A Brugia malayi Antigen Specifically Recognized by Infected Individuals N. Rahmah,* ,1 A. Khairul Anuar,† A. Noor A’shikin,* B. H. Lim,* R. Mehdi,* B. Abdullah,* and M. N. Zurainee† *Department of Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; and †Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Received August 12, 1998 Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individ- uals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti- filarial IgG4 antibodies, 200 sera from healthy city- dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10 –15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solu- tion, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in be- tween each incubation step. Luminol chemilumines- cence detection was then used to develop the blots. An antigenic band with the MW of 37 kDa was found to be consistently present in the Western blots of all mi- crofilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated pa- tients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individ- uals, and city dwellers. Therefore the B. malayi anti- gen of with the MW of 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application. © 1998 Academic Press Lymphatic filariasis caused by Brugia malayi and Brugia timori affects about 12.5 million people in South East Asia (1) and is still endemic in several states in Malaysia. The routine diagnostic test em- ployed to detect brugian filariasis is by night blood examination for microfilaria. This method is specific but insensitive (2) and require night blood sampling which is unpopular with patients, communities and health workers. A more sensitive assay which obviate night blood sampling is required; thus this study was aimed at identifying B. malayi antigen that may be specific for serological diagnosis of brugian filariasis. MATERIALS AND METHOD Antigen preparation. Gerbils were infected intraperitoneally with the third stage larvae of B. malayi. After 3-4 months, the animals were dissected for collection of adult worms. The worms were washed, cut, homogenised, sonicated and freeze-thawed. The preparation was then concentrated, centrifuged and the supernatant kept (-20°C) as soluble B. malayi antigen. The protein content of the antigen was determined to be 2000 g/ml by the Bio-Rad assay. Study population. Sera from a brugian filariasis endemic area in two states in North Eastern Malaysia was used for this study. Informed consent was obtained from all subjects before the blood sampling. Microfilaria detection was performed using stained thick blood smear and Knott concentration technique. A sandwich ELISA to detect anti-filarial IgG4 antibodies in sera from this area had previously been performed and the cut-off optical density value for detection of active infection was determined to be 0.420 (3). 444 serum samples from the following groups of individuals were em- ployed: 1. Individuals with circulating microfilaria (n = 40) 2. Ami- crofilaraemic individuals with elephantiasis (n = 10). 3. Previously microfilaraemic individuals i.e. they have been treated and were amicrofilareamic at time of sampling (n = 24 ) 4. Endemic normals i.e. endemic area individuals who demonstrated no anti-filarial IgG4 antibodies towards adult worm and microfilaria antigens (previously determined; n = 50). 5. Amicrofilaraemic individuals in endemic areas with high titres of anti-filarial IgG4 antibodies (n = 20). 6. City-dwellers (non-endemic samples; n = 200). 7. Non-endemic area individuals infected with soil-transmitted helminths (STH; n = 100). Western blotting. Phast Electrophoresis System (Pharmacia, Sweden) was employed for running of the SDS-PAGE gel and for electroblotting. 2 l antigen per well was electrophoresed on 10-15% gradient gels and the protein bands transferred onto a PVDF mem- brane. The membrane was then cut into strips, the lane containing the molecular weight marker was stained in amido black and the 1 Corresponding author. Department of Microbiology and Parasi- tology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Fax: 60-9-765 3370. E-mail: rahmah@kb.usm.my. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 586 –588 (1998) ARTICLE NO. RC989360 586 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.