GB2045432A

GB2045432A - Improved indicator system and method for determination of antibody presence by means of an antigen-antibody reaction

Info

Publication number: GB2045432A
Application number: GB8005893A
Authority: GB
Original assignee: Orion Yhtyma Oy
Current assignee: Orion Oyj
Priority date: 1979-02-22
Filing date: 1980-02-21
Publication date: 1980-10-29
Also published as: FR2449894A1; FR2449894B1; GB2045432B; SE8000236L; DE2951248A1; FI58566C; FI58566B

Abstract

The presence or absence of antibodies to a particular pathogenic factor in a test sample can be determined by means of an antigen- antibody reaction according to the hemolysis in gel method using an indicator system comprising a buffered agarose gel plate containing erythrocytes to which antigens reactive with the antibodies to be tested for are bound, and a separate carrier, e.g. a membrane, impregnated with a complement for the antigen- antibody system causing hemolysis of the erythrocytes in the presence of the system. The carrier bearing the complement is applied to the gel plate only after the latter has been treated with the test sample.

Description

SPECIFICATION Improved indicator system and method for determination of antibody presence by means of an antigen-antibody reaction The present invention is directed to an improved indicator system for determining the presence or absence of antibodies to a particular pathogenic factor in a test sample by means of an antigen-antibody reaction according to the hemolysis in gel method.

The hemolysis in gel (HIG) method is used in hospitals and laboratories to indicate whether a person suffers or has suffered from a certain disease, by way of demonstrating the presence of antibodies to a given pathogenic factor, and more specifically the antigen in question, in the patient's serum or blood. The HIG method may further be used for identifying a certain antigen with the aid of a known antibody.

The HIG method is carried out with an agarose plate or gel prepared in a buffered milieu and containing erythrocytes to which antigens or antibodies are bound. The erythrocyte-antigen or erythrocyte-antibody bond may be made firmer by the use of chemicals such as chromiumtrichioride. The gel also contains a complement.

The test sample, e.g. a serum, is placed in a well in the agarose gel and incubated at 370C, If hemolysis occurs around the well, it shows that the test sample contains the antibody or antigen determined. The hemolysus is based on the following mechanism: The antibody or antigen in the test sample diffuses into the gel and reacts with the antigen or antibody in the gel plate thus activating the complement which leads to hemolysis of the erythocytes. The hemolysis is visible as a clear, circular area around the well.

Indicator systems based on the HIG method have been previously described, for instance in British Patent Specification 1 501395 and in German Patent Application DE 2732554 by the same applicant and in a paper by Schildt et al.: Intern. Symp. on Immunity to Infections of Respiratory Systems in Man and Animals. Devel.

Biol. Standard Vol. 28, pp. 253-272. In the indicator system described in these publications the complement is included in the gel. This reduces the shelf life of the agar plate to 3-4 weeks at the most. By leaving out the complement and adding it to the plate only just before use, as in the present method, the shelf-life of the indicator system can be prolonged up to 3-4 months.

Skaug et al.: Acta Path. Microbiol Scand. Sect.

B Vol. 83, pp. 367-372, and Strannegard et al.: J. Clin. Microbiol. Vol. (6), pp. 491-494 have described a method where the complement solution is poured over the plate. According to the method of Skaug the test sample is added at first, and incubated for a few hours, after which the complement solution is added by pouring it on the plate and thereafter incubating for about 20 hours.

In the method of Strannegärd the plate is incubated for 7-24 hours after addition of the serum and about two hours after the addition of complement. When the complement is added as a solution, it is difficult to get it evenly spread over the gel, which is often higher at the edges. Thus the zones of hemolysis may be different in size in different parts of the plate. In addition, the two incubations delay the answer.

To overcome these disadvantages, an improved indicator system has been developed according to the present invention as well as a method for preparing this rapid, reliabie, and more stable system.

According to the invention there is provided an indicator system for determining the presence or absence of antibodies to a particular pathogenic factor in a test sample by means of an antigenantibody reaction according to the hemolysis in gel method, comprising a buffered agarose gel plate containing erythrocytes to which antigens reactive with the antibodies to tested for are bound and a separate carrier impregnated with a complement for the antigen-antibody system causing hemolysis of the erythrocytes in the presence of. that system, the carrier being designed for application to the gel plate after the latter has been treated with the test sample.

Thus, for use in this system a buffered agarose gel plate is prepared containing erythrocytes and, bound to them, antigens or antibodies. The bond can be made firmer by adding a chemical such as chromium trichloride. The complement belonging to the system is suitably applied to a membrane of pore-size of 0.5-5,u, preferably 1.2 u, acting as carrier, for example by impregnating the membrane with the complement solution and drying it, e.g. by freeze-drying. The membrane may be made of a cellulose-ester mixture or cellulose triacetate. The impregnated dried membrane is preferably kept in a metalfoil-plastic bag at +4 to +60C until it is used.Alternatively, the complement may be applied to a filter paper as carrier, but a membrane is easier to standardize and the complement is more evenly diffused from the membrane than from a filter paper.

The invention also provides a complement impregnated carrier for use in such a system and a combination of such carrier and a buffered agarose gel plate.

A method of determining the presence or absence of antibodies to a particular pathogenic factor in a serum test sample according to the invention comprises providing an indicator system according to the invention having bound antigens reactive with the antibodies to be tested for, applying the serum test sample to the gel plate, thereafter applying the complement-impregnated carrier and incubating the system to cause hemolyses to occur if the test sample contains the antibodies in question so that zones of hemolysis are visually observable.

Usually, the test sample is applied to the plate and incubated at 370C for an hour, after which the carrier, e.g. membrane, impregnated with the complement is applied to the plate and the incubation is continued for 20 hours at +35at.

The zones of hemolysis are regular and easy to measure, particularly against a white membrane.

Systems according to the invention are easy to use and require no specially trained staff or specially equipped laboratory; they may be used where needed, as in hospitals and health centres.

The test is rapid and simple to perform and the result is clear The improved stability of the indicator system means that plates can be prepared in large series and stored. This facilitates industrial production and distribution. If the stability is poor, the plates must be prepared just before use and delivered by special transport. The improved stability furthermore renders the system practically available to those who need it only occasionally.

Thus, it is evident that this indicator system with its good stability means a considerable technical and economic improvement.

The invention is illustrated by the following Examples concerning tests for influenza, mumps and rubella.

EXAMPLE 1 PREPARATION OF AN HIG PLATE WITH INFLUENZA VIRUS ANTIGEN a) Preparation of the plate In a test tube, 0.5 ml of rooster erythrocytes are mixed with 0.5 ml of influenza virus-containing aliantoic fluid having a hemagglutination titer of 640 HAU/ml and 0.5 ml of 0.1% CrCI3-6H20 in 0.1 5 M NaCI.

After 5 minutes 30 ml of 0.05 M phosphate buffer pH 7.2 containing 0.1 M NaCI, 0.003 M CaCI2, 0.001 M MgCI2 and 0.2% bovine albumin is added into the mixture.

After centrifugation the red blood cells are washed once more with the same buffer and centrifuged again. The pellet of red bood cells is thereafter suspended to 10% in the abovementioned buffer without bovine albumin.

0.6 g of agarose is mixed with 39 ml of 0.05 M phosphate buffer pH 7.2, containing 0.15 M NaCI.

The mixture is heated on a water bath until the agarose has melted. The agarose is cooled to 430C and the above-mentioned 10% cell preparation (5 ml) is added. A suitable amount of the agarose-erythrocyte mixture, e.g. 7.5 ml, is poured immediately on a plastic plate which has a 1.5 mm deep and 6 x 8 cm wide depression.

When the agarose has solidified wells of 2 mm are punched for the samples.

b) Preparation of the complement membrane Complement (guinea-pig serum) is diluted 1:3 with 0.05 M phosphate buffer, pH 7.2, containing 0.15 M NaCI.

A filtration membrane with a pore size of 1.2 8, prepared of a cellulose-ester mixture, is cut to 6 x 8 cm large pieces which are placed in turn to float on the surface of the complement solution.

When the complement is adsorbed (about 0.7 ml/piece), the membrane is placed on a steel plate and transferred to a freeze-drier. The freeze-dried membranes may be stored for instance in a heatsealed metalfoil-plastic bag in a refrigerator.

c) Use 5,ul of serum is pipetted into the well in the HIG plate and allowed to diffuse for 1 hour, after which a complement membrane is placed on the agarose surface and the lid of the plate is replaced. After incubation for 18 hours in an incubator at 370C, the zone of hemolysis around the well, which is clearly visible against the white membrane, is measured. The diameter of the zone of hemolysis correlates with the concentration of antibodies in the sample.

EXAMPLE 2 AN HIG PLATE FOR DETERMINATION OF RUBELLA ANTIBODIES The plate is prepared as in Example 1a, but with rubella virus culture medium, the hemagglutination titer of which is 512 HAU/ml, instead of influenza antigen.

The complement membrane may also be made of a cellulose triacetate membrane, pore size 1.2,u, prepared as in Example 1. This membrane absorbs about 0.85 ml of complement.

The plate and the complement membrane are used as in Example 1 c.

EXAMPLE 3 AN HIG PLATE FOR DETERMINATION OF MUMPS ANTIBODIES The HIG plate is prepared as in Example 1 a, but with the influenza antigen replaced by allantoic fluid which contains mumps virus and has a hemagglutination titer of 640 HAU/ml.

As the complement membrane there may also be used a filtration membrane made of a celluloseester mixture and with a pore size of 0.8 u. This membrane absorbs about 0.65 ml of complement.

The plate and the complement membrane are used as in Example 1 c.

Claims

1. An indicator system for determining the presence or absence of antibodies to a particular pathogenic factor of antibodies to a particular pathogenic factor in a test sample by means of an antigen-antibody reaction according to the hemolysis in gel method, comprising a buffered agarose gel plate containing erythrocytes to which antigens reactive with the antibodies to be tested for are bound and a separate carrier impregnated with a complement for the antigen-antibody system causing hemolysis of the erythrocytes in the presence of that system, the carrier being designed for application to the gel plate after the latter has been treated with the test sample.

2. An indicator system according to claim 1 wherein the carrier is made of a cellulose ester mixture or cellulose triacetate.

3. An indicator system according to claim 1 or 2 wherein the carrier is a membrane which has a pore size of from 0.5 to 5 y.

4. An indicator system according to claim 3 wherein the membrane has a pore size of about 1.2 y.

5. An indicator system according to any of claims 1 to 4 wherein the impregnated carrier has been made by impregnating a membrane with a solution containing the complement followed by freeze-drying.

6. An indicator system according to any of claims 1 to 5 wherein the gel plate contains chromium trichloride to improve the erythrocyteantigen bond.

7. An indicator system according to any of claims 1 to 6 wherein the antigens are antigens for influenza, rubella or mumps antibodies.

8. An indicator system according to claim 1 and substantially as hereinbefore described or exemplified.

9. A complement-impregnated carrier for use in an indicator system according to any of claims 1 to 8.

10. A system comprising a complementimpregnated carrier according to claim 9 and a buffered agarose gel plate.

11. A method of determining the presence or absence of antibodies to a particular pathogenic factor in a serum test sample, comprising providing an indicator system according to any of claims 1 to 8 having bound antigens reactive with the antibodies to be tested for, applying the serum test sample to the gel plate, thereafter applying the complement-impregnated carrier and incubating the system to cause hemolysis to occur if the test sample contains the antibodies in question so that zones of hemolysis are visually observable.

New claims or amendments to claims filed on 10 June 1980.

New or amended claims:

12. A method of preparing an indicator system for the determination of antigen-antibody reactions according to the hemolysis in gel method, wherein the complement in the indicator system is applied to a membrane and lyophilized and added separately to the indicator system.