An assay for quantification of white spot syndrome virus using a capture ELISA

Abstract

Keywords: capture ELISA, diagnosis, quantification,WSSV.White spot syndrome virus (WSSV) is the causativeagent of a shrimp viral disease which has severelyimpacted most of the shrimp farming regions ofsouth-eastern Asia and presently overshadows allother disease agents as the leading cause ofproduction losses in Asia (Flegel 1997). AlthoughWSSV has been known since 1993, viral quantifi-cation in infected animals was hindered by the lackof a continuous cell culture system for shrimp.Recently, an in vitro system using primary shrimplymphoid cells was used for the quantification ofthe Chinese baculo-like virus (CBV, now known asWSSV) from the haemolymph and gill samples ofexperimentally infected Penaeus stylirostris (Tapay,Lu, Gose, Nadala, Brock & Loh 1997). Acompetitive polymerase chain reaction (PCR)method was developed for quantification of theWSSV genome and successfully applied in analy-sing changes in virus quantity in the haemolymphduring the course of infection (Tang & Lightner2000). A monoclonal antibody (MAb), designated6E1, recognizing an epitope on a major envelopeprotein, VP 28 of WSSV, has been produced andcharacterized (Shih, Wang, Tan & Chen 2001). Inthe present note, we describe the development of adouble antibody sandwich, i.e. capture enzyme-linked immunosorbent assay (ELISA) combiningMAb 6E1 with polyclonal antibodies affinity-purified from WSSV-immunized rat sera. Themethod was applied to monitor changes in con-centration of WSSV proteins in the haemolymphduring the progression of white spot syndrome.The WSSV used in this study was obtained frominfected P. japonicus collected from Ilan in north-eastern Taiwan. The head soft tissues were homo-genized at 4 C in TNE buffer [50 mm Tris,100 mm NaCl, 1 mm ethylene diamine tetraaceticacid (EDTA), pH 7.4] and ultracentrifuged topurify WSSV as described previously (Shih et al.2001). An aliquot of tissue homogenate was dilutedwith TNE buffer and used as the inoculum for thesubsequent infection trial. The purified WSSV wasassayed for protein using a protein assay reagent(BIORAD Laboratories, NY, USA). Adult Wistarrats were immunized and boosted 3 weeks laterwith 100 lg purified WSSV. Immunoglobulin G(IgG) was purified from pooled rat sera using aHiTrap protein A HP column (Pharmacia Biotech,Uppsala, Sweden). The MAb 6E1, isotyped toIgG