Journal of Virological Methods, 38 (1992) 145-l 52 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00


VIRMET 01339

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections S. Sumathya, S.P. Thyagarajana, Rauf LatiQ, N. Madanagoaalanb, K. Raguramb, P. Rajasambandam” and Eric Gowans aDepartment of Microbiology. Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, Taramani, Madras, India bDepartment of Digestive Health and Diseases, Annanagar Peripheral Hospital Madras, and ‘Department of Gastroenterology, Government General Hospital, Madras, India, and ‘Division of Medical Virology. Institute of Medical and Veterinary Science, Adelaide, S. Australia


13 January



A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and antibodies to hepatitis delta virus (anti-HDV), utilizing cellulose nitrate membrane is described. Screening of 8 15 serum specimens for HBsAg by DIA and micro ELISA revealed a positivity of 22.69% and 22.94% respectively. In the detection of antibodies to delta antigen, DIA was compared with an indirect immunofluorescence technique using As cell line as antigen substrate and a commercial macro ELISA. Of the 143 HBsAg positive sera tested for anti-HDV, 59 (41.25%) were positive by both immunofluorescence and macro ELISA and 61 (42.65%) by DIA. While the positive and negative predictive values of DIA for HBsAg were 100% and 99.6%, for anti-HDV by DIA these were 96.7% and 100% respectively. Based on the simplicity of performance and the economical nature of the test system, DIA is recommended as a diagnostic tool for field surveys and small laboratories in developing countries. Dipstick immunobinding-enzyme-linked immunosorbent assay; Hepatitis B surface antigen; Hepatitis delta antigen; Conventional enzyme-linked immunosorbent assay

Correspondence to: S.P. Thyagarajan, Department of Microbiology, Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, Taramani, Madras 113, India.


Introduction Enzyme immunoassay (EIA) is widely accepted for the diagnosis of infectious diseases (Bommeli et al., 1983; Charan et al., 1984) and has thus revolutionized the field of microbial diagnosis (Kleeman et al., 1983; Ngo et al., 1985). The major drawbacks of the conventional ELISA are its high cost, time taken to complete the test, requirement ef sophisticated equipment and utilization of large amounts of the reagents. In addition to these, the requirement of purified antigens, monoclonal antibodies (Sarov et al., 1989); the varying binding characteristics of the solid phases for different proteins influence the reproducibility and standardization of ELISA (Burt et al., 1979). In recent years, growing medical costs have prompted a distinct trend toward simplified diagnostic immunoassays which require minimal cost, time and equipment to perform, more especially in developing countries. Based on the reported use of nitrocellulose as a solid phase for spot ELISA testing (Hawkes et al., 1982; Pappas et al., 1983; Towbin et al., 1984), we describe the development and evaluation of a DIA using cellulose nitrate membrane for the rapid, specific and sensitive serological detection of HBsAg and anti-HDV in comparison with the conventional ELISA and immunofluorescence techniques.

Materials and Methods DIA for HBsAg Antisera Affinity purified horse-antibody to HBsAg (Anti-HBs, Wellcome Burroughs) was used for coating the discs. Protein determination was carried out with bovine serum albumin as the standard (Lowry et al., 1951). 80 nanograms (ng) of anti-HBs was determined as the optimal coating concentration by the checkerboard method. 815 sera, comprising 120 acute viral hepatitis (AVH), 150 Clinical specimens chronic liver diseases (CLD), 15 hepatoma and 530 voluntary blood donors were included in screening for HBsAg by DIA and standard micro ELISA. Control sera 16 coded panel sera obtained from Centre de Nationale Transfusion Sanguine, Paris, served as positive and negative controls. DIA Technique Cellulose nitrate membrane filters (Sartorius 0.2 ,u) were punched to a diameter of 5 mm and stuck with synthetic rubber based adhesive (Fevicol, Pidelite Industries Ltd., India) to one end of the plastic strips (5 cm length). Dots of 1 ~1 (80 ng) of polyclonal anti-HBs in phosphate buffered saline (PBS) were applied on the discs. After overnight incubation at 4°C the dried discs were incubated with 200 ~1 of 2% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20 at room temperature (rt) for 2 h with gentle


agitation to block the remaining protein binding sites. After washing twice in PBS-Tween, the discs were stored at 4°C until further use. 10 ~1 of test sera were spotted on the disc and incubated in moist chamber at rt for 30 min. The discs were washed thoroughly three times with PBS for 10 min. After washing, they were blotted dry and 10 ,~l of sheep raised anti-HBs antibody bound to horse radish peroxidase (HRP) in PBS-tween of appropriate dilution were spotted on the discs and incubated for 30 min at rt. The discs were washed three times with three changes of PBS-tween and incubated with the substrate containing hydrogen peroxide and 3-3, Diamino benzidine tetrahydrochloride (Sigma) for 10 min. The reaction was stopped in tap water and read. Positive reactions appeared as brown dots on the discs and negative was colourless. Standard


Micro ELISA for HBsAg was carried out using commercial Hepanostika ELISA kits supplied by Organon Teknika. The time required to complete this assay was 4 h. DIA for anti-HD V Delta antigen (HDAg)

Purification of liver derived delta antigen was carried out according to the procedure of Rizetto et al. (1980). 200 ng was determined as the optimal coating concentration by the checkerboard method.

Anti-HD V and A3 cell line

Affinity purified human anti-HDV and A3 cell line (continuous hepatoma cell line expressing HDAg) were used in indirect immunofluorescent study for the serological detection of anti-HDV, after the procedure of Gowans et al. (1990). Clinical specimens 143 HBsAg positive

sera (acute viral hepatitis 76; chronic liver diseases 67), and 10 HBsAg negative liver disease cases formed the study material. Control sera 34 coded panel

Science, Adelaide,

sera obtained from Institute of Medical Australia were used in the study.

and Veterinary

DIA technique

Preparation of dipsticks coated with delta antigen. Dots of 1 ~1 containing 200 ng of delta antigen were applied allowed to dry at rt. After overnight incubation at 4°C the blocked with 2% BSA in PBS-tween for 2 h at rt with gentle washing in PBS-tween, the discs were stored dry at 4°C until

on the discs and dried discs were agitation. After use.

Test procedure

10 ~1 of sera were spotted on the discs and incubated for 12 h at room temperature in a moist chamber. The discs were washed in 3 changes of PBS-


tween for 10 min and blotted dry. 10 ,nl of l/3000 dilution of rabbit raised antihuman IgG conjugated to HRP (DAKOPATTS) in PBS-tween was spotted and incubated for 1 h at rt. The discs were washed in PBS-tween and incubated with the substrate containing hydrogen peroxide and 3-3, diaminobenzidine for 10 min. The reaction was stopped in tap water and read. Appropriate controls were also included. Positive reactions were visualised as brown dots and negative was colourless. Standard ELISA Commercially available Sorin Bio-medica anti-HDV ELISA kits were used. This required 22 h to complete the test. Check tests for specificity by blocking experiments HBsAg positive sera were absorbed with anti-HBs and PBS (control) and tested for reactivity by DIA for HBsAg. Similarly anti-HDV positive sera were absorbed with HDAg and PBS and tested for reactivity by DIA for anti-HDV. Statistical analysis Statistical analysis for evaluating the sensitivity, specificity and clinical performance of the test was carried out using predictive value formula (Browner et al., 1988). Results

The results of the coded sera for HBsAg by DIA and ELISA (Table 1) revealed no discrepancies. However, anti-HDV screening has detected the same four samples in excess by all the three techniques adopted in the study (Table 2), although no titration was carried out. TABLE 1 Screening of coded panel sera obtained from centre de Nationale Transfusion HBsAg by DIA and Micro ELISA Type

Coded Panel sera

No. tested


Sanguine (CNTS) for

HBs Ag positivity CNTS result






TABLE 2 Screening of coded panel sera for anti-HDV Type

Coded panel sera

No. tested


by DIA, Immunofluorescence


and Macro ELISA


Panel result








149 TABLE 3 HbsAg Screening by DIA and Micro ELISA in various clinical groups


No. tested

HBsAg positivity DIA

Acute viral hepatitis Chronic liver diseases Hepatoma Voluntary blood donors Total

120 150 15 530 815

16 66 6 31 185 (22.69%)

Micro ELISA 76 67 6 38 187 (22.94%)

Predictive value of DIA

On screening 8 15 serum samples comprising of acute viral hepatitis, chronic liver diseases, hepatoma and voluntary blood donors for HBsAg, 22.69% were positive by DIA and 22.94% by ELISA (Table 3). The DIA technique (Fig. 1) for HBsAg was shown to have a sensitivity of 98.9% and specificity of 100% with a positive and negative predictive value of 100% and 99.6% respectively. The efficiency of this DIA technique was 99.75%.0f the 143 HBsAg positive cases screened for anti-HDV, 59 (41.25%) were positive by immunofluorescence and ELISA and 61 (42.6%) by DIA (Table 4). The sensitivity and specificity of this DIA was 100% and 97.6% respectively. The positive and

Fig. 1. DIA technique showing positive and negative reactions (from right) negative, weak positive and strong positive.

150 TABLE 4 Anti-HDV screening by DIA, Immunofluorescence clinical groups No. tested


Acute viral hepatitis Chronic liver diseases Total

76 67 143


and macro ELISA in various HBsAg positive positivity




19 20 40 59 (41.25%) :; (41.25%)

:f (42.6%)


negative predictive values being 96.7% and loo%, respectively. The efficiency of this DIA system was 98.6%. The 10 HBsAg negative liver disease cases screened by these assays remained negative for anti-HDV by any of the 3 techniques described. Blocking experiments confirmed the specificity of DIA reactions, using specific antigen or antibody, respectively. Intra-assay

and inter-assay evaluation

The reproducibility of the DIA was 100% on the basis of visual interpretations by more than five investigators. In within-run-tests (intra-assay) of four positive sera (one weak and three moderately strong) and four negative sera involving 32 replicate tests, all positive sera were always positive and all negative sera were always negative. Similarly run-to-run tests (inter-assay) of reproducibility performed extremely well. In the latter studies 6 positive sera (4 weak and 2 moderately strong) and 4 negative sera were analyzed. In the total of 72 tests, all positive sera were always positive and all negative sera were always negative. Minimum

detection limit of DIA

The minimum detection limit of DIA for HBsAg was evaluated using sera of known concentration of HBsAg, 1.5 ng/ml (HBsAg standard preparation from Paul Ehrlich Institute, Germany), which was assayed at varying concentrations. The results were compared with those observed by testing the same samples in micro ELISA. The DIA detected 450 pg and micro ELISA 150 pg of HBsAg.

Discussion Several studies have shown the usefulness of dot ELISA for serodiagnosis of infectious diseases. Chan et al. (1988) demonstrated the accurate detection of trichinosis by dot-ELISA with no nonspecific reactions. The dot ELISA has also been applied successfully for rapid serodiagnosis of human visceral leishmaniasis (Pappas et al., 1983), human leptospirosis (Pappas et al., 1985)


and human hydatidosis (Sorice et al., 1985). A disc ELISA reported by Gandhi et al. (1989) for serological detection of HBsAg had 100% correlation with commercial micro ELISA. The use of a plastic strip attached to cellulose nitrate disc in the study was preferred because of the difficulty encountered in handling the fragile nitrocellulose (NC) membrane blots for routine purposes in hospital and field laboratories and to avoid wastage of NC blots. The DIA was shown to utilize small volumes of reagents besides being useful in screening small number of specimens. We used routinely 10 ~1 of sample, although even smaller volumes may be used with good results (Hawkes et al., 1982). Whereas, the commercial ELISA required on average of 100 ~1 of sample and other reagents. The high cost of NC membranes, its non-availability in developing countries and also the reported use of cellulose acetate paper (Schaltmann et al., 1980) has prompted us to use cellulose nitrate membrane as a suitable substitute for NC membrane. The present day requirement for sero-surveillance of infectious diseases in developing countries especially for hepatitis and AIDS is an economical assay system preserving the sensitivity and specificity factors of the conventional ELISA. The present technique standardized in this laboratory has achieved the specificity and sensitivity anticipated. An analysis of the cost effectiveness of this technique in comparison with the commercially available ELISA kits has shown that an HBsAg test costs approximately ten cents (US) and the approximate cost of one anti-HDV assay is US $1.00. Another advantage of this technique is that an ELISA washer and reading system and electricity are not required. The DIA technique may be the most suitable technique for field studies and small laboratories in the underdeveloped countries.

Acknowledgements The financial support of the Council of Scientific and Industrial Research, New Delhi, India and Indian Council of Medical Research through Indo-UK Project on Viral Hepatitis is gratefully acknowledged. References Bommeli, W.R. (1983) Use of enzyme immunoassay in veterinary medicine. In: S. Avrameas, P. Druet, R. Masseyeff and G. Feldmann (Eds), Immunoenzymatic Techniques, Elsevier, Amsterdam, pp. 349-362. Browner, W.S., Newman, T.B. and Cummings, S.R. (1988) Designing a new study: Diagnostic tests. In: S.B. Hulley and S.R. Cummings (Eds), Designing Clinical Research, Williams and Wilkins, Baltimore, U.S.A. pp. 87-97. Burt, SM., Carter, T.J.N. and Kricka, L.J. (1979) Thermal characteristics of microtitre plates used in immunological assays. J. Immunol. Methods. 31, 231-236. Chan, S.W. and Ronald, C.K. (1988) Comparison between standard ELISA and dot-ELISA for

152 serodiagnosis of human trichinosis. Trans. R. Sot. Trop. Med. Hyg. 82, 892-894. Charan, S. and Gautam, O.P. (1984) Application of ELISA in veterinary medicine. A bibliography. Vet. Res. Commun. 8, 255-267. Gandhi, B.M., Irshad, M. and Tandon, B.N. (1989) A low budget disc-ELISA for hepatitis-B surface antigen (HBsAg). Trop. Gastroenterol. 10, 11l-1 16. Gowans, E.J., Macnaughton, T.B., Mickan, L., Jilbert, A.R. and Burell, C.J. (1990) Use of recombinant hepatitis delta antigen in diagnostic assays for HDV antibody. J. Virol. Methods 27, 69-78. Hawkes, R., Niday, E. and Gorden, J. (1982) A dot immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147. Kleeman, K.T., Kiefer, D.J. and Halbert, S.P. (1983) Rubella antibodies detected by several commercial immunoassays in haemagglutination inhibition negative sera. J. Clin. Microbial. 18, 1131-1137. Lowry, O.H., Rosebrough, N.J., Farr, L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265. Ngo, T.T. and Lenhott, H.M. (1985) Enzyme mediated immunoassay. Plenum Publishing Corp., New York. Pappas, M.G., Hajkowski, R. and Hockmeyer, W.T. (1983) Dot enzyme-linked immunosorbent assay (dot-ELISA): a microtechnique for the rapid diagnosis of visceral leishmaniasis. J. M.G., Hajkowski, R., Cannon, L.T., Sr. and Immunol. Methods. 64, 205-214.Pappas Hockmeyer, W.T. (1984) Dot enzyme-linked immunosorbent assay (dot ELISA): Comparison with standard ELISA and complement fixation assays for the diagnosis of human visceral leishmaniasis. Vet. Parasitol. 14, 239-249. Pappas, M.G., Ballou, W.R., Gray, M.R., Takafuji, E.T., Miller, R.N. and Hockmeyer, W.T. (1985) Rapid serodiagnosis of leptospirosis using the IgM-specific dot-ELISA: comparison with the microscopic agglutination test. Am. J. Trop. Med. Hyg. 34, 346354. Rizzetto, M., Shih, J.W.K. and Gerin, J.L. (1980) The hepatitis B virus-associated delta antigen (6): isolation from liver, development of solid-phase radioimmunoassays for delta antigen and antidelta and partial characterisation of antigen. J. Immunol. 125, 318-324. Sarov, I., Anderson, P. and Anderson, H.K. (1980) Enzyme-linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus. Acta. Pathol. Microbial. Stand. Sec. B88, I-9. Schaltmann, K. and Pongs, 0. (1980) A simple procedure for blotting of proteins to study antibody specificity and antigen structure. Hoppe-Seylers Z. Physiol. Chem. 361, 207-210. Sorice, F., Vullo, V., Contiui, C., Mastroianni, C.M., Massetti, A.P., Monaco, M.T. and Delia, S. (1985) Use of dot immunobinding assay for the rapid diagnosis of human hydatidosis. BolletinoInstituto Sieroterapico Milanese Serafino Belfanti. 64, 414418. Towbin, H., Staehelin, T. and Gorden, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc. Nat]. Acad. Sci. USA 17, 596600. Towbin, H. and Gorden, J. (1984) Immunoblotting and dot immunobinding-current status and outlook. J. Immunol. Methods, 72, 313-340.

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections.

A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and...
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