(World Health Organization, 1975). For a number of years our laboratory has worked on the development of a new and sensitive immunochemical technique, the enzymeimmunoassay (EIA) Schuurs, 1971, 1972;van Weemen et al, 1974). This method has also been studied by several other groups of investigators, particularly by Perlmann (1971, 1972) and . The present paper describes the application of a solid-phase enzyme-immunoassay for the detection of IHBsAg. injections of purified HBsAg in complete Freund's adjuvant at two-week intervals until a reciprocal titre of antibody to HBsAg (anti-HBs) of > 512 in a standard immunodiffusion set-up was attained with both subtype ay and ad. The resulting antiserum was tested by immunodiffusion against normal human serum (undiluted and diluted from 1:2 to 1:32). The antiserum was absorbed with insolubilized normal human serum proteins until no antibodies to human serum proteins could be detected. The gammaglobulin fraction was precipitated with 14% (w/v) Na2SO4, and the precipitate was dissolved in 0-0175 M phosphate buffer pH 7-6 and dialysed against the same buffer. ANTIBODY-COATED MICROTITRE PLATESThe wells of Microtiter's plates (Cooke) were filled with 0-1 ml sheep anti-HBs gammaglobulin fraction having a protein concentration of 0 03 mg/ml. The liquid was removed from the plates after overnight incubation at 4°C, the plates were washed four times with 0'04 M TRIS-buffer pH 7 4, dried over silicagel, and stored at 4VC in the presence of silicagel.ANTIBODY-ENZYME CONJUGATE Horse-radish peroxidase (Miles; RZ 3-0) was linked to sheep anti-HB8 gammaglobulin in a protein ratio of 4:1 by the 'two-step' method of Avrameas 873 on 12 May 2018 by guest. Protected by copyright.

A solid-phase enzyme-linked immunosorbent assay (ELISA), based on the "sandwich" principle with use of microtiter plates, was developed for the detection of hepatitis B surface antigen (HBSAg). Results could be read within one day by the naked eye or by colorimeter. The detection level was less than or equal to 5-10 ng of HBSAg/ml. The sensitivities of ELISA and radioimmunoassay were about the same in dilution series and in a follow-up study of 19 patients with acute hepatitis B infection. In 11 European medical centers where greater than 50,000 samples were tested, ELISA detected significantly more HBSAg-positive samples than a reversed hemagglutinatiom test. No significant difference in sensitivity between ELISA and radioimmunoassay could be demonstrated. On the average, 2.2% of readings were false-positive reactions. Falsely positive samples were identified by a confirmatory test.

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The results of a multi-centre trial of a simple reversed haemagglutination (rHA) technique for hepatitis B antigen (HB Ag) screening are presented. rHA was compared with counter-immunoelectrophoresis (CEP), immunodiffusion (ID) and microcomplement hation (CF). Radioimmunoassay (RIA) was used as a reference test.In a group of serum panels consisting of 3,700 samples, rHA was generally found more sensitive than CEP and ID, and about as sensitive as CF. In terms of specificity, rHA was found to be equal to CEP and CF and less specific than ID. An absorption procedure proved to eliminate practically all false positives. In a second phase, 41,355 samples from blood donors were tested. On the average, the sensitivity of rHA was superior to that of CEP. rHA detected, with a high degree of specificity, about 40% more positive samples than CEP.These results allow the conclusion that rHA is very suitable for mass HB Ag screening of blood donors. SCHUURS/KACAKI serum. The haemagglutination inhibition test of VYAS and SHULMAN [ 161, although highly sensitive and simple, uses a reagent of limited stability. Radioimmunoassay (RIA) has also been applied for HB Ag screening [I, 5, 171. This undoubtedly is a very sensitive technique, but it requires special equipment and well-trained personnel.All these considerations pointed to the desirability of developing a sensitive, quick and simple method for HB Ag detection, applicable for iarge-scale screening of blood donors. A technique of reversed haemagglutination (rHA) for HB Ag detection has been developed in our laboratory. The reagent system (Hepanosticon @, Organon Teknika, Oss, Holland) consists of stable sheep erythrocytes, 'sensitized' with sheep hepatitis B antibody (HB Ab). An integral part of this system is an absorbent consisting of sheep erythrocytes 'sensitized' with sheep immunoglobulin not containing HB Ab. After extensive testing in our laboratory, the rHA method was subjected to a trial in collaboration with 16 blood transfusion and clinical centres in Europe and the USA1. This paper deals with the set-up and results of the trial.

The results obtained in a multicentre clinical trial of an enzyme immunoassay (EIA) method for hepatitis B surface antigen (HBsAg) (65,451 sera tested) have demonstrated that this new test has a significantly higher sensitivity than a reversed haemagglutination test (rHA). In a part of the trial, EIA was also compared with radioimmunoassay (RIA). Only a small number of discrepant results was obtained with these two tests, indicating similar sensitivities. No definite conclusion about a difference in sensitivities could be drawn from these results. Although the specificity of the EIA screening test is lower than that of rHA and RIA, the mean percentage of false positives was 2.2% of the total number of donor samples screened. Presumptive positives in EIA were subjected to a confirmatory test based on neutralization with human antibodies to HBsAg. After elimination of false positives in EIA screening, there was excellent agreement between EIA and RIA results.