacted with the binding protein is
coupled -- via this protein -- to these insoluble antibodies. In
proportion as there is more bindable substance present in the
sample, less enzyme conjugate will react with the specific binding
protein and ultimately get into the insoluble phase; the result is
that more unbound enzyme conjugate remains in the liquid phase,
which can simply be determined there.
The specific binding protein can be determined by incubating the
sample or a dilution series of same with a known amount of enzyme
conjugate and with an amount of the insoluble-made antibodies
against the specific binding protein. The enzyme activity can only
pass into the insoluble phase if the conjugate has reacted with the
specific binding protein: the more specific binding protein there
is in the sample, the less of bound enzyme conjugate remains in the
liquid phase.
The sensitivity of the test systems described can be varied by
altering the quantities of the reagents (whether or not in the same
ratio). However, the amount of enzyme conjugate that can be applied
is limited at the lower end by the requirement that its enzyme
activity can be measured reasonably, so that the sensitivity of the
test systems has a limit. The minimum measurable enzyme activity
depends inter alia on the nature of the enzyme used for the
coupling and on the nature of the substrate and of the incubation
period of the enzyme reaction. Further the affinity of the specific
binding proteins strongly influences the sensitivity of the
determination. For a highly sensitive test system specific binding
proteins with a higher affinity are required.
The quanitities of reagent required per determination are
established empirically.
For the determination of the bindable substance the quantity of
enzyme conjugate will be determined with the help of the enzyme
activity; then this quantity is incubated with a dilution series of
the specific binding protein to determine the required quantity of
this protein. By preference a quantity of specific binding protein
is chosen, which binds 50-90% of the enzyme conjugate. Finally it
is checked whether the desired sensitivity has indeed been reached,
by testing a dilution series of the substance under test in the
system. For the determination of the specific binding protein
another consideration regarding the dosage of the enzyme conjugate
is its activity, which should be reasonably determinable.
The insoluble-made antibodies against the specific binding protein
are preferably added in excess in the two types of determination;
the dosing of same is determined in preliminary tests. The
advantages of this method over the existing are:
a. working with radioisotopes can be replaced by working with
enzymes. This requires considerably less laboratory facilities and
apparatus, whilst the staff can be less highly qualified. Moreover,
working with radioactive isotopes is highly limited because of
legal regulations. Further the reagents according to the invention
will keep long and the safety is increased;
b. the combination of "Double Antibody" and "Solid Phase" methods,
for ease of reference called DASP method below, offers several
advantages over the existing methods. Thus the performing of the
found method, viz.: addition of insoluble-made antibodies against
the specific binding protein, incubation, centrifuging and
measuring, is very simple. The dosing is often easier than in the
solid phase methods because it suffices to add an excess of
insoluble material, whereas in the solid phase methods an
accurately measured amount of material has to be used. Moreover,
the affinity of the binding protein for the bindable substance is
not impaired by the binding to the carrier material, which can be
the case in the solid phase method. A further advantage over the
latter method is the rapid equilibrium adjustment of the reaction
between specific binding protein and bindable substance (both in
solution). A further advantage is that in the DASP method the
insoluble-made antibody, called immunoadsorbent below, can be used
in any system in which antibodies are used as specific binding
protein, provided that these antibodies have been prepared in the
same animal species. On the other hand, for each antigen or hapten
to be determined with the solid phase method, the antibodies have
to be made insoluble. The double antibody method is very sensitive
for relatively low variations in salt concentrations, pH and the
like, which makes a rigid control of the conditions necessary.
Moreover, the method requires the addition of "carrier"
.gamma.-globuline and consequently much second antibody to obtain
an immune precipitate. In addition to being a simpler procedure,
the DASP method, which does not require "carrier"
.gamma.-globuline, thus leads to material saving. Further it be
added that a double antibody-like separation applied to transport
or receptor proteins is not possible, as no suitable "carrier"
protein is available for this purpose. The method according to the
invention will therefore offer a unique opportunity in this
respect.
c. The method according to the present invention can easily be
automatized. In principle it is possible to bring the reaction
components together at once or to add them in any sequence. It was
found, however, that the determination acquires a higher
sensitivity if the immunoadsorbent is added after the incubation of
the other components.
The reagent required for the invention, viz. the coupling product
of antigen, hapten or bindable substance with an enzyme, can be
prepared in a known manner. These methods can also be used to bind
a hapten or a low-molecular bindable substance to an enzyme,
provided that one substance possesses one or more amino-groups and
the other one or more carboxyl-groups. If the latter is not the
case then it is possible to introduce the desired group into the
molecule to be coupled with the help of known organo-chemical
processes. Methods are also known to bind amino or carboxyl-groups
together, whether or not by introducing a bridge. Finally compounds
as glutaric aldehyde, difluorodinitrodiphenyl-sulphon and di- and
tri-chloro-s-triazine can often be used for the coupling in
question. It can be necessary to separate the prepared enzyme
conjugates from non-converted substances or from substances that
have become inactive. To this end the known biochemical methods can
be used, such as precipitation with organic solvents,
gelfiltration, and centrifugation at a density gradient.
The choice of the enzyme which is taken up in the coupling product,
is determined by a number of properties of that enzyme. It is, of
course, essential that the enzyme should be resistant to the
coupling with another molecule, i.e., modification of one or more
aminoacid side chains. Also of great importance is the specific
activity of the enzyme. As less enzyme conjugate needs to be added
to reach a measurable enzyme effect, the test system grows more
sensitive. Further those enzymes are to be preferred, of which the
determination of the activity can be made in a simple manner. In
the first place those enzymes are considered that can be determined
colorimetrically, spectrophotometrically or fluorimetrically. This
kind of determinations is suitable for automation, which is an
additional advantage.
Colorimetrically those enzymes can be determined that catalyze a
reaction in which a coloured substance appears or disappears,
either in the primary or in the secundary reaction.
As enzymes considered to act as enzymatically active component in
conjugates, are mentioned catalase, peroxidase,
.beta.-glucoronidase, .beta.-D-glucosidase, .beta.-D-galactosidase,
urease, glucose oxidase, galactose oxidase, and alkaline
phosphatase.
At the end of the reaction between the components and the reagents
according to the invention the enzyme activity of the liquid or
solid phase of the reaction mixture or of the two phases can be
determined. Most simple is, however, to determine the enzyme
activity of the liquid phase.
The insoluble-made antibodies against the specific binding
proteins, which are also an essential reagent for the process of
the invention, can also be prepared in a known way. The antibodies
can be prepared by taking a purified preparation of the specific
binding protein, or of proteins which have at least partly the same
antigen properties as the specific binding protein, and injecting
this in a known way into another animal species than from which it
was obtained. The serum of the treated animal, or the
gammaglobuline fraction thereof, can be made insoluble by
cross-linking with compounds such as glutaric aldehyde and
chloroformic acid ethyl ester, or by binding to solid carrier
particles, either physically by adsorption, or chemically by the
formation of covalent bonds. As solid carriers can be considered
materials such as cellulose (modified or not), agarose,
cross-linked dextran, polystyrene and the like. Covalent binding of
the antibodies to these materials can be effected with the help of
substances such as carbodiimides, di- and tri-chloro-s-triazines,
glutaric aldehyde, cyanogenbromide, and e.g., by diazotation.
The advantages of the method of determination found are done full
justice if an excess of insoluble antibodies is applied, so that
the specific binding protein passes completely into the solid
phase.
The forms in which the reagents can be used are manifold. The
enzyme-conjugated component of the reaction system can be
freeze-dried, or dissolved in a buffer. Also a solid carrier can be
used, e.g., a strip of paper impregnated with the conjugate. This
applies equally to the required specific binding proteins.
The insoluble component can be brought in the form of particles of
different dimensions, such as granules, flakes, rods, or in the
form of a strip of some carrier material.
For the performing of the procedure according to the invention a
test pack is applied by preference. This consists mainly of:
1. a known amount of a conjugate of an antigen, hapten or
low-molecular bindable substance with an enzyme,
2. a corresponding amount of specific binding protein (antibodies
or transport or receptor proteins),
3. a known amount of insoluble-made antibodies directed against the
specific binding protein used. The test pack can further contain
the reagents required for the enzyme determinations, and also
auxiliary means for conducting the test, such as test tubes,
pipettes and bottles with dilution liquid. Such a test pack is
suitable for the determination of a bindable substance, but also
for the determination of a specific binding protein, in which case
the specific binding protein contained in the test pack need not be
used.
The test packs are especially frequently and with advantage used
for the detection and determination of an antigen or hapten and to
this end they contain mainly:
a. a known amount of a conjugate of the antigen or hapten and an
enzyme,
b. a corresponding amount of corresponding antibodies,
c. a known amount of insoluble-made antibodies directed against the
antibodies used. When applying such test packs for the detection
and determination of antibodies the antibodies mentioned under (b)
are not required.
An important embodiment of a test pack according to the present
invention is a test pack to be used for the determination of
gonadotropic hormones, and particularly for the determination of
HCG (Human Chorionic Gonadotropin) as a means to diagnose pregnancy
already in a very early stage, which test pack consists of an
ampoule, tube, bottle or other container containing as essential
ingredients, in separate lyophilized layers, pre-determined amounts
of:
a. a conjugate of HCG with an enzyme, e.g., HCG-peroxidase.
b. anti-HCG.
c. antibodies against anti-HCG, made insoluble.
d. possibly other ingredients like a buffer.
By adding a certain amount of urine of a possibly pregnant woman to
this test kit and to incubate the urine with the components of the
kit a mixture of insoluble material is formed whereas the
supernatant contains the remaining soluble HCG-enzyme conjugate.
The amount of the latter is dependent on the amount of HCG in the
urine to be tested. By determining the enzyme activity of this
remaining HCG-enzyme conjugate it can be ascertained whether the
urine is coming from a pregnant woman or not.
A preferably applied method for the determination of the enzyme
activity consists in contacting an indicator-paper impregnated with
enzyme reagents, e.g., in case use is made of a peroxidase, a
H.sub.2 O.sub.2 -supplier like urea-H.sub.2 O.sub.2, and a
colour-reagent like o-tolidine.
By a correct choice of the amounts of each of the reagents it will
be possible to ascertain pregnancy already in a very early stage
and in a simple, rapid and very reliable way to be performed even
by unskilled persons.
Example I
Determination of human choriongonadotrophine (HCG)
a. Preparation of HCG-HRP. 5 mg HCG and 20 mg horse radish
peroxidase (HRP) were dissolved in 2 ml 0.05 M phosphate buffer of
pH 6.2. After addition of 40 .mu.l 25% glutaric aldehyde solution
the mixture was shaken for 2 hours at room temperature. After 5
minutes centrifugation at 250 g, the liquid was fractionated over
Sephadex G-200 in 0.05 M phosphate buffer of pH 6.2. The fractions
of which the highest percentage enzyme activity was bound by
antibodies against HCG were used in the test system.
b. Preparation of antibodies against HCG. Antibodies against HCG
were induced in rabbits as described by Schuurs et al. Acta Endocr.
(Kbh.) 59, 120 (1968).
c. Preparation of antibodies against rabbit-.gamma.-globuline.
Rabbit-.gamma.-globuline was isolated from normal rabbit serum by
precipitation with 18% w/v solid sodium sulphate. Antibodies
against this were prepared by immunizing a sheep according to the
following schedule:
day amount Freund's adjuvans injection manner
______________________________________ 0 0.5 mg + intramuscular 14
0.5 mg + intramuscular 28 1 mg + intramuscular 42 1 mg -
intravenous 56 1 mg - intravenous
______________________________________
On day 70 the sheep was bled.
d. Preparation of the immunoadsorbent
[sheep-anti-(rabbit-.gamma.-globuline)] cellulose. The
.gamma.-globuline fraction of the sheep serum described under 1 c)
was prepared by precipitation with 16% w/v solid sodium sulphate.
After washing, the precipitate was taken up in so much 0.05 M
borate buffer of pH 8.6 that the resulting protein concentration
amounted to 10 mg/ml. 350 mg m-aminobenzyloxymethylcellulose was
suspended in 50 ml distilled water, and diazotized by adding 10 ml
36% hydrochloric acid and, dropwise, 10 ml 10% NaNO.sub.2 solution
at 0.degree.C. The suspension was centrifuged, washed and the
precipitate re-suspended in 43 ml 0.05 M sodium borate of pH 8.6.
Then 7 ml of the prepared .gamma.-globuline solution was added. The
mixture was stirred for 26 hours at 4.degree.C, then centrifuged
and washed with 0.02 M phosphate buffer of pH 6.0.
e. Determination of HCG. A dilution series (32-16-8-4-2-1-0.5-0
IU/ml) of HCG in 0.02 M phosphate buffer with pH 6.0 was prepared,
which contained 2% v/v normal sheep serum. 0.5 ml of each of the
HCG-containing samples was incubated with 0.1 ml rabbit-(anti-HCG)
serum and 0.1 ml HCG-HRP conjugate, both in suitable dilution, for
half an hour at room temperature. Then 0.3 ml of the
immunoadsorbent (10 mg/ml) prepared acccording to d) was added, and
the resulting mixture was rotated at room temperature for 1 hour.
After centrifugation the enzyme activity in the supernatant was
measured by mixing 0.5 ml of this liquid with 1.5 ml substrate (10
.mu.l 30% H.sub.2 O.sub.2 and 20 mg 5-aminosalicylic acid in 150 ml
0.02 M phosphate buffer of pH 6.0) and after 30 minutes at
25.degree.C measuring the extinction at 460 nm. In this way it
proved possible to detect a HCG concentration from 0.5 to 1 IU/ml
in the sample. With this method also urine samples could be tested;
the test is therefore suitable for a pregnancy check. The
correlation with an existing method of test, a haemagglutination
inhibiting test, was good. It proved possible to raise the
sensitivity of the system by the application of a pre-incubation.
Here, first the sample only was incubated with the antiserum, and
then the HCG-HRP conjugate was added.
Example II
Determination of insuline and anti-insuline.
a. Preparation of insuline-(glucose oxidase). 5 mg pig insuline and
25 mg glucose oxidase were dissolved in 2 ml 0.05 M phosphate
buffer of pH 6.5. To this, 5 .mu.l 25% glutaric aldehyde solution
was added, after which the mixture was shaken for 90 minutes at
room temperature. The mixture was fractionated over Sephadex G-200
in 0.05 M phosphate buffer of pH 6.5. The fractions of which the
highest percentage of enzyme activity could be bound by antibodies
against insuline, were used in the test system.
b. Preparation of antibodies against insuline. 10 guinea pigs were
given a weekly intramuscular injection with 1 mg pig insuline in
complete Freund's adjuvans over a period from 4-8 weeks. After two
weeks' rest the animals were given 1 mg additional insuline by
intravenous injection without adjuvans. 2 weeks after that the
animals were bled. Hypoglycaemia occurring at times was
counteracted by intraperitoneal administration of glucose.
c. Preparation of antibodies against guinea pig .gamma.-globuline.
Guinea pig .gamma.-globuline was prepared by adding 1 volume
saturated ammonium sulphate solution to 2 volumes guinea pig serum.
The precipitate formed was twice washed with 33% saturated ammonium
sulphate solution, and then taken up in a physiological salt
solution. A sheep was immunized with increasing doses of the
prepared .gamma.-globuline: 0.5, 1 and 2 mg. The injections were
given every two weeks, whilst the immunogen was mixed with complete
Freund's adjuvans. Two weeks after the last injection an additional
2 mg .gamma.-globuline in a physiological salt solution were given,
and 1 week later the animal was bled.
d. Preparation of insoluble antibodies against guinea pig
.gamma.-globuline. 10 g microcrystalline cellulose was activated by
adding it whilst stirring to 400 ml 2.5% w/v CNBr-solution, after
which the pH was brought to 10.5 with 1 N NaOH solution and
maintained thus for 2 minutes. Then the cellulose was washed with
ice water and with 0.1 M NaHCO.sub.3. To 10 ml sheep-anti(guinea
pig .gamma.-globuline) serum 1.6 g Na.sub.2 SO.sub.4 was added.
After 1 hour stirring at room temperature the precipitate was
centrifuged, twice washed with 20 ml 16% w/v Na.sub.2 SO.sub.4
solution, and subsequently taken up in 10 ml 0.1 M NaHCO.sub.3. The
activated cellulose was mixed with 40 ml 0.1 M NaHCO.sub.3 solution
and the 10 ml .gamma.-globuline solution. This suspension was
rotated for 40 hours at 4.degree.C, and in succession washed with
twice 500 ml 0.5 M NaHCO.sub.3, twice 500 ml 0.05 M citrate pH 1.1,
and twice 500 ml 0.05 M phosphate of pH 6.2.
e. Determination of antibodies against insuline. 0.1 ml
insuline-(glucose oxidase), in a suitable dilution, was incubated
with 0.4 ml of a dilution series of a guinea pig anti-insuline
serum for 4 hours. The dilution series was made with 0.05 M
phosphate buffer of pH 6.0. Then 0.3 ml immunoadsorbent (15 mg/ml)
and 0.2 ml buffer were added and the mixture was rotated during the
night at 4.degree.C. After centrifuging the enzyme activity of the
supernatant was determined by incubating 0.5 ml of same with 2.5 ml
substrate for 30 minutes, and then measuring the extinction at 460
nm. The substrate contained 50 mg glucose, 10 .mu.g peroxidase and
1 mg 5-aminosalicyclic acid per 2.5 ml 0.05 M phosphate buffer of
pH 6.0. By means of this system the antibody content of the
different serums could be intercompared. As reference point was
chosen the serum dilution at which 50% of the total combinable
enzyme activity is bound.
f. Determination of insuline. 0.2 ml of a dilution series of
insuline was incubated for 2 hours with 0.4 ml anti-insuline serum
in such a dilution that it could bind 60% of the enzyme conjugate
to be added. Then 0.1 ml insuline-(glucose oxidase) was added in
the corresponding dilution, and incubated for 4 hours. Finally 0.3
ml immunoadsorbent (15 mg/ml) was added. The mixture was rotated
during the night at 4.degree.C. After centrifuging the enzyme
activity of the supernatant was measured as described under (e).
The sensitivity of the determination, which depends on the
antiserum used, lies in the nanogram range: 20-100 ng/ml, i.e.,
0.5-2.5 mU/ml.
Example III
Determination of oestradiol.
a. Preparation of oestradiol-17-succinyl-HRP. 50 mg
oestradiol-17-hemisuccinate and 0.08 ml tri-n-butylamine were
dissolved in 2.5 ml dioxane. To the cold (2.degree.C) solution 15
.mu.l isobutylchlorocarbonate was added. After 30 minutes this
solution was mixed with 100 mg horse radish peroxidase (HRP) in 7.5
ml of a dioxane/water mixture (2:3) which had been adjusted to pH
9.5 with sodium hydroxide. The solution was stirred for 4 hours at
2.degree.C, and then dialyzed for 18 hours. The precipitate formed
after the pH of the dialysate had been adjusted to 4.6, was
centrifuged, washed and taken up in 5 ml distilled water that had
been adjusted to pH 8. The material was further purified by twice
precipitating with 10 ml acetone. The finished product was taken up
in 10 ml 0.05 M phosphate buffer of pH 7.8.
b. Preparation of oestradiol-17-succinyl-BSA. The preparation was
prepared according to the mixed anhydride method, as described in
Example IIIa. This preparation was made starting from 100 mg
oestradiol-17-hemisuccinate and 150 mg bovine serum albumine
(BSA).
c. Preparation of antibodies against oestradiol. A sheep was
injected once in four weeks with 4 mg oestradiol-17-succinyl-BSA in
complete Freund's adjuvans. At regular intervals blood was taken
from the sheep. The serum was absorbed with BSA that was made
insoluble.
d. Preparation of antibodies against sheep-.gamma.-globuline.
Sheep-.gamma.-globuline was prepared as described in example I, but
now with 16% w/v sodium sulphate. Rabbits were immunized with this
sheep-.gamma.-globuline according to the following schedule:
day amount Freund's adjuvans injection manner
______________________________________ 0 200 .mu.g + intramuscular
14 400 .mu.g + intramuscular 28 800 .mu.g + intramuscular 42 800
.mu.g - intravenous ______________________________________
2 weeks after the last injection the animals were bled.
e. Preparation of the immunoadsorbent
[rabbit-anti(-sheep-.gamma.-globuline)]-cellulose. The
.gamma.-globuline fraction of the antiserums, described under (d),
was prepared by precipitation with 18% w/v Na.sub.2 SO.sub.4. The
product obtained was coupled to cellulose according to the Gurvich
method, described in example I.
f. Determination of oestradiol. The immune reaction was made in
0.02 M phosphate buffer of pH 6.0, which contained 2% BSA: 0.5 ml
sample was mixed with 0.1 ml of the sheep-anti-oestradiol serum in
the desired dilution. After 30 minutes incubation at room
temperature, 0.1 ml oestradiol-17-succinyl-HRP was added in a
suitable dilution, after which another 30 minutes incubation at
room temperature followed. Then 0.3 ml immunoadsorbent suspension
(30 mg/ml) was added and the mixture was rotated for 2 hours at
room temperature. Then the liquid and solid phases were separated
from each other by centrifuging. The enzyme activity in the
supernatant was measured as described in example I. The
sheep-anti-oestradiol serum could be used in dilutions from 1:1600
to 1:12,800, depending on the quality of the
oestradiol-17-succinyl-HRP used. With a dilution of 1:12,800 of the
antiserum an oestradiol concentration of 10 ng/ml could be detected
in the sample. Oestriol and oestron showed a cross reaction in this
system.
Example IV
Determination of cortisol and corticoid-binding globuline.
a. Preparation of cortisol-21-(galactose oxidase). 50 mg
cortisol-21 -hemisuccinate and 100 mg galatose oxidase were coupled
by means of the mixed anhydride technique, as described in example
IIIa.
b. Corticoid-binding globuline (CBG) was isolated from the serum of
men by means of chromatography in succession over DEAE-cellulose
and hydroxyl apatite. Antibodies against this were prepared by
injecting rabbits at 14-day intervals with 500 .mu.g CBG in
complete Freund's adjuvans. After 3 months the animals were
injected with 1 mg CBG and 2 weeks later they were bled.
c. The .gamma.-globuline fraction of anti-CBG serum was coupled to
m-aminobenzyloxymethylcellulose, as described in example III.
d. Determination of cortisol. 0.5 ml of a cortisol-containing
sample (standard solution) was extracted with 2 .times. 3 ml
methylenechloride. The combined extraction liquids were evaporated
to dryness. The residue was taken up in 0.5 ml 0.05 M phosphate
buffer of pH 6.2, then mixed with 0.1 ml of a solution of CBG in
the same buffer in a suitable concentration, and incubated for 30
minutes at 4.degree.C. Then 0.1 ml cortisol-21-(galactose oxidase),
also in a suitable dilution, and 0.3 ml of the immunoadsorbent
prepared under (c) with a concentration of 5 mg/ml were added. The
mixture obtained was rotated for 2 hours at 4.degree.C and then
centrifuged, after which the enzyme activity was measured in the
supernatant. To this end 0.5 ml of same was added to 1.5 ml
substrate consisting of 100 mg D-galactose, 20 mg 5-aminosalicyclic
acid and 10 .mu.g peroxidase in 150 ml 0.02 M phosphate buffer of
pH 6.0. After 30 minutes the extinction was measured at 460 nm.
When applying a CBG concentration of 0.4 .mu.g/ml and so much
cortisol-21-(galactose oxidase) that without the addition of
steroids 80% of the enzyme conjugate was bound to the
immunoadsorbent, it proved possible to determine amounts from 3-30
ng cortisol.
e. Determination of CBG was also possible with the reagents
described. From a dilution series of transcortine, ranging from
0-1280 ng/ml, 0.5 ml was incubated for 15 minutes with 0.2 ml
cortisol-21-(galactose oxidase) in a suitable dilution. Then 0.3 ml
immunoadsorbent suspension (5 mg/ml) was added, and the mixture
rotated for 15 minutes. The two incubations were conducted at
4.degree.C. Then the enzyme activity in the supernatant was
measured as under d). The sensitivity of the test system proved to
be 50 ng/ml.
Example V
In a bottle the following reagents were subsequently lyophilized in
separate layers:
1. 0.3 ml of the immunoadsorbent suspension (10 mg/ml) as described
in Example I a).
2. 0.1 ml of a 1% mannitol solution.
3. 0.1 ml of HCG-HRP as described in Example I (a).
4. a second layer of 0.1 ml of a 1% mannitol solution.
5. 0.1 ml of rabbit (anti-HCG) serum as described in Example I
(b).
To this lyophilized mixture 0.5 ml of a urine sample and
subsequently 0.5 ml distilled water was added. After 10 minutes the
enzyme activity in the supernatant was measured by means of a slip
of paper impregnated with urea-hydrogenperoxide and o-tolidine.
If the urine sample was coming from a pregnant woman (>2 IU
HCG/ml) a blue colour appeared within 5 minutes, whereas in case of
urine of a non-pregnant woman no discoloration took place within
the same period.
* * * * *