Development of EIA for detection of Chlamydia trachomatis in genital specimens.

A double antibody sandwich enzyme immunoassay (EIA) for chlamydial antigen detection was developed using a monoclonal antibody against lipopolysaccharide (LPS) of Chlamydia trachomatis as a coating antibody. Polyclonal rabbit antiserum against partially purified antigen from elementary body (EB) antibody and horse-radish peroxidase conjugated goat anti-rabbit antibody were used as the primary and secondary antibody respectively. The developed EIA could detect protein of partially purified EB at the lowest concentration of 250 ng/ml. The assay was evaluated against the cell culture (CC), DNA hybridization assay (PACE2 system: Gen-Probe, San Diego, CA, USA) and a commercial enzyme immunoassay (kEIA) (Bioquest, NSW, Australia). The sensitivity, specificity, positive and negative predictive values of the developed EIA (dEIA) were 87, 96.2, 80, 97.7 for the specimens from females and 90.9, 90.7, 71.4, 97.5 for the specimens from males repectively. Cross reaction was not found with Escherichia coli, Acinetobacter anitratus, beta-Streptococcus group A, Enterobacter spp, Enterococcus, Lactobacillus spp, Neisseria spp, but it was found with Candida albicans and herpes simplex virus type 1. The developed EIA can be applied successfully for both genders, particularly males. The cost per test is less than those for CC, kEIA and PACE2.

Immunodiagnosis of group-A streptococci by latex agglutination assays with monoclonal or monospecific polyvalent antibodies.

Seven clones of murine monoclonal antibodies specific for group-A streptococci were generated. All of them were of IgM isotypes and recognized trypsinized as well as nontrypsinized group-A streptococci and polysaccharide. These were devoid of reactivity with streptococci-B, -C, -G and Staphylococcus aureus. Polyclonal antibodies against group-A polysaccharide (APS) were also raised in rabbits by linking APS to bovine serum albumin, and rendered monospecific by adsorption. Latex agglutination assays were developed employing both types of antibodies. The assay employing monospecific polyvalent antibodies had a sensitivity of 12.5 ng APS/ml as compared to 1 microgram APS/ml for latex sensitized with monoclonal antibodies. Both assays were specific, as no agglutination was observed with polysaccharides obtained from streptococci-B, -C, -G, Staph. aureus, Cornybacterium diptheriae, Candida albicans, Candida spp., Morexella catarrhalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Streptococcus agalactiae, Strep. pneumoniae. Salmonella typhi and S. paratyphi A and B. Throat swabs from children obtained in duplicate, when tested for the presence of streptococci-A, showed a good correlation of the results obtained by latex agglutination assay with the microbial culture test and serogrouping.

Gel-filtration chromatography of Salmonella protein antigens and their implication in immunodiagnosis.

Crude Barber protein (Bp) antigens were prepared from Salmonella typhi, S. krefeld and S. derby by an original method that has been described previously. These antigens were subjected to gel-filtration chromatography using Sephadex G-200. A sharp peak that eluted together with the void volume was thus separated from a broad second peak that eluted from the column at positions equivalent to 118,000 to 12,000 daltons. The proteins eluted in the latter peak were arbitrarily divided into 5 fractions and, together with the first peak, subjected to polyacrylamide gel electrophoresis and immunoprecipitation with both homologous and heterologous rabbit antisera. The extent of immunological cross reactivities was determined by enzyme-linked immunosorbent assay. The preliminary results obtained by this technique showed species-specific protein antigens to have molecular weights ranging between 36,000 and 68,000 daltons.