Development of a dot-ELISA for swine trichinellosis using isolated specific antigens and molecular cloning of specific antigen gene

A dot-enzyme-linked immunosorbent assay (dot-ELISA) using multiple, denatured, specific antigens from Trichinella spiralis to detect anti-Trichinella IgG in swine was developed. Specific antigens were identified by western blot (WB). Excretory-secretory product (ESP) antigens were resolved on 12% SDS-PAGE and incubated with over 500 sera which were collected from farm-raised swine and had high optical density (O.D.) in ELISA. Antigens with molecular masses of 49, 52, 54, 60 kD and 25 to 30 kD were shown to be specific for T. spiralis.; Monoclonal antibodies (Mab) were generated to isolate specific antigens identified by WB. From one fusion, 101 hybridomas were identified by ELISA. A glycoprotein of about 60 kD was isolated by Mab column 7G6-2. Two antigen fractions, P49/57 and 52/62, were eluted from 10B6-1 column by buffers of pH 2.5 and pH 11.5, respectively.; A dot-ELISA using ESP, P49/57, and P52/62, was developed by individual application of the three antigens on nitrocellulose paper. The sensitivity and specificity of the dot-ELISA was evaluated by comparing the dot-ELISA with ELISA and WB using ESP as antigens. The dot-ELISA was as sensitive as ELISA and WB in testing 20 low level experimental infections. The dot-ELISA gave similar results as the WB in confirming 87 sera from farm-raised swine which tested positive 4X by ELISA. Nearly 2000 (1960) sera collected from farm-raised swine were tested by ELISA, dot-ELISA, and WB to evaluate the efficacy of the dot-ELISA in testing field samples. Two hundred and sixty two (13.5%) were positive by ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by WB. These results indicate that the dot-ELISA was as sensitive as ELISA and WB, and nearly as specific as WB.; Antigen P49 was purified by Mab affinity chromatography and reversed phase high performance liquid chromatography (HPLC). Twenty seven 5{dollar}spprime{dollar}-end amino acids of the protein were sequenced. To overcome codon usage degeneracy, a sequence of 40 nucleotides was amplified by mixed oligonucleotides primed amplification of cDNA. An internal probe and a new 5{dollar}spprime{dollar}-end polymerase chain reaction (PCR) primer were synthesized based on the amplified sequence. A 1.2 Kb cDNA was again amplified from cDNA ends by PCR using 5{dollar}spprime{dollar} specific primer and adaptor-oligo (dT) was 3{dollar}spprime{dollar} primer and cloned to PUC{dollar}sb{lcub}19{rcub}{dollar}. About 10% of the bacterial colonies were identified by the internal probe. The 1.2 Kb fragment was confirmed to be the cDNA encoding P49 by DNA sequencing. A fusion protein of 66 kD was expressed after the cDNA was ligated to plasmid vector pGEMEX2 and transformed into Escherichia coli (JM109, DE3). The cDNA product was recognized by sera from mice immunized with P49 and sera from swine infected with T. spiralis.