cDNA coding for Brugia malayi pepsin inhibitor homolog (Bm-33) from the human filarial parasite was cloned in pRSET for large-scale expression and functional characterization. The pRSET-B cloned gene did not yield recombinant protein expression and the reason was attributed to the presence of an N-terminal signal peptide. The gene was subcloned in pRSET-A without a signal peptide and the 33 kDa histidine-tagged recombinant protein was purified by IMAC. All individuals from an endemic area generated IgG responses against Bm-33 in the order MF>CP>EN. Isotype analysis indicated an elevated IgG4 reactivity in the order MF>EN>CP. Bm-33-specific IgE levels were elevated in MF, CP and EN compared to non-endemic normals with no significant differences among the groups. Paraffinembedded sections of Setaria digitata (cattle filarial parasite) stained with mouse anti-Bm-33 antibodies exhibited the hypodermal nature of Bm-33. These findings suggest that Bm-33 is an immunodominant antigen and contributes to filarial pathogenesis. Key words antibody, Brugia malayi, ELISA, immunolocalization, isotype.Human lymphatic filariasis is caused by lymph-dwelling, parasitic nematodes such as Wuchereria bancrofti, Brugia malayi and Brugia timori which are prevalent in tropical and subtropical countries of the world. The initiatives of the Filarial Genome Project to combat this nematode scourge have enabled the identification and characterization of genes with diagnostic and prophylactic capabilities such as WbSXP-1, Bm-ALT etc. (1, 2). Despite these and a few more, there is an urgent need to identify and characterize more antigens involved in parasite-specific immune modulation, a major factor that contributes to filarial pathogenesis.The cDNA coding for B. malayi, pepsin inhibitor homolog (Bm-33), a 33 kDa protein (3, 4), was cloned Correspondence Rangarajan B. Narayanan, Centre For Biotechnology, Anna University, Chennai-600 025, Tamil Nadu, India. Tel: +91 044 22350772; fax: +91 044 22350299; email: rbn@annauniv.edu List of Abbreviations ALP, alkaline phosphatase; anti-BmA, antibodies against Brugia malayi crude protein; BCIP, bromochloroindoloyl phosphate; CP, chronic pathology; DAB, diaminobenzidine; EN, endemic normal; HLA, human leukocyte antigen; HRP, horseradish peroxidase; IgG, immunoglobulin G; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl beta thiogalactosidase; MF, microfilaremic; NBT, nitroblue tetrazolium; NEN, non-endemic normal; pNPP, para nitrophenol phosphate. and serologically characterized. Earlier, a similar homolog from Onchocerca volvulus (Ov33) was cloned in pGEX-2T and the GST tagged-recombinant protein was used for immunodiagnosis based on its elevated IgG4 reactivity in microfilaremic patients (5, 6). Homologs were also identified from related parasites such as Dirofilaria immitis and Acanthocheilonema viteae and the respective recombinant proteins (Di33 and Av33) were used for the development of immunodiagnostic assays (7,8). In the case of the Brugian homolog, the 750 bp cDNA cod...