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10.3

Z. Anal. Chem., Band 279, Heft 2 (1976)

Enzyme-Immunoassay (EIA) of Hepatitis B Surface Antigen (HBsAg) in Microtiter Plates

G. Wolters, L. P. C. Kuijpers, A. H. W. M. Schuurs, and J. Ka~aki

Biochem. R & D Laboratories, Organon Scientific Development Group, Oss, The Netherlands

Enzymimmunoassay ( EIA ) des Hepatitis-B-Surface-Antigens ( HB~Ag) in Mikrotiter-Platten

Best. yon Hepatitis-B-Surface-Antigen in Serum; Enzym-Immunoassay

Enzyme-immunoassays (EIA's) have been described for a number of antigens and antibodies [2, 3]. The sensitivity approaches that of radio-immunoassays (RIA's). We therefore set out to develop an EIA for HB~Ag, the detection of which is important for the screening of donor blood.

In this assay we used the sandwich technique with a solid-phase antibody and an enzyme-labelled anti- body.

Methods. Solid-phase antibody: the wells of Microtiter plates (Cooke) were coated with sheep anti-HBs gamma- globulin.

Enzyme-labelled antibody (conjugate): sheep anti-HBs gammaglobulin was coupled to horse radish peroxidase with glutaraldehyde by the "two-step" method [1]. After purifica- tion the conjugate was properly diluted so that a negative control gave just no visible colour in the test.

Test performance: To each well of the microtiter plate 0.1 ml of test sample was added. Pooled human serum free from HBsAg, and a mixture of HBsAg positive sera of sub- types ad and ay served as negative and positive controls, respectively. After incubation for 2 h at 37~ each well was aspirated and washed. Thereafter 0.1 ml of conjugate was added and the plate was incubated again for 2 h at 37~ followed by aspiration and washing. Peroxidase activity bound to the solid phase was determined by (a) incubating each well with 0.1 ml of a solution of peroxide and o-phenylene diamine at room temperature for 50 rain, (b) stopping the enzyme reaction with 0.05 ml of diluted sulphuric acid, (c) reading the colour qualitatively by the naked eye and quantitatively by measuring E492 ,m.

Colorimetric results were considered positive if the extinc- tion was at least 2.1 times the average extinction of 5 negative controls.

Presumptive positives were checked by neutralisation with human anti-HBs.

Results and Discussion. Dose-response curves of EIA and RIA (Ausria II): Four sera containing HB~Ag (two of subtype ad and two of ay) were diluted in two different human sera, free from both HB~Ag and anti-HBs. Both tests appeared to have their positive cut-off at the same dilutions for each dilution series, but EIA had a steeper dose-response curve than RIA with subtype ay. Disappearance of HB~Ag after acute hepatitis B: 14 patients were followed during reconvalescence up to 15 weeks upon hospitalization. Serum samples were tested both by EIA and RIA. The latter was in no case positive where EIA was negative.

EIA detected all positives labelled A, (A), B, (B) and C of the BOB Reference panel no. 2.

EIA detected all 70 true positives (including 24 missed by CEP, one of which was not detectable by any usual technique except Ausria II) of a panel of the Central Laboratory of the Blood Transfusion Service of the Netherlands Red Cross.

The results with the panel of the German National Reference Centre for Viral Hepatitis (Prof. Thorns- sen, G6ttingen, West Germany) indicated, that the detection level of HB~Ag in EIA is 3 - 5 ng/ml, which corresponds to the sensitivity of RIA.

Donor sera. In a series of 679 fresh donor sera, negative in reversed haemagglutination, no true positives and 3 false positives were found in the screening with EIA.

These results indicate that EIA can be used as a highly sensitive test (comparable with RIA) for the detection of HBsAg in sera of blood donors and hepatitis patients.

References

1. Avrameas, S., Ternynck, T.: Immunochemistry 8, 1175 (1971)

2. Engvall, E., Perlmann, P. : J. Immunol. 109, 129 (1972) 3. van Weemen, B. K., Schuurs, A. H. W. M. : FEBS Letters

15, 232 (1971)