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past infection. With the help of direct methods, e.g. mouse isolation test, direct microscopical examination or pathological examination, the diagnosis can be made directly. However, the latter tests are, in general, time-consuming and laborious, while the chance of finding toxoplasma is rather poor. The use of immunological methods to visualise the parasite itself seems very attractive. The introduction of the direct immunofluorescent technique in order to visualise free parasites in patient material was an important advantage. A new direct approach is the detection of circulating antigen by means of the enzyme-linked immunosorbent assay (ELISA). The test is carried out in polystyrene microtitre plates. Immunoglobulins from a rabit previously infected with toxoplasma were used to bind antigens in the patients serum. To complete the sandwich the same rabbit immunoglobulins, now labelled with the enzyme peroxidase, were used. After adding the specific substrate, 5-aminosalicylic acid plus hydrogen peroxide, the change of colour from yellow to brown indicates the presence of circulating toxoplasma antigen. In experimentally infected mice we could already detect antigens one day before the presence of parasitaemia. In sera from 1116 patients suspected of having toxoplasmosis 5.7% gave positive reactions in the antigen test. Patients were divided into two groups, those with a known history of toxoplasmosis and those without. Two other serological tests were used to evaluate the finding of circulating antigen, namely the endpoint titre in the indirect immunofluorescence test, and the presence or absence of specific lgM antibodies as measured by ELISA. In conclusion, it can be said that in both groups of patients toxoplasma antigen could be detected in combination with high or low, or even negligible, antibody titres. In a relatively high percentage of patients IgM antibodies were detectable, suggesting acute primary toxoplasmosis. The fact that antigens are not often found in patients suggests that they do not circulate for a long period. This could be confirmed by repeated examinations of individuals. The advantage of testing both antibody level and circulating antigen is the possibility of diagnosing an active toxoplasmosis on the basis of one serumsample only, instead of using paired sera. Even in ocular toxoplasmosis, circulating antigen was detected where detection of antibodies failed to confirm the diagnosis. Also, the screening of donor serum before blood is transfused into immunosuppressed recipients seems a worthy application.

T h e h e p a t i t i s B - r e l a t e d ' e ' a n t i g e n - a n t i b o d y s y s t e m as m e a s u r e d b y e n z y m e lmmunoassay

M. VAN DER WAART 1, A. SNELTING 1, J. CICHY 1, P. NIERMEIJER 2, C. H . GIPS 3, J. R . HU|ZENGA 3 AND A . SCHUURS 1

l Organon Seientific Development Group, Oss, The Netherlands 2Majella Hospital, Bussum, The Netherlands aDepartment of Medicine, Division of Hepatology, University Hospital, Groningen, The Netherlands Recently, we have developed an enzyme-immunoassay (EIA) for the detection of the hepatitis Brelated'e'antigen(HBeAg)anditsantibody(anti-HBe)(VanderWaartetal, 1978). This EIA is 26 28 times more sensitive than the immunodiffusion (ID) technique. In a series of 259 hepatitis B surface antigen (HBsAg)-carriers, 88 ~ of the sera were positive in EIA for either HBeAg or anti-HBe. With ID only 30~ of the HBsAg-carriers were found to be positive for HBeAg or anti-HBe. HBeAg could only be detected in HBsAg-positive sera and a correlation was found between the titre of HBsAg and the presence of HBeAg. No correlation was observed between either HBeAg or anti-HBe on the one hand or rheumatoid factor on the other. Anti-HBe could be detected in HBsAg-positive sera but also in HBsAg-negative sera in patients convalescing from acute type B hepatitis. In a series of 22 longitudinally followed acute hepatitis B patients, simultaneous

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HBsAg and HBeAg positivity and enhanced alanine aminotransferase (ALT) activity were observed. On the average, HBeAg, HBsAg and increased ALT activity were observed for 1, 9 and 11 weeks respectively. The activity was maximal just after disappearance of HBeAg. Anti-HBe appeared some weeks before the simultaneous clearance of HBsAg and ALT. In most individuals recovering from an acute type B hepatitis, anti-HBe persisted for several years (Niermeijer et al., to be published). VAN DER WAART, M., SNELTING,A., CICHV, J., WOLTERS,G. and ScnutJRs, A. H. W. M. 1978.-J. Med. Virol., in press. NIERME1JER, P., GIPS, C. H., HU1ZENGA,J. R., v. O. WAART, M., SNELTING, A. 1978.--Abstract European Association for the Study of the Liver, Sept.

Costs and benefit of the mechanisation of tests on serially diluted samples E. ENGELBRECHT

Department of Microbiology, Stichting Samenwerking Delftse Ziekenhuizen, Reynier de Graefweg 7, Delft, The Netherlands Referring to the report of Kwantes (1976), we here communicate our experience with the automation of serology by means of the BIOREACTOR(Engelbrecht, 1974a, 1975, 1978), and the mechanisation of various small scale investigations by way of the BIODILUTOR (Engelbrecht, 1974a, 1974b, 1978). Considering the fact that the share of the employer in the premiums for social insurances amounts to 47.5% of the salary, the total annual expenses for labour in Holland amount to aboutf35,000.(Dutch Guilders) for a low graded technical laboratory assistant and about f44,000.- for a licenced technician with several years of experience. Allowing for weekends, holydays, sick and special leaves, coffee and tea breaks, there are 210 effective working days of 7.5 hours per year. Hence, the labour costs are f22,25 and f 2 8 , - per effective hour respectively. In the average serological laboratory about 8000 titrations are performed annually, that is about 2000 samples for virus serology or 4000 samples for general serology. This means 40 titrations per effective working day. Allowing for at least 3 rain per titration in 6 to 8 dilutions, and for 2 • 15 min for preparing the tests and arranging and cleaning the materials afterwards, their manual execution require 2.5 hours. With the BIOREACTORthese 40 titrations are performed in 17 min. Since the preparation and cleaning-up of the machine require 2 • 4 min, the total time involved is 25 min only. Hence, 2 hours at least are gained each effective working day or 420 working hours per year, that is f9,345.- in the case ofthe assistant a n d f l 1,760, in the case of the technician. If these yearly savings are used to pay off a loan taken for the purchase of the machine, an additional saving of 11% is obtained. Over a 5-years' period the total savings will thus amount to f57,000.- and f72,000.respectively. The net price of the BIOREACTORbeingf38,500.-, and allowing for an exchange value offS,000.- after 5 years, the annual depreciation will be f6,700.- orf7,906.- if 18% Value Addition Tax has to be included. With a loan against an interest of 11% of the yearly remaining sum, the total interest to be paid amounts t o f l 6,920.-. No costs of maintenance need to be accounted for, since these are similar to the savings on the pipettes and auxiliary materials needed for manual titrations. Hence, the total costs are f 56,000.- for the first 5 years. So, with 8000 titrations annually the investment made in a BIOREACTORis fully recovered in 5 years under Dutch conditions and in case of operation by a low graded technical assistant. A profit offl 6,000.-is obtained in the case of the licenced technician. In fact the profit is even higher because of the reduction in fall-out as a result of the improvement in reproducibility and failure level.