ELISA in the diagnosis of Wuchereria bancrofti infection in man: a technique for reducing cross-reactivity

Bulletin of the World Health Organization, 58 (4): 655-657 (1980) ELISA in the diagnosis of Wuchereria bancrofti infection in man: a technique for reducing cross- reactivity S. DISSANAYAKE,1 & M. M. ISMAIL,2 The cross-reactivity offilarial heteroantigens, the non-availability ofadult Wuchereria bancrofti for antigen extraction, and the lack ofstructural information on purifiedfilarial heteroantigens make the serological diagnosis ofW. bancrofti infection in man a difficult problem. Theuseofimmuneserafromcattleinfected with Setariadigitata whencarryingout the enzyme-linked immunosorbent assay (ELISA) largely eliminatedthe cross-reactions with serum antibodies generally seen in subjects showing no evidence ofW. bancrofti infection. The complexity of filarial antigens, the lack of information about their structure, and the non- availability of adult Wuchereria bancrofti for antigen extraction demand the use of unpurified filarial heteroantigens for the serological diagnosis of W. bancrofti infection in man (1). While the sero- logical cross-reactivity of filariae (2-4) permits the use of heteroantigens for this purpose, the same property constitutes a serious limitation in filarial serology (1). Setaria digitata has been shown to contain antigens that cross-react with serum antibodies of W. ban- crofti-infected subjects (5). However, when the fraction showing the highest reactivity was used in the enzyme-linked immunosorbent assay (ELISA), low levels of antibodies were detected in the serum ofsome subjects showing no evidence of W. bancrofti infec- tion. The results of this study demonstrate that immune sera from cattle infected with S. digitata can be used to block selectively the cross-reactions with serum antibodies in subjects who show no evidence of W. bancrofti infection. MATERIALS AND METHODS The rationale of the method described in this communication is to use the S. digitata antigen SD24 (5) in ELISA and to block the cross-reactions with I Lecturer, Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka. 2 Head, Department of Parasitology, The Medical Research Institute, Colombo, Sri Lanka. 'non- W. bancrofti antibodies' in the test serum by the use of immune sera from cattle infected with S. digitata. S. digitata antigen SD2-4 was prepared and used in ELISA as described previously (5). Immune cattle serum (ICS) was collected from 15 cattle slaughtered at the municipal abbatoir, Colombo, Sri Lanka, and the adult S. digitata worms were obtained from the animals' peritoneal cavity. Unfortunately, we were unable to characterize this cattle serum in immuno- logical terms as very little is known about the S. digitata antigens that elicit antibodies in cattle. However, these sera showed precipitation in Ouchter- lony analysis with the crude extract of S. digitata. S. digitata fraction SD2-4 coated microtitration plates were washed repeatedly with phosphate- buffered saline (PBS) containing 50o ICS. Sera from W. bancrofti-infected patients and non-fiarial subjects were diluted in PBS containing 5o ICS and used in the ELISA, thereby blocking the non-fiarial antibodies with antibodies in ICS. The subjects in this study could be placed in 4 groups. Group 1 comprised 127 filariasis subjects showing clinical signs of filarial infection and circu- lating antibody to W. bancrofti microfilariae (by the indirect immunofluorescent antibody (IFA) test). Group 2 comprised 23 asymptomatic microfilaraemic subjects with no circulating antibody to microfiariae. In group 3, there were 361 asymptomatic amicrofil- araemic subjects from an area not free of filariasis but not endemic, and group 4 included 141 subjects from an area completely free of filariasis. Groups 1 and 2 were considered as filarial and groups 3 and 4 as non- filarial. 3985 - 655 - S. DISSANAYAKE & M. M. ISMAIL Table 1. Serological examinations: IFA test, ELISA, and effect of ICS on ELISA No. positive for IFA test ELISA ELISA Subject groups No. micro-filariae positive positive positive in ICS (1) Symptomatic amicrofilaraemic, from endemic areas 127 1 127 (100%) 120 (94.5%) 118 (92.9%) (2) Asymptomatic microfilaraemic, from endemic areas 23 23 0 22 (95.7%) 22 (95.7%) (3) Asymptomatic amicrofilaraemic, from non-endemic 361 0 28 (7.8%) 81 (22.4%) 30 (8.3%) areas (4) Asymptomatic amicrofilaraemic, from areas completely 141 0 4 (2.8%) 24.17.0%) 3 (2.1%) free of filariasis. RESULTS DISCUSSION In the IFA test, positive immunofluorescence at a serum dilution of 1:8 was considered as evidence of the presence of circulating antibody to W. bancrofti microfilariae. The results of IFA screening are shown in Table 1. Ideal serum dilution and antigen concentration for use in the ELISA were determined by chequerboard titrations. With I Mg of antigen (coating concen- tration), the highest serum dilution that resulted in best discrimination between positive and negative sera was 1:40. Therefore, all sera showing an ELISA titre greater than 40 were considered as ELISA-positive. ELISA titres for subjects in groups I and 2 were generally in the range of 160-2560. In contrast, positive subjects in groups 3 and 4 had ELISA titres less than 320. Incorporation of ICS in the assay did not change the percentage of ELISA positives in groups I and 2 (Table 1). However, there was a marked reduction in the number ofELISA positives in groups 3 and 4, upon incorporation of ICS (Table 1). Filariasis in Sri Lanka is predominantly due to W. bancrofti (6). Animal filariae are common and man is probably exposed to their vectors. In addition, various parasitic infections, such as Ancylostoma duodenale, Ascaris lumbricoides, Enterobious vermi- cularis, and Trichuris trichura are prevalent. Cross- reactions due to serum antibodies in these infections, if present, probably contributed to the observed ELISA titres in both 'filarial' and 'non-filarial' sub- jects. The use of ICS in the ELISA largely eliminated cross-reactions with antibodies in sera of 'non-filarial' subjects, but not in those of 'filarial' subjects. The positivity rates for ELISA in ICS were comparable to those for the IFA test. This suggests that ICS used in this study did not contain antibodies to W. bancrofti specific antigens-probably because W. bancrofti specific antigens of S. digitatado not elicit an antibody response in cattle. This system, if adapted to filariae that can be grown in laboratory animals, may provide a useful source of antigen and a test system for the diagnosis of filarial infections. ACKNOWLEDGEMENTS This study received support from the filariasis component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. RESUME LA MtTHODE ELISA DANS LE DIAGNOSTIC DE L'INFECTION A WUCHERERIA BANCROFTI CHEZ L'HOMME: UNE TECHNIQUE POUR RtDUIRE LA REACTIVITE CROIStE On a utilis un extrait partiellement purifie de vers adultes corps circulant a et decel. chez 95%1 environ des sujets Setaria digitata dans la methode de titrage avec immuno- asymptomatiques presentant une microfilaremie et des adsorbant lie a une enzyme (ELISA) pour le diagnostic de sujets symptomatiques sans microfilaremie. Cependant, des l'infection a Wuchereria bancrofti chez I'homme. Un anti- reactions ELISA positives ont ete observees chez environ 656 ELISA IN THE DIAGNOSIS OF FILARIASIS 657 25% de sujets non filariens. L'utilisation d'immunserums de bovins infectes de S. digitata lors de 1'execution de 1'epreuve ELISA permettait de bloquer selectivement les reactions croisees avec I'anticorps serique des sujets qui n'avaient aucun signe d'infection a W. bancrofti, et de r6duire a 8% approximativement le pourcentage de reactions faussement positives. REFERENCES 1. AMBROISE-THOMAS, P. Acta tropica, 31: 108-128 (1974). 2. FUJITA, K. ET AL. Japanese journal of experimental medicine, 40: 67-77 (1970). 3. TANAKA, H. ET AL. Japanese journal of experimental medicine, 40: 47-58 (1970). 4. BARTLET, A. ET AL. Tropenmedizin und Parasitologie, 26: 370-374 (1975). 5. DISSANAYAKE, S. & ISMAIL, M. M. Bulletin ofthe World Health Organization, 58: 649-654 (1980). 6. HAWKING, F. Tropical disease bulletin, 73: 967-1016 (1976).