148 SA MEDIESE TYDSKRIF DEEL 63 29 JANUARIE 1983 Hepatitis B surface antigen in donated blood ­ screening and confirmation by enzyme-linked immunosorbent assay

M. O. BUBB, T. MARIMUTHU, J. D. CONRADIE

Material and methods Summary Isolation of HBsAg . Principles of and test procedures for enzyme-linked HBsAg was prepared from donated blood heavily: contami­ immunosorbent assay (ELlSA) screening and con­ nated with hepatitis B virus. Typically, HBsAg in I litre ofserum firmatory tests for hepatitis B surface antigen was precipitated by addition of PEG 6000 (11% w/v) and the (HBsAg) in donated blood are described. The assay precipitate recovered by centrifugation at I 000 g for 10 minutes. is of 'third-generation' sensitivity and is practical The precipitate was redissolved in 500 ml 0,05M tris-O, IM NaCI and economical for large-scale screening of blood buffer, pH 8,0 (TS), and centrifuged at 176 OOOg for 90 minutes donors. Results from 119000 tests show ELlSA to in a Spinco rotor 60Ti. 11 Pellets were resuspended and pooled in be superior to the passive haemagglutination inhibi­ a total of 100 ml TS and centrifuged again at 176 000 g for 90 tion (PHAI) test for HBsAg. The ELlSA described. minutes, after which pellets were collected and resuspended in here is considerably cheaper than any comparable 12 ml NaBr at p = 1,30. Gradient centrifugation on an NaBr commercially available isotopic kit for HBsAg gradient between density 1,28 and 1,15 took place at 82 OOOg for screening but is slightly less sensitive. 16 hours in a Spinco SW27 rotor. 12 Fractions of 0,5 ml were collected after bottom-puncture ofthe centrifuge tubes and the S Air Med J 1983; 63: 148-151. density ofeach fraction was determined. Those fractions having a density between 1,20 and 1,24 were pooled and dialysed against TS, and the hepatitis B virus was concentrated by centrifugation at 176000 g as before. The resulting pellet was resuspended in The discovery of what is today known as hepatitis B surface TS, carefully layered onto a 7,5 - 20% (w/v) sucrose gradient and antigen (HBsAg),1 a surface component of hepatitis B virus,2,3 centrifuged in a Spinco SW27 rotor for 16 hours at 82 000 g. The was a major breakthrough in the history of viral hepatitis. Pre­ pellet resulting from this was resuspended in 65% sucrose in TS transfusion screening of donated blood, introduced in the late and overlayered by a 20 - 60% (w/v) sucrose gradient. After 1960s, reduced the incidence ofpost-transfusion hepatitis." This centrifugation at 82 OOOg for 16 hours (SW27 rotor) the position disease has been one of the major hazards connected to blood of the HBsAg band was determined by measuring the absor- transfusion. - bency at 280 nm. Purified HBsAg was pooled, dialysed against Considerable advances have been made in the last 10 years in TS and stored at -20°C. An f~Jd~m = 3,73 12 was used to the development ofsensitive, rapid and practical methods for the determine the concentration of HBsAg. detection of HBsAg. Today 'third-generation' assays are freely available and are widely used for the screening ofdonated blood. Of the 'third-generation' methods, immunoradiometric assay (IRMA), which employs a 'sandwich' J:;rinciple, is gene­ Production of sheep anti-HBsAg rally accepted as the most sensitive method. ,6 Unfortunately the Sheep were immunized by intramuscular injection at multiple cost of commercially available kits and equipment makes this sites with 50 J..Lg HBsAg in Freund's complete adjuvant for 4 assay prohibitive for most blood transfusion services in South weeks at weekly intervals. After 6 weeks test blood samples were Africa. obtained and sera were examined for the presence of anti-HBs. The development in 1971 of enzyme-linked immunosorbent Sera produced in this manner were also tested by immuno­ assay7.8 (ELISA) and its use for the detection of HBsAg9 offer a electrophoresis for the presence of antibodies against human cheaper alternative to the isotopic assays. The popularity of serum proteins. Such antibodies were routinely removed from ELlSA stems mostly from the possibility that this kind ofassay immune serum by passage through a column (5 x 90 cm) of can be developed 'in house'. Furthermore, less expensive human serum insolubilized onto Sepharose 6B .13 The immuno­ equipment is required and the enzyme-labelled antibody has a globulin fraction of antiserum was recovered by precipitating longer shelf-life than its isotope-labelled equivalent. 10 three times with 15% (w/v) sodium sulphate. The final precipi­ We describe ELISA screening and confirmatory tests for tate was dissolved in a minimum volume of distilled water and HBsAg in donated blood which have been in use since July 1980, extensively dialysed against distilled water. Euglobulins which present results obtained from preliminary studies and compare formed were removed by centrifugation and the clear superna­ ELISA with two other 'third-generation' methods. tant was freeze-dried.

Immunochemistry Department, Natal Institute of Immu­ Conjugate preparation nology, Pinetown, Natal Highly purified horseradish peroxidase (HRPO type VI; Sigma) M. O. BUBB, PH.D. T. MARIMUTHU, DIP. MED. TECH. was used. Antibody HRPO conjugate was prepared according to 14 J D. CONRADIE, PHD the method of Wilson and Nakane. Normally 8 mg sheep IgG was conjugated to 4 mg horseradish peroxidase. After conjuga­ Dale recei\·eJ: 10 May 1982. tion the mixture was chromatographed on a column (2,6 x 83 cm) SA MEDICAL JOURNAL VOLUME 63 29 JANUARY 1983 149 of Sephacryl S-300 in TS. The absorbancy was monitored spec­ Prime 11000 computer was linked to the photometer and was trophotometrically at 280 nm and 403 nm. Selected· fractions used to directly calculate positive/negative (P/N) values obtained containing conjugate were pooled and mixed with an equal in the assay. Results were printed our on a Texas Instruments volume ofglycerol containing 0,02% (w/v) NaN J . Conjugate was 820 printer. stored at 4°C (it should not be deep-frozen). This assay normally takes 3 hours.

Antibody coating of microtitre plates Confirmation assay The concentration of sheep antibody used in coating plates I. Dispense 50 /-Ll ofpresumptive HBsAg-positive sample into was established by developing ELISA colour in a plate coated each of two adjacent wells of an anti-HBs-coated plate. with increasing amounts ofantibody. The concentration ofanti­ 2. Cover the plate with a lid, place in a humidified container body at which a plateau in ELISA colour was reached was used and incubate overnight (18 hours) at room temperature. for coating subsequent plates. Throughout this srudy plates were 3. Wash plate once with TST using the washing 'shower'. coated at a concentration of 80 /-Lg/ml. Dynatech l29B plates 4. Add SO /-LI ofhuman anti-HBs antiserum to the first well and were coated with 100 /-LI sheep antibody using a semi-automatic SO /-LI of 4% (w/v) human serum albumin in TS to the second dispenser (Titertech Aurodrop; Flow Laboratories). Antibody well. was dissolved in 0,05M glycine-O, lM NaCl buffer, pH 2,5, and 5. Incubate in a humidified container at 45°C for 90 minutes. after 5 - 10 minutes was brought to a pH of7 - 7,5 by the addition 6. Follow procedure as for screening assay starting at point 4. ofsolid tris before the plates were coated. 15 Plates were allowed A sample is confirmed positive if the absorbancy of the well to stand for 2 hours at room temperature before washing by treated with human anti-HBs antiserum is reduced by 50% or means of a specially constructed washing 'shower',16 first with more relative to the absorbancy in the companion well treated TS containing 0,05% Tween 20 (TST) and then with distilled with HSA. water. Coated plates were dried under vacuum and packed and sealed into airtight plastic-metal-foil laminate packets contain­ ing a small bag of silica gel desiccant. lmmunoradiometric assay for HBsAg Commercial kits using 1251 as label were purchased from Tra­ venol and samples were tested for the presence ofHBsAg accord­ ELISA methodology ing to the manufacturers' instructions. The ELISA for the detection of HBsAg in donated blood consists ofa screening assay which is followed by a confirmation Passive haemagglutination inhibition (PHAI for assay on positive samples. detecting HBsAg) This assay was developed at the Natal Blood Transfusion l7 Screening assay Service and has been in routine use for screening donated blood since 1977. Dispensing ofconjugate, chromogenic substrate and acid-stop was done with a Titertech Autodrop using separate syringe heads for each reagent, as follows: Results I. Pipene accurately SO /-Ll of each of two positive controls (positive +++, positive +) into each of three adjacent wells and Determination ofthe cut-offpoint ofthe ELISA SO /-Ll of a negative control serum into 5 additional wells. Serum samples from 2 946 blood donors known to be free from 2. Dispense 50 /-Ll ofindividual samples to be tested into avail­ HBsAg were collected and tested by ELISA in groups of 200 able wells. -300 together with IS - 20 negative controls (2% (v/v) neutral 3. Cover the plate with a lid and place in a sealed humidified sheep serum"in TS) per group. Fig. I shows the distribution of container. Incubate at 45°C for 90 minutes. the PIN ratio of the serum samples. Because of the skewed 4. Wash plate once with TST using the washing 'shower'. nature of this distribution it was apparent that the high PIN 5. Dispense SO /-Ll of diluted ami-HBs-HRPO conjugate to ratio of certain negative samples would overlap with that of each individual well. Conjugate is diluted in TS containing 10% weakly positive samples (Fig. 2). In order to determine the (v/v) neutral sheep serum and 5% (v/v) negative human serum. lowest detectable P/N ratio ofa positive sample, which is signifi- 6. Cover the plate with a lid, place in a humidified container and incubate at 45°C for 60 minutes. 7. Wash plate as before with TST. Dispense SO /-Ll chromo­ genic subsr.rate* to each well and aJ.low colour to develop in the dark for 30 - 40 minutes at room temperarure (20 - 22°C). 320 8. Stop the enzymic reaction by adding into each well 100 /-LI of 300 1,5 HCl. .. 9. Measure the intensity of developed colour (492 nm) in a C.'" E Dynatech MR 580 Microelisa Autoreader. Serum samples ..'" 200 giving an absorbancy three times greater than that ofthe mean of (; the negative controls are presumed to be positive for HBsAg. A <; .c E ·Chromogenic substrate was prepared from anhydrous borax (Na,B,O,) 93 mg, <:" 100 succinic acid 93 mg; urea hydrogen peroxide 10 mg, and distilled water 25 m!. To this was added 30 mg a-phenylenediamine dihydrochloride (Fluka) which had been washed in methylene chloride containing 5% (v/v) MeOH until a clear filtrate was obtained. This ratio ofborax and succinic acid should yield a pH of5,0. Fine white 20 particles in the solution originate in the urea hydrogen peroxide and need not be removed. Because ofthe catalvtic breakdown ofH,O, by metals it is important that 3·0 4·0 5·0 6·0 contact with metals be completely avoided during"illsteps ofthe assay, and buffer PIN ratio salts used in the substrate should be of sufficient purity to avoid development of high background colour. The purity of the hydrochloric acid used is particularly important because acid of low quality will cause spurious colour development" Fig. 1. Frequency histogram of PIN ratios in an HBsAg-negative Merck Suprapur HO was found to be excellent. population. 150 SA MEDIESE TYDSKRIF DE EL 63 29 JANUARIE 1983

320 TABLE I. RANDOM BLOOD DONOR SAMPLES TESTED BY PHAI AND ELlSA PHAI EL/SA 300 No. tested 9701 9701 No. negative 8880 9531 No. retested 821 (8,46%) 170 (1,75%) 44 No. positive 52 51 No. repeatedly false positive o 40 'Confirmed negative by lAMA.

36

TABLE 11. LARGE-SCALE SCREENING BY EllSA: FOR 32 HBsAg IN DONATED BLOOD % of total No. tested 118532 100 28 No. negative 111931 94,4 0 No. retested by -l1l... confirmatory assay 6601 5,6 24 Z ~ donors, males and females of the four racial groups constituting 20 the donor population in Natal. In this series 118532 samples were tested by 45 individual technicians over a period of 18 months. Of these samples 94,4% were negative by the ELISA 16 screening test and 5,6% were presumed positive and had to be retested by the ELISA confirmatory test (Table Ill). This test showed that 5121 (77,6%) of the samples were in fact negative 12 and that 910 were positive. The remaining 570 were considered 'inconclusive' because oftechnical problems or 'borderline' PIN values. Upon retesting these 570 samples with IRMA, 16 \vere 8 found to be positive.

4 TABLE Ill. SAMPLES WITH PIN ~ 3,0 RETESTED BY ELlSA ------1 S.D. CONFIMATORY ASSAY (BLOCKING ASSAY) %% of total

2048 256 64 16 8 4 2 No. tested 6601 100 5,6 2 No. negative 5121 77,6 4,3 HBs Ag Dilution-'( X 10 ) No. positive 910 13,8 0,8 No. inconclusive 570 8,6 0,5 Fig. 2. Standard curve showing PIN ratio v. HBsAg dilution. candy higher than the mean negative control, an HBsAg-positive serum sample was serially diluted and tested by ELlSA. The The sensitivities of ELISA and IRMA were compared by dose-response curve obtained is shown in Fig. 2. Analysis of testing serial doubling dilutions oftwo strongly positive HBsAg replicate samples at each dilution on this curve show that the sera ofthe two main subrypes 'ad' and 'ay'. The lowest dilutions absorbancies differed by more than I SD from the negative mean which still gave a positive result are shownin Table IV. Although from dilutions of 1/25600 or less. In this study values above I IRMA detected both subtypes with equal facility, ELISA dis­ SD or approximately three times the negative control mean played different sensitivities towards the subtypes and was also (PIN = 3) were therefore regarded as positive. less sensitive than IRMA.

Comparison of PHAI and ELISA Discussion In order to evaluate the ELISA described here 9701 blood donor samples were screened for the presence of HBsAg by The ideal assay for detecting HBsAg in blood should be: (a) PHAI and ELISA (Table I). This preliminary trial clearly sensitive; (b) rapid; (c) objective, i.e.leaving no room for techni­ showed the ELISA to be as specific as the PHAI but also cian error; (d) inexpensive; and (e) stable, i.e. having a long underlined the fact that it was necessary to retest fewer samples. shelf-life. This preliminary comparison indicated that the ELISA was a No single assay satisfies the above requirements, and for that more objective and reliable assay than PHAI and that a further reason several different types ofassays using different detection trial was warranted. principles have been developed. Immunoassays using isotopic Table Il shows results from a much larger series of blood labels are known to be very sensitive, but they have the drawback samples tested by ELlSA. These included new and old blood of being relatively expensive and special laboratory equipment SA MEDICAL JOURNAL VOLUME 63 29 JANUARY 1983 151

po sibilities are impracticable in a blood bank where the creen­ TABLE IV. SENSITIVITY OF ELISA AND IRMA (TRAVENOL) ing for HBsAg of donated blood must be completed as soon as IN DETECTING HBsAg OF SUBTYPES ad AND ay possible. Yet another po sibility exists, and this lies in the pro­ duction of monoclonal antibodies of high avidity. \,(Te have ELlSA IRMA HBsAg titre- titre-I recently developed a mouse hybridoma producing monoclonal ' IgG, antibodies, and preliminary results with this material are ad 8000 (3,5) 16000 (5,4) \·ery encouraging. ay 4000 (4,1) 16000 (3,2) PIN values in parentheses.

REFERE~CES

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