U.S. patent number RE32,696 [Application Number 06/972,411] was granted by the patent office on 1988-06-14 for enzymatic immunological method for determination of antigens and antibodies.
This patent grant is currently assigned to Akzona Incorporated. Invention is credited to Antonius H. W. M. Schuurs, Bauke K. Van Weemen, Gerrit Wolters.
United States Patent |
RE32,696 |
Schuurs , et al. |
June 14, 1988 |
**Please see images for:
( Certificate of Correction ) ** |
Enzymatic immunological method for determination of antigens and
antibodies
Abstract
The present invention relates.Iadd., inter alia, .Iaddend.to
improvements in the sandwich technique for the determination of a
component of an antigen-antibody reaction in a liquid sample to be
tested, utilizing as reagents (a) one component of said reaction
bound to the surface of a water-insoluble, water-insuspensible,
solid carrier, and (b) a component having the same immunological
properties covalently linked to an enzyme. .Iadd.If and only if the
component of the reagent that is water-insoluble and
water-insuspensible has the same immunochemical properties as the
component to be determined, is a predetermined amount of a binding
partner for said first reagent (e.g., the reagent that is
water-insoluble and water-insuspensible) added to the liquid
sample. .Iaddend.The liquid sample is contacted and incubated with
the reagent(s) to form a reaction mixture, the enzyme activity of
either the liquid or solid phase of which is a measure of the
presence and quantity of the component to be determined. The method
is especially useful for diagnostic testing for hepatitis or
rubella antibodies.
Inventors: |
Schuurs; Antonius H. W. M.
(Oss, NL), Van Weemen; Bauke K. (Oss, NL),
Wolters; Gerrit (Oss, NL) |
Assignee: |
Akzona Incorporated (Asheville,
NC)
|
Family
ID: |
27086257 |
Appl.
No.: |
06/972,411 |
Filed: |
December 22, 1978 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
610469 |
Sep 4, 1975 |
04016043 |
Apr 5, 1977 |
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Current U.S.
Class: |
435/5; 435/7.93;
435/810; 436/518; 436/531; 436/532; 436/808; 436/820 |
Current CPC
Class: |
G01N
33/54306 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 033/53 (); G01N 033/535 ();
G01N 033/545 () |
Field of
Search: |
;435/5,7,810
;436/518,531,532,808,820 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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7101728 |
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Feb 1971 |
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NL |
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1387625 |
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Mar 1975 |
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GB |
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|
Primary Examiner: Marantz; Sidney
Attorney, Agent or Firm: Wegner & Bretschneider
Claims
What is claimed is: .[.
1. A method for the detection and determination of a component of
an antigen-antibody reaction in a liquid sample containing the
component to be determined, comprising the steps of:
a. providing a given quantity of a first reagent consisting of one
component of said reaction selected from the group consisting of an
antigen and an antibody bound to the surface of a water-insoluble,
water-insuspensible, solid carrier;
b. providing a given amount of a second reagent consisting of a
component having the same immunological properties as the component
in the said first reagent covalently linked to an enzyme, and also
providing a predetermined amount of the binding partner for the
component to be determined, when the component in said liquid
sample and the component bound to said solid carrier have the same
immuno-chemical properties;
c. contacting a given quantity of said liquid sample with said
reagents forming a reaction mixture having a solid phase and a
liquid phase; and
d. determining the enzyme activity of either the solid or the
liquid phase which is a measure of the presence and quantity of the
component to be determined..]. .[.2. A method for determining the
presence of a component of an antigen-antibody reaction in a liquid
sample containing the component to be determined comprising the
steps of:
a. providing a given quantity of a first reagent consisting of one
component of said reaction selected from the group consisting of an
antigen and an antibody bound to the surface of water-insoluble,
water-insuspensible, solid carrier, and also providing a
predetermined amount of the binding partner for the component to be
determined when the component in said liquid sample and the
component bound to said solid carrier have the same immunochemical
properties;
b. contacting and incubating a given quantity of said liquid sample
with said first reagent;
c. washing said solid carrier;
d. providing a given quantity of a substance having the same
immunological properties as the component previously bound to the
solid carrier, said substance being covalently linked to an
enzyme;
e. contacting and incubating the solid phase from step (c) with
said enzyme-linked substance;
f. washing the solid carrer; and
g. determining the enzyme activity substance bound to the solid
phase, which is a measure of the presence and quantity of the
component to be determined..]. .[.3. The method of claim 2 wherein
said liquid sample and the binding partner of the component to be
determined are preincubated with each other, before contacting the
sample with said solid carrier..].
.[.4. A method for the detection and determination of a component
in the reaction of a hepatitis antigen and the associated antibody
in a liquid sample containing said component, comprising the steps
of:
a. providing a given quantity of a reagent consisting of one
component of said reaction selected from the group consisting of
said antigen and said antibody bound to a water-insoluble,
water-insuspensible, solid carrier;
b. contacting and incubating said liquid sample with said solid
carrier of step (a) to form a reaction mixture;
c. providing a given quantity of a substance having the same
immunological properties as the component bound to said solid
carrier, said substance being covalently linked to an enzyme, and
also providing a predetermined amount of the binding partner for
the component to be determined when the component in said liquid
sample and the component bound to said solid carrier have the same
immunochemical properties;
d. contacting and incubating the reaction mixture with said
enzyme-linked substance; and p1 e. determining the enzyme activity
of substance bound to the solid phase, which is a measure of the
presence and quantity of the component to be determined..]. .[.5.
The method of claim 4 in which said hepatitis antigen is hepatitis
B surface antigen, and the antibody is its associated antibody..].
.[.6. The method of claim 4 in which said solid carrier is washed
after the incubation of the liquid sample with the immunological
component bound to the solid carrier..]. .[.7. A diagnostic pack
for the detection and determination of a component of an
antigen-antibody reaction selected from the group consisting of an
antigen and an antibody, consisting of:
a. one of said components coupled to a water-insoluble,
water-insuspensible, solid carrier;
b. a substance having the same immunological properties as the
component in (a) covalently linked to an enzyme; and
c. the binding partner of the component to be determined if the
component to be determined has the same immunological properties as
the component in (a)..]. .[.8. A diagnostic pack for the detection
and determination of hepatitis B consisting of:
a. the antibody against Hepatitis B surface antigen coupled to a
water-insoluble, water-insuspensible, solid carrier;
b. said antibody covalently linked to an enzyme; and
c. Hepatitis B surface antigen in case the component to be
determined is
the antibody against Hepatitis antigen..]. .Iadd.9. A method for
the detection and determination of an antibody in a liquid sample
containing said antibody to be detected and to be determined,
comprising the steps of:
a. providing a given quantity of an antigen to said antibody, said
antigen bound to the surface of a water-insoluble,
water-insuspensible, solid carrier;
b. contacting and incubating a given quantity of said liquid sample
having the antibody to be detected and determined with bound
antigen of step (a), forming a reaction mixture having a solid
phase and a liquid phase;
c. separating the solid phase from the liquid phase;
d. contacting and incubating with said solid phase a given quantity
of a coupling product obtained by covalently binding a component
having the same immunological properties as said antigen, to an
enzyme, which quantity of coupling product is at least
immunochemically equivalent to said antigen provided in step (a),
in order to form a second solid phase and a second liquid phase;
and
e. detecting and determining the enzyme activity of either the
second solid or the second liquid phase of step (d) after
separating the solid and liquid phases formed thereby, which
detection and determination is a measure of the presence and
quantity of said antibody to be detected and to be
determined..Iaddend. .Iadd.10. The method of claim 9, wherein the
quantity of said bound antigen in step (a) is at least sufficient
to react with all of the antibody to be determined..Iaddend.
.Iadd.11. The method of claim 9, wherein an additional step of
separating the solid phase from the liquid phase takes place
between steps (d) and (e)..Iaddend. .Iadd.12. The method of claim
11, wherein both separation steps are accomplished by
washing the solid carrier..Iaddend. .Iadd.13. A method for the
detection and determination of a component of an antigen-antibody
reaction in a liquid sample containing said component to be
detected and determined, comprising the steps of:
a. providing a given quantity of a first reagent consisting of one
component of said reaction selected from the group consisting of an
antigen and an antibody bound to the surface of a water-insoluble,
water-insuspensible, solid carrier with the proviso that said first
reagent has the same immunochemical properties as the component to
be detected and determined;
b. providing a predetermined amount of a binding partner for said
first reagent, which amount is less than or is immunochemically
equivalent to the given quantity provided in step (a) of the
insolubilized component,
c. contacting and incubating a given quantity of said liquid sample
having the component to be detected and determined with said
reagents of step (a) and step (b), forming a reaction mixture
having a solid phase and a liquid phase;
d. separating the solid phase from the liquid phase;
e. contacting and incubating with said solid phase a given quantity
of a coupling product obtained by covalently binding a component
having the same immunological properties as the first reagent, to
an enzyme, which quantity of coupling product is at least
immunochemically equivalent to the first reagent provided in step
(a), in order to form a second solid phase and a second liquid
phase; and
f. detecting and determining the enzyme activity of either the
second solid phase or the second liquid phase of step (e) after
separating the solid and liquid phases formed thereby, which
detection and determination is a measure of the presence and
quantity of the component to be detected and to be determined.
.Iaddend. .Iadd.14. The method of claim 13, wherein said liquid
sample and the binding partner of the component to be determined
are preincubated with each other before contacting the sample with
said
solid carrier..Iaddend. .Iadd.15. A method for the detection and
determination of an antibody in a liquid sample containing the
antibody to be detected and determined, comprising the steps
of:
a. providing a given quantity of an antigen to said antibody, which
antigen is bound to the surface of a water-insoluble,
water-insuspensible, solid carrier;
b. contacting and incubating a given quantity of said liquid sample
having said antibody to be detected and to be determined with said
bound antigen of step (a), forming a reaction mixture having a
solid phase and a liquid phase;
c. washing said solid carrier to separate the solid phase from the
liquid phase;
d. contacting and incubating with said solid phase a given quantity
of a coupling product, obtained by binding covalently a component
having the same immunological properties as the antigen previously
bound to the solid carrier, to an enzyme, which quantity of
coupling product is at least immunochemically equivalent to said
antigen provided in step (a) to form a second solid and second
liquid phase;
e. washing the solid carrier to separate the solid phase from the
liquid phase of step (d); and
f. detecting and determining the enzyme activity of the solid phase
of step (d) after said washing, which detection and determination
is a measure of the presence and quantity of said antibody to be
detected and to be determined..Iaddend. .Iadd.16. The method of
claim 15, wherein said liquid sample and the binding partner of the
antibody to be determined are preincubated with each other, before
contacting the sample with said solid
carrier..Iaddend. .Iadd.17. A method for the detection and
determination of a component of an antigen-antibody reaction in a
liquid sample containing said component to be detected and
determined, comprising the steps of:
a. providing a given quantity of a first reagent consisting of one
component of said reaction selected from the group consisting of an
antigen and an antibody, which component is bound to the surface of
a water-insoluble, water-insuspensible, solid carrier, with the
proviso that said first reagent has the same immunochemical
properties as the component to be detected and determined;
b. providing a predetermined amount of a binding partner for the
first reagent, which amount is less than or is immunochemically
equivalent to the given quantity provided in step (a) of the
insolubilized component,
c. contacting and incubating a given quantity of said liquid sample
having the component to be detected and to be determined with said
first reagent of step (a) and step (b), forming a reaction mixture
having a solid phase and a liquid phase;
d. washing said solid carrier to separate the solid phase from the
liquid phase;
e. contacting and incubating with said solid phase a given quantity
of a coupling product, obtained by binding covalently a component
having the same immunological properties as the component
previously bound to the solid carrier, to an enzyme, which quantity
of coupling product is at least immunochemically equivalent to the
component of the first reagent provided in step (a) to form a
second solid and second liquid phase;
f. washing the solid carrier to separate the solid phase from the
liquid phase of step (e); and
g. detecting and determining the enzyme activity of the solid phase
of step (e) after said washing, which detection and determination
is a measure of the presence and quantity of the component to be
detected and to be
determined..Iaddend. .Iadd.18. A method for the detection and
determination of an antibody specific to an hepatitis antigen in a
liquid sample containing said antibody, comprising the steps
of:
a. providing a given quantity of an hepatitis antigen, which
antigen is bound to a water-insoluble, water-insuspensible, solid
carrier;
b. contacting and incubating said liquid sample having the antigen
to be detected and determined with said bound antigen of (a) to
form a reaction mixture;
c. separating the first solid phase from the first liquid
phase;
d. contacting and incubating with said solid phase a given quantity
of a coupling product obtained by covalently binding a component
having the same immunological properties as the antigen bound to
said carrier, to an enzyme, which quantity of coupling product is
at least immunochemically equivalent to the first reagent provided
in step (a) to form a second solid and second liquid phase;
e. contacting and incubating the reaction mixture to form a solid
phase and a liquid phase; and
f. detecting and determing the enzyme activity of the solid phase
of step (d) after separating the solid and liquid phases formed
therein, which detection and determination is a measure of the
presence and quantity of the antibody to be detected and
determined..Iaddend. .Iadd.19. The method of claim 18 in which said
hepatitis antigen is hepatitis B surface antigen
and the antibody is its associated antibody..Iaddend. .Iadd.20. A
method of the detection and determination of a component in the
reaction of a hepatitis antigen and the associated antibody
specific to said antigen in a liquid sample containing said
component, comprising the steps of:
a. providing a given quantity of a first reagent consisting of one
component of said reaction selected from the group consisting of
said antigen and said antibody, which component is bound to a
water-insoluble, water-insuspensible, solid carrier with the
proviso that said first reagent has the same immunochemical
properties as the component to be detected and determined;
b. providing a predetermined amount of a binding partner for the
first reagent, which amount is less than or is immunochemically
equivalent to the given quantity provided in step (a) of the
insolubilized component;
c. contacting and incubating said liquid sample having the
component to be detected and determined with said reagent of step
(a) and step (b), to form a reaction mixture;
d. separating the first solid phase from the first liquid
phase;
d. contacting and incubating with said solid phase a given quantity
of a coupling product obtained by covalently binding a component
having the same immunological properties as the component bound to
said carrier, to an enzyme, which quantity of coupling product is
at least immunochemically equivalent to the first reagent provided
in step (a) to form a second solid and second liquid phase;
f. contacting and incubating the reaction mixture to form a solid
phase and a liquid phase; and
g. detecting and determining the enzyme activity of the solid phase
of step (e) after separating the solid and liquid phases formed
therein, which detection and determination is a measure of the
presence and quantity of the component to be detected and
determined..Iaddend. .Iadd.21. The method of claim 20 in which said
hepatitis antigen is hepatitis B surface antigen
and the antibody is its associated antibody..Iaddend. .Iadd.22. A
diagnostic pack for the detection and determination of a component
of an antigen-antibody reaction selected from the group consisting
of an antigen and an antibody, consisting of:
a. a given quantity of a first reagent consisting of one component
of said reaction selected from the group consisting of an antigen
and an antibody bound to the surface of a water-insoluble,
water-insuspensible, solid carrier;
b. a given quantity of a coupling product obtained by covalently
binding a component having the same immunological properties as the
component in (a) to an enzyme, which quantity of coupling product
is at least immunochemically equivalent to the component of the
first reagent provided in (a); and
c. a predetermined amount of a binding partner of the first
reagent, which amount is less than or is immunochemically
equivalent to the given
quantity (a) of the insolubilized component..Iaddend. .Iadd.23. A
diagnostic pack for the detection and determination of an antibody,
consisting of:
a. a given quantity of an antigen bound to the surface of a
water-insoluble, water-insuspensible, solid carrier; and
b. a given quantity of an antigen having the same immunological
properties as the antigen in (a) coupled to an enzyme, which
quantity of antigen is at least immunochemically equivalent to said
antigen provided in (a)..Iaddend. .Iadd.24. A diagnostic pack for
the detection and determination of antibody directed against
hepatitis B, consisting of:
a. a given quantity of hepatitis B surface antigen coupled to a
water-insoluble, water-insuspensible, solid carrier; and
b. a given quantity of a coupling product obtained by covalently
binding hepatitis B surface antigen to an enzyme, which quantity is
at least immunochemically equivalent to the antigen in
(a)..Iaddend.
Description
BACKGROUND OF THE INVENTION
This invention relates to a diagnostic method for the
.[.direction.]. .Iadd.detection .Iaddend.and determination of
antigens and antibodies. More particularly, this invention relates
to diagnostic methods for the detection and determination of
pathogenic disease antigens and antibodies, such as hepatitis and
rubella antigens and their associated antibodies.
A number of immunological methods have been developed for the
determination of antigens and antibodies, including methods for the
determination of hepatitis B surface antigen (HB.sub.2 Ag), a
component of hepatitis B virus and its associated antibodies.
In this field it is important that whatever methods are used are as
sensitive and reliable as possible since an incorrect diagnosis can
have serious consequences. This is especially true of the
determination of hepatitis antigens and their associated antibodies
since the transmission of the hepatitis virus via blood donors
constitutes a significant public health risk.
Up to the present time, the radio-immunoassay (RIA) method in its
various forms has been the most sensitive system available. This
method has several disadvantages, however, including the
requirement of special equipment, trained staff, the need for extra
safety measures to protect against harmful radiation, and the short
half-life span of the radioactive labelling element. The
possibility of replacing the radioactive label with an enzyme label
was proposed in 1968 in an article by L. E. M. Miles and C. N.
Hales, entitled "Labelled Antibodies and Immunological Assay
Systems," Lancet, London 1968 II, page 492; Nature, Vol. 219, pages
186-189 (July 13, 1968), but no procedural details were provided,
the article failing to offer more than the general idea, leaving it
to future workers to determine the basic steps and to perform the
extensive experimentation needed to establish a practical operative
enzymic immunoassay method.
The pioneering work on enzyme-immunoassay (EIA) methodology was
performed by Schuurs and coworkers, and is disclosed in a series of
their U.S. Pat. Nos. 3,654,090, 3,791,932, 3,850,752, 3,839,153,
and 3,879,262.
In the course of the further evolution of the radio-immunoassay
procedures by numerous workers, it was pointed out out in an
article by E. Habermann, entitled "A new Principle for the
Quantitative Determination of High Molecular Antigens (Junction
Test), etc." published in Z. klin. Chem. u. klin. Biochem., 8th
year, January, 1970, pages 51-55, that those methods based on the
competition of radio-labelled and unlabelled antigens for a limited
amount of antibodies gave better results than other methods.
The Habermann article proposed an RIA method .[.in.]. which .[.in a
first step,.]. .Iadd.involved reacting an unknown bindable
substance with an insolubilized binding partner to insolubilized
the unknown. The insolubilized unknown was then, in turn, reacted
with a radioactively labelled binding partner, and the quantity of
insolubilized radioactivity determined. More specifically,
Habermann proposed an RIA method whereby .Iaddend.the antigen is
adsorbed on an antibody fixed with a covalent bond by a solid
phase, e.g., celluose; a second step consists .[.in.]. .Iadd.of
.Iaddend.fixing .[.by.]. labelled antibodies .Iadd.to
.Iaddend.those antigen determinants remaining accessible. The
non-fixed component of the antibody preparation is removed by
washing. The radioactivity of the solid phase is correlated with
the antigen content of the original solution. Since the labelled
and solid phase antibodies are joined via the antigen, the author
called the technique the "junction test." This arrangement is also
known in the art as the "sandwich technique or method." The
Habermann article, referred to the earlier suggestion of Miles and
Hales (loc. cit.), and contained a passing suggestion that the test
could be improved in sensitivity by coupling the antibody molecule
with an easily detectable enzyme, but also offered no further
suggestions, leaving it to others to develop such an enzyme method.
.Iadd.Haberman does not set forth any specific assay employing
enzymes. Apart from the general statement referred to above,
Habermann merely cites a supposed suggestion by Miles et al about
how to use enzymes--but the referenced Miles et al article contains
no mention of enzymes..Iaddend.
In Schuurs et al. U.S. Pat. No. 3,791,932, there is disclosed, in
Example III, .[.a rudimentary.]. .Iadd.first-generation
.Iaddend.sandwich-type procedure for the determination of human
chorionic gonadotropin (HCG) and luteinizing hormone (LH) in low
concentrations by means of an enzyme-antibody coupling component,
in accordance with which a predetermined amount of .[.HCG.].
.Iadd.anti-HCG .Iaddend. is first coupled to a solid
immuno-adsorbent (m-aminobenzyloxymethyl cellulose).
.[.Antibodies.]. .Iadd.Purified Antibodies .Iaddend.from rabbit
anti-HCG serum are then coupled to an enzyme (horse radish
peroxidase, HRP) .[.and the coupling product is bound to HCG
cellulose.].. .Iadd.The test proceeds when an .Iaddend.HCG
.[.and.]. .Iadd.or .Iaddend.LH dilution series .[.are.]. .Iadd.is
.Iaddend.mixed with anti-HCG cellulose .[.to form an
immunoadsorbent, to which.]. .Iadd.immunoadsorbent forming an
antigen-antibody complex. Then .Iaddend.a given amount of the
antibody-enzyme coupling product is added, and the enzyme activity
of the supernatant liquid is determined. This procedure is somewhat
involved and requires a number of coordinated operating steps.
It is an object of the present invention to develop and further
improve and simplify the early version of the sandwich technique as
applied to the determination of antigens and antibodies.
.Iadd.It is an object of the present invention to use a
water-insoluble, water-insuspenssible solid carrier for binding one
component of an antigen-antibody reaction.
It is another object of the present invention to provide a
technique for the determination of antigens and antibodies that has
sensitivity as good as the prior art RIA methods..Iaddend.
GENERAL DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a
novel, very simple and sensitive enzyme immunoassay (EIA) test
system especially adapted for the detection and determination of a
component of an antigen-antibody reaction. The general principle of
the procedure of the invention is that of employing as a test
reagent a given amount of one component of said reaction bound to
the surface of a water-insoluble, water-insuspensible solid
carrier, and as a second reagent, a given amount of the same
component covalently linked to an enzyme. The liquid sample
containing the component to be determined is contacted with these
reagents. In case the component to be determined and the component
bound to the solid carrier are the same, a given amount of a
binding partner of said component is added to the liquid sample.
Upon mixing the reagents and the sample, there is formed a reaction
mixture having a solid phase and a liquid phase. Finally the enzyme
activity of the solid or liquid phase is determined, said activity
being a measure of the presence and quantity of the component to be
determined.
The enzyme-immunoassay (EIA) method according to the present
invention does not possess the disadvantages of a radioimmunoassay
method, and is simpler to set up and perform than the earlier EIA
methods, but, surprisingly, is as sensitive as the RIA method. One
of the advantages of the EIA according to the present invention is
that by using an enzyme as a labelling means the immunological
reaction can be measured by a color reaction. Either a colored
substrate or a colored end-product should participate in the
enzyme-catalyzed reaction so that the detection can take place by
reading with the naked eye or by measurement of the extinction.
More specifically, the method of the present invention comprises
contacting a liquid sample, for example serum or plasma, containing
an unknown amount of antigen or antibody, with a predetermined
amount of either the .[.antigen.]. .Iadd.antibody .Iaddend.or the
.[.antibody.]., .Iadd.antigen respectively, .Iaddend.which is bound
to the surface of a water-insoluble, water-insuspensible, solid
carrier. The liquid sample is then incubated with this coated solid
carrier for a period of 0.5 to 35 hours, usually at a temperature
between 4.degree. and 50.degree. C. After aspiration and washing,
the foregoing solid phase is contacted with an amount of the same
component covalently linked to an enzyme. If the component to be
determined in the liquid sample, and the component coupled to the
solid carrier are the same, the solid phase is contacted with a
binding partner for the component to be determined.
The binding partner is employed in an insoluble form. The
determination can take place by adding the binding partner in an
insoluble form, or it can be added in a dissolved form, and
insolubilized afterwards. The binding partner is preferably a
protein capable of binding the antigen or antibody
specifically.
After an incubation period, usually from 0.5 to 25 hours, and at a
temperature between 4.degree. and 50.degree. C., aspiration and
washing, the enzymatic activity bound to the solid phase is
determined, which activity is a measure of the pressure of the
antigen or antibody component in the liquid sample tested.
Both for the detection and quantitative determination of the
antigen or antibody, the foregoing reagents are employed in
predetermined amounts.
The solid carrier to which one of the components is bound may be
any water-insoluble, water-insuspensible, solid carrier. Examples
of suitable solid carriers include large beads, e.g., of
polystyrene, filter paper, test tubes, and microtiter plates. The
immunological component may be bound to the solid carrier by
covalent bonds or by adsorption. The advantage of the use of a
solid carrier is that no centrifugation step is needed for the
separation of solid and liquid phase.
As a solid carrier, use is preferably made of a test tube of a
microtiter plate the inner walls of which are coated with the
immunological component.
The antigen or antibody enzyme conjugate consists of the
immunological component covalently linked to one or more enzyme
molecules. Such linking can be achieved either by direct
condensation or by using external bridging molecules, in accordance
with methods known to those skilled in the art.
Thus, the production of enzyme coupling products employing a
covalent bond can be effected by reagents such as carbodiimides,
diisocyanates, glutaric aldehyde, and bis-diazobenzidine.
The choice of the enzyme that is to form a part of the coupling
product is determined by properties such as the specific binding
activity (a high conversion rate increases the sensitivity of the
test system) and the simplicity of determination of the enzyme. The
determination of an enzyme catalyzing a conversion in which colored
reaction components are involved, is simple. Such colorimetric
determinations can be automatic in a simple manner. It is also
possible to employ enzymes catalyzing those conversions in which
reaction components are involved that can be determined
spectrophotometrically or fluorimetrically. These determinations
are also suitable for automation, which is an additional
advantage.
As enzymes suitable for the method of the invention there can be
employed catalase, peroxidase, urease, glucose oxidase, alkaline
phosphatase. Horse radish peroxidase is preferred.
The method according to the present invention can be used for the
detection and determination of antigens and antibodies in general,
including bacteria, viruses, hormones, proteins and their
associated antibodies, but is particularly suited for the detection
and determination of hepatitis B surface antigen and rubella and
their associated antibodies. The invention will be illustrated with
respect to these examples but is not to be considered as limited
thereto.
In order to compare the enzyme-immunoassay of the invention with
the corresponding radio-immunoassay, for the detection of HB.sub.S
Ag, two HB.sub.S Ag-containing sera (1 subtype ad, 1 subtype ay)
were taken and were diluted in two different normal sera, free from
HB.sub.S Ag and anti-HB.sub.S. The results from these four dilution
series are indicated in the accompanying drawing. They demonstrate
that EIA and RIA have about the same sensitivity, but that the EIA
gives a steeper dose-response curve with the serum containing
subtype ay.
The enzyme immunoassay for HB.sub.S Ag according to the present
invention detected all antigen-positive samples "A," "B" and "C" of
the NIH reference panel No. 2, both by eye reading and extinction
measurement. This means that the assay system according to the
present invention has at least "third generation" sensitivity as
defined in the Federal Register, Vol. 39, No. 132, of July 9,
1974.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples serve to illustrate the practice of the
invention, but are not to be regarded as limiting:
EXAMPLE I
Detection of Hepatitis B Surface Antigen In Human Serum or
Plasma
0.1 ml test sample (human serum or plasma) is added to each well of
a polystyrene microtiter plate to the inner walls of which sheep
anti-(Hepatitis B surface antigen) is bound. The plate is incubated
for 2 hours at 37.degree. C. The wells are emptied by aspiration.
Each well is washed three times with 0.2 ml 0.2 M TRIS
(trihydroxy-methylaminomethane;
2-amino-2-hydroxymethyl-1,3-propanediol) buffer, pH 7.4 (buffer A).
0.1 ml Horse radish peroxidase coupled to sheep anti-(Hepatitis B
surface antigen) in a predetermined dilution in Buffer A is added.
The plate is incubated for 2 hours at 37.degree. C. The wells are
emptied by aspiration. Each well is washed four times with 0.2 ml
buffer A. 0.1 ml of a substrate solution of:
0.4 mg orthophenylene diamine per ml
0.2 mg urea-peroxidase per ml
in a McIlvain buffer of pH 5.0. is added to each well. The plate is
incubated for 60 minutes at room temperature. The reaction is
stopped by adding 0.05 ml of 0.5 N sulphuric acid. The color is
read by eye or measured colorimetrically at 492 nm.
A result is called positive if the ratio is .gtoreq.2.1, i.e., the
average extinction is at least 2.1 times the average extinction of
six negative control sera.
Result A
Titration of two Hepatitis B surface antigen-positive sera of the
ay and ad subtypes in enzyme immunoassay and a radio-immunoassay
licensed by the U.S. Federal Drug Administration. The positive sera
were diluted in two sera negative for Hepatitis B surface antigen
and for anti-(Hepatitis B surface antigen), so that the serum
concentration was the same in all dilutions. The curves of the ad
subtype in both tests are represented by one line. (See
accompanying drawing).
Result B
Reaction of the NIH Reference panel No. 2 in the abovementioned
assay system.
______________________________________ lot No. ratio eye-reading
lot No. ratio eye-reading ______________________________________
201 >26. + 229 1.51 - 202 >26. + 230 1.72 - 203 0.97 - 231
1.51 - 204 10.33 + 232 >26. + 205 >26. + 233 1.15 - 206
>26. + 234 >26. + 207 1.79 - 235 >26. + 208 >26. + 236
1.56 - 209 >26. + 237 1.16 - 210 >26. + 238 >26. + 211
1.11 - 239 >26. + 212 1.25 - 240 1.39 - 213 >26. + 241 1.38 -
214 >26. + 242 1.23 - 215 2.05 - 243 >26. + 216 1.32 - 244
>26. + 217 >26. + 250 >26. + 218 >26. + 251 >26. +
219 >26. + 252 >26. + 220 >26. + 253 >26. + 224 1.04 -
254 >26. + 225 1.16 - 255 >26. + 226 >26. + 256 >26. +
227 1.36 - 260 >26. + 228 >26. + 261 >26. +
______________________________________
EXAMPLE II
Detection of antibodies against hepatitis B surface antigen in
human serum of plasma
0.1 ml test sample (human serum or plasma) is added to each well of
a polystyrene microtiter plate to the inner walls of which sheep
anti-(Hepatitis B surface antigen) is bound. 0.05 ml of a
predetermined dilution of hepatitis B surface antigen in 0.2 M TRIS
buffer pH 7.4 is added to each well. The plate is incubated for 2
hours at 37.degree. C. and treated further in the same way as in
Example 1.
Results
Extinction after testing a dilution series of an anti-(hepatitis B
surface antigen)-containing serum in antigen-and antibody-negative
serum and six antigen- and antibody-negative control sera. Serum
positive for anti-(Hepatitis B surface antigen)
__________________________________________________________________________
Undiluted 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 0.050 0.051
0.049 0.062 0.060 0.058 0.168 0.200 0.203 0.198
__________________________________________________________________________
Negative control sera undiluted: serum No. 1 2 3 4 5 6
__________________________________________________________________________
0.201 0.197 0.232 0.205 0.212 0.218
__________________________________________________________________________
A reaction is called positive is the extinction is .ltoreq.50% of
the average extinction of 6 negative control sera.
EXAMPLE III
Detection of antibodies against Rubella virus in human serum or
plasma
0.5 ml of test sample (human serum or plasma) is added to
polystyrene tubes .phi. 1 cm to the inner walls of which
inactivated Rubella virus is coupled. The tubes are incubated for 1
hour at 37.degree. C. The tubes are emptied by aspiration. Each
tube is washed three times with 2 ml 0.15 M phosphate buffer. 0.5
ml horse radish peroxidase coupled to inactivated Rubella virus in
a predetermined dilution in 0.2 M TRIS buffer +0.1% triton
X.sub.100 is added. The tubes are incubated for 3 hours at
37.degree. C. Each tube is washed three times with 2 ml 0.2 M TRIS
buffer+0.1% triton X.sub.100. The horse radish peroxidase activity
is determined by adding 0.5 ml substrate solution (see example I)
and incubating for 60 minutes at 37.degree. C. The reaction is
stopped by adding 0.25 ml of 0.5 N sulphuric acid. The color is
measured colorimetrically at 494 nm. A result is called positive if
the ratio is .gtoreq.2.9, i.e., the average extinction is at least
2.9 times the average extinction of 8 negative control sera.
Results
Extinction after testing a dilution series of an
anti-(Rubella-virus)-containing serum in serum negative for Rubella
virus and anti-(rubella virus), and eight negative control sera.
Serum positive for anti-rubella-virus:
__________________________________________________________________________
1/32 1/64 1/128 1/256 1/512 1/1024 1/2048 1/4096 1/8192 1/6384
>2.000 >2.000 >2.000 1.653 0.985 0.499 0.285 0.175 0.103
0.095
__________________________________________________________________________
Negative control sera undiluted: serum No. 1 2 3 4 5 6 7 8
__________________________________________________________________________
0.058 0.092 0.070 0.101 0.085 0.063 0.058 0.082
__________________________________________________________________________
EXAMPLE IV
Detection of rubella virus in humn serum or plasma
0.5 ml of test sample (human serum or plasma) is added to 0.1 ml of
a predetermined solution of rabbit anti-rubella-virus. The mixture
is incubated for 1 hour at 37.degree. C. 0.5 ml of this mixture is
added to a polystyrene tube .phi.1 cm to the inner walls of which
inactivated rubella virus is bound. The tubes are incubated for 1
hour at 37.degree. C. and treated further inthe same way as in
Example III.
Results
Extinctions after testing a dilution series of a rubella
virus-containing serum in serum negative for rubella-virus and
anti-rubella-virus. Serum positive for rubella virus:
__________________________________________________________________________
1/100 1/200 1/400 1/800 1/1600 1/3200 1/6400 1/12800 1/25600 0.075
0.076 0.073 0.090 0.088 0.097 0.201 0.252 0.0249
__________________________________________________________________________
Negative control sera undiluted: serum No. 1 2 3 4 5 6 7 8
__________________________________________________________________________
0.250 0.238 0.278 0.275 0.263 0.291 0.0258 0.261
__________________________________________________________________________
A reaction is called positive if the extinction is .ltoreq.50% of
the average extinction of 8 negative control sera.
For the performance of the method according to the invention, a
test pack or kit of the reagents is preferably employed, chiefly
composed of:
a. a given quantity of a component of the antigen-antibody reaction
bound to a water-insoluble, water-insuspensible solid carrier;
b. a substance having the same immunological properties as said
component, covalently linked to an enzyme;
c. the binding partner of the component to be determined if the
component has the same immunological properties as the component in
(a). According to their nature, these reagents can be preserved by
freeze-drying or dissolved in a buffer. Thus, a specific test pack
may comprise (a) the antibody against hepatitis B surface antigen
coupled to a water-insoluble, water-insuspensible, solid carrier;
(b) said antibody covalently linked to an enzyme; and (c) hepatitis
B surface antigen in case the component to be determined is the
antibody against hepatitis antigen.
* * * * *