CA2021048A1

CA2021048A1 - Method of determining antigens and/or antibodies in human body liquids

Info

Publication number: CA2021048A1
Application number: CA002021048A
Authority: CA
Inventors: Helmut Aicher; Ewald Molinari
Original assignee: Immuno AG fuer Chemisch Medizinische Produkte
Current assignee: Oesterreichisches Institut fuer Haemoderivate
Priority date: 1989-07-13
Filing date: 1990-07-12
Publication date: 1991-01-14
Also published as: DK0408542T3; EP0408542A2; EP0408542B1; FI903504A0; EP0408542A3; ATE128232T1; ES2081363T3; AT394114B; JPH0353166A; ATA169989A; NO903107D0; NO903107L; DE59009676D1

Abstract

ABSTRACT OF THE DISCLOSURE:

To simultaneously determine a plurality of antibodies Aka1, Aka2, ...Akb1, Akb2, ... contained in a sample of human body liquid, the sample is introduced into a microtiter plate whose wells are coated with antigens Aga1, Aga2, ... complementary to one portion of the antibodies Aka1, Aka2, ... to be detected, and a plurality of solid supports, which are coated with antigens Agb1, Agb2, ... complementary to another portion of the antibodies Akb1, Akb2, ... to be detected are contacted with the sample; then, after complexing has been completed, the wells and the solid supports are washed free of non-adsorbed proteins, the complexes Aga1-Aka1 ... and/or Agb1-Akb1 ... adsorbed in the wells of the microtiter plate and/or on the solid supports are incubated with a mixture of labeled antigen enzymes Aga3E* ... Agb1E* ...complementary to the antibodies Aka1, Aka2, ... Akb1, Akb2, ... to be detected, and the activities of the enzymes Aga1-Aka1-Aga1E* ... Agb1-Akb1-Agb1E*, ... is photometrically determined upon reaction with a staining substrate.

Description

The invention relates to a method of determining antigens and/or antibodies in human body liquids by the formation of an antigen/antibody complex, wherein the sample to be examined for its antigen or antibody content is contacted with a complexing antibody or ant;gen, respectively, whlch is adsorbed on a solid support, as well as to a test kit for carrying out this method.
From DE-C-24 24 465, a method for the simultaneous determination of hepatitis B antigen and its antibody in sera is known. The assay principle resides in an inhibitory reaction, which, naturally, largely reduces the sensibility of the system.
Canadian Patent No. 1,148,859 describes an assay method for simultaneously detecting various hepatitis indicators, i.e., hepatitis-B surface antigen and an antibody to hepatitis-B core antigen. The disadvantage of this method consists in that the two substances to be assayed have to be detected by way of two completely different types of reaction, one reaction partner having to be labeled with a radioactive isotope and the other one being assayed enzymatically.
In general, a definite sequence of method steps must be strictly observed when assaying at least two immunoreactants, which, of course, strongly increases the risk of false determinations. The source of errors increases the more the larger the number of 2~2~

immunoreactants to be assayed. Add to this that, when assaying a plurality of substances, the sample must be pipetted accordingly frequently, thus adversely affecting the accuracy of the individual values.
The invention has as its object to provide a simple method of determining a plurality of antigens and/or antibodies in samples of human body liquids, which does not involve the disadvantages cited above and which can be realized without radiolabeling.
In the simultaneous assay of a plurality of antigens, this object is achieved according to the invention in that the sample to be examined, which contains the antigens Agal, Aga2, ... Agbl, Agb2, ....
is introduced into a microtiter plate whose wells are coated with antibodies Akal, Aka2, ...directed against one portion of the antigens Agal, Aga2, ... to be detected, and that a plurality of solid supports, preferably in the form of a comb, which are coated with antibodies Akbl, Akb2, ... directed against another portion of the antigens Agbl, Agb2, ... to be detected, are contacted with the sample; that, after complexing has been completed, the wells and the solid supports are washed free of non-adsorbed proteins, the complexes Akal-Agal ... and/or Akbl-Agbl ... adsorbed in the wells of the microtiter plate and/or on the solid supports are incubatecl with a mixture of labeled antibody enzymes AkalE*, ..., AkblE~, ...directed against the antigens 2~0~

Agal, Aga2, ... Agbl, Agb2, ... to be detected, and the activities of Lhe enzymes Akal-Agal-AkalE*, ....
Akbl-Agbl-AkblE*, ... bound to the complexes are photometrically determined upon reaction with a staining substrate.
To simultaneously determine a plurality of antibodies Akal, Aka2, ...Akbl, Akb2, ... contained in the sample to be assayed, the sample is introduced into a microtiter plate whose wells are coated with antigens Agal, Aga2, ... complementary to one portion of the antibodies Akal, Aka2, ... to be detected, and a plurality of solid supports, preferably in the form of a comb, which are coated with antigens Agbl, Agb2, ...
complementary to another portion of the antibodies Akbl, Akb2, ... to be detected are contacted with the sample;
then, after complexing has been completed, the wells and the solid supports are washed free of non-adsorbed proteins, the complexes Agal-Akal ... and/or Agbl-Akbl ... adsorbed in the wells of the microtiter plate and/or on the solid supports are incubated with a mi~ture of labeled antigen enzymes Aga3E* ... AgblE*
...complementary to the antibodies Akal, Aka2, ... Akbl, Akb2, ... to be detected, and the activities of the enzymeS Asal-Akal-A9alE* -- A9bl Akbl Ag l photometrically determined upon reaction with a staining substrate.
By the method according to the invention, it is 2~2~

also possible to simultaneously detect at least one antibody Akal, ... and at least one antigen Agbl, ... by coating the wells of the microtiter plate employed as well as the solid supports employed, with the antigen(s) Agal, ... or antibody(ies) Akbl, ... complementary to the antibody(ies) Akal, ... or antigen(s) Agbl, ....
respectively, to be detected.
Thus, the method according to the invention enables a plurality of antibodies and/or antigens to be assayed in one sample, the sample having to be pipetted just once. Therefore, the risk of pipetting errors faced in daily routine operation is minimized with the method according to the invention.
The invention also relates to a test kit for carrying out this method, which comprises a microtiter plate and a solid support fitting into the wells of the microtiter plate, in particular a comb-like support, and which is characterized in that the wells of the microtiter plate and the solid supports are coated with one or several antigen(s) Agal, Agbl, ... or with one or several antibody(ies) Akal, Akbl, ... that form complexes with the antigen(s) and/or antibody(ies), respectively, to be detected.
A kit comprised of a microtiter plate and a comb-like support for the simultaneous radioimmunochemicalassay of an antigen and an antibody is known from U.S.
Patent No. 4,276,259.

2 ~

The invention will be explained in more detail by the following examples.
Exa_~le 1.
Simultaneous assay of apolipoprotein AI (antiathero-sclerotic HD lipoprotein) and apoprotein B (athero-sclerotic LD lipoprotein) in biological liquids.
Wells of a microtiter plate were treated with 200 myl (per well) of a solution of an affinity-purified sheep anti-apo-A antibody in a 0.1 m PBS buffer (p~l 7.4) at an antibody concentration of l myg/ml for 12 hours at room temperature and coated with the antibody. After this, the wells were washed at least three times with A 250 myl PBS buffer containing 0.05 % Tween 20.
Unoccupied binding sites on the polystyrene wall of the wells, if present, were saturated by the addition of an 0.2 ~ w/v albumin solution in PBS buffer for two hours at room temperature, whereupon the plates were washed another time and subjected to thorough drying under vacuum. The plates were kept under silicagel.
In a similar manner, the plates of the test comb were coated with an affinity-purified anti-apo-B
antibody.
The samples and the standards to be assayed were ~ pre-diluted in PBS Tween buffer l : 500 and incubated in the microtiter plate upon insertion of the test comb, for two hours at room temperature. The sample volume was 200 myl. After this, the wells of the microtiter plate ~ T~

~2~

as well as the test combs were thoroughly washed in PBS-Tween buffer and 200 myl of a mixture of purified anti-apo-AI and anti~apo-B antiboclies tagged with horseradish peroxidase were pipetted into the wells. The test combs were inserted and the kit was incubated for one hour at room temperature. After a further washing step, 200 myl of a substrate containing tetramethylbenæidine were each pipetted into the microtiter plate coated with anti-apo-AI as well as into a second microtiter plate, which was uncoated.
~he test combs were inserted into the second microtiter plate and the two plates were incubated for 30 minutes at room temperature. After this, the enzymatic substrate reaction in the two plates was stopped by the addition of 50 myl 4 n sulfuric acid. By the aid of a photometer, the ex~inctions of the individual samples in the two plates were measured at 450 nm and the concentrations of apo-AI and apo-B in the samples ~ere evaluated by comparison with the standards employed.
According to the method of the invention, it is also possible to form the quotient of the concentrations of the respective samples, which is of relevance to medical diagnosis and prognosis.
E~a_ple 2.
Simultaneous assay of IgM antibodies to T~E (tick-borne encephalitis), IgG-antibodies to TBE and antibodies to Borrelia burgdorferie.

~2~

Wells of a microtite~ plate were coated with a purified virus suspension of TBE antigen in a manner analogous to Example l, and the lamellae of the combs were coated with a suspension of Borrelia burgdorferie antigen. 200 myl of a l : 50 dilution of the samples were introduced into the microtiter plate, the test combs were inserted and the kit was incubated at 45C
for two hours. The wells of the microtiter plate and the test combs were washed, and into the coated microtiter plate there was pipetted a mixture of anti-human-IgG
labelled with alkaline phosphatase (enzyme l) and of TBE
antigen tagged with horseradish peroxidase (enzyme 2).
Into a second, uncoated microtiter plate, anti-human-IgG labeled with enzyme l was charged in a similar manner, and the test combs were inserted. Both plates were incubated at 45C for one hour, the plates and the test combs were washed a second time, and into the coated microtiter plate there was pipetted a mixture of o-phenylenediamine and p-nitrophenyl phosphate (PNPP) in a suitably buffered solution.
PNPP was introduced into the second plate and the test combs were inserted. Both plates were incubated at room temperature for 30 minutes and subsequently the test combs were discarded. The reaction occurring in plate 2 was stopped by the addition of 50 myl 4 n sulfuric acid. With plate l, the extinctions in the individual wells were measured by a suitable photometer ~2~8 (measurement of the enzymatic substrate extinction of enzyme l at 405 nm; after this, 50 myl 4n sulfuric acid were added and the enzymatic substrate extinction of enzyme 2 was measured at 492 nm).
The extinctions of enzymatic substrate No. l correspond to the anti-TBE-IgG content of the sample, the extinctions of enzymatic substrate No. 2 corresp~nd to the anti-TBE-IgM content of the sample.
In the next step, the measurement of the extinctions in plate 2 was effected at the wave length suitable for enzyme l (405 nm) In plate No. 2, the measured extinctions correspond to the content of IgG-antibodies to Borrelia burgdorferie.
Thus, it is possible to get knowledge of possible infections by TBE and/or Borrelia burgdorferie in a single assay.

g

Claims

1. In a method of determining antigens in human body liquids by forming an antigen/antibody complex, wherein a sample to be examined for its antigen content is contacted with a complexing antibody adsorbed on a solid support, the improvement which, for simultaneously determining a plurality of antigens contained in said sample to be examined, comprises providing a microtiter plate having wells coated with first antibodies directed against a first portion of antigens to be detected, introducing said sample to be examined into said microtiter plate, providing a plurality of solid supports coated with second antibodies directed against a second portion of antigens to be detected, contacting said plurality of solid supports with said sample to be examined so as to cause complexes to be adsorbed on at least one of said wells of said microtiter plate and said solid supports, washing said wells and said solid supports free of non-adsorbed proteins, providing a mixture of labeled antibody enzymes directed against the respective ones of said first and said second antigens to be detected, incubating said complexes with said mixture so as to cause said labeled antibody enzymes to bind to said complexes, and photometrically determining the activities of said enzymes bound to said complexes upon reaction with a staining substrate.

2. In a method of determining antibodies in human body liquids by forming an antibody/antigen complex, wherein a sample to be examined for its antibody content is contacted with a complexing antigen adsorbed on a solid support, the improvement which, for simultaneously determining a plurality of antibodies contained in said sample to be examined, comprises providing a microtiter plate having wells coated with first antigens complementary to a first portion of antibodies to be detected, introducing said sample to be examined into said microtiter plate, providing a plurality of solid supports coated with second antigens complementary to a second portion of antibodies to be detected, contacting said plurality of solid supports with said sample to be examined so as to cause complexes to be adsorbed on at least one of said wells of said microtiter plate and said solid supports, washing said wells and said solid supports free of non-adsorbed proteins, providing a mixture of labeled antigen enzymes complementary to the respective ones of said first and said second antibodies to be detected, incubating said complexes with said mixture so as to cause said labeled antigen enzymes to bind to said complexes, and photometrically determining the activities of said enzymes bound to said complexes upon reaction with a staining substrate.

3. A method as set forth in claim 1 or 2, wherein said solid supports are comb-shaped.

4. A method as set forth in Claims 1 or 2, for simultaneously determining at least one antibody and at least one antigen contained in a sample, wherein said wells of said microtiter plate are coated with either one of said at least one first antigen and said at least one first antibody and said solid supports are coated with the respective other one of said at least one second antigen and said at least one second antibody complementary to said at least one first antibody and said at least one first antigen to be detected.

5. A test kit for carrying out the method set forth in claim 1 or 2, comprising a microtiter plate including a plurality of wells and a solid support fitting into said wells, and wherein said wells of said microtiter plate and said solid support are coated with one of at least one antigen and at least one antibody adapted to form complexes with the respective other one of at least one antigen and at least one antibody to be detected.

6. A test kit as set forth in claim 5, wherein said solid support is designed like a comb.

7. A test kit for carrying out the method set forth in claim 4, comprising a microtiter plate including a plurality of wells and a solid support fitting into said wells, and wherein said wells of said microtiter plate and said solid support are coated with one of at least one antigen and at least one antibody adapted to form complexes with the respective other one of at least one antigen and at least one antibody to be detected.

8. A test kit as set forth in claim 7, wherein said solid support is designed like a comb.