(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.

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.

Pre-S proteins may have an important role in virus assembly and virus entry into the host cell. The presence of pre-S proteins in serum has also been thought to correlate with active viral replication. To investigate whether pre-S proteins in serum might have additional diagnostic and/or predictive value for liver sequelae in HBV infection, sera from six different serological groups of patients with HBV markers (total number 363) and different manifestations of liver histology were examined for the presence of pre-S1 and pre-S2 proteins using micro-ELISAs. Pre-S1 and pre-S2 proteins were detected significantly more often in HBV-DNA-positive than in HBV-DNA-negative sera from HBsAg carriers. However, pre-S1 and pre-S2 proteins were also found in HBV-DNA-negative HBsAg carriers irrespective of serum HBeAg/anti-HBe or liver histologic findings. These results suggest that the presence of the pre-S1 and or pre-S2 proteins in serum either does not seem to reflect the presence of active viral replication and active liver disease or pre-S proteins are more readily detectable than HBeAg and HB-DNA as measured by a dot-blot technique. Furthermore, the presence of pre-S proteins in serum is strongly correlated with that of HBsAg.

Two reactive sequences of the pre-S regions of hepatitis B surface antigen were synthesized chemically and used in micro-ELISAs for the assay of pre-S1 and pre-S2 antigens in serum from patients with acute and chronic hepatitis B. Pre-S1 antigen correlated well with the presence of HBV-DNA and was no longer detectable on cessation of viral replication, after natural recovery and after successful treatment with alpha-interferon. Pre-S2 proteins were also lost after treatment with alpha-interferon. The results show that the assay of pre-S1 and pre-S2 proteins in serum provides additional useful markers for assessing patients with acute and chronic hepatitis B infection and for monitoring the response to treatment with interferon.