Determination of purified gonococcal pili antigen in clinica isolated neisseria

Abstract

The present investigations attempt to examine the purified gonococcal pili antigen in clinical isolated Neisseria. Thirteen piliated gonococci were selected from thirty-one clinical isolates on the basis of colony typing system and electron microscopy. The former method remains the useful test for sorting out piliagted gonococci from gonococcal population. Purification of pili was encountered with protein contamination, poor out put and time consumption. Only five pili preparations out of twelve piliated strains could be obtained, at a low yield of 0.22 to 1.03 mg/10 g wet weight of bacteria. Visualization of gonococcal pili under electron microscopy revealed long filamentous strands with a diameter of 7 mm. The 5 pili preparations retain their morphology after purification process and are indistinguishable both among themselves and from E.coli pili. Most pili preparations showed a single hand upon SDS-Polyacrylamide Gel Electrophoresis from which the sub-unit molecular weights were calculated (ranging from 18,000 to 22,500 dal-tons). Antigenicity and immunogenicity of pilin were explored by raising anti-pili antibody for ELISA test. Immunization of one pili preparation in rabbits could elicit a good antibody response with a titer of 1:81920 by Indirect Hemagglutination. This antibody afforded high specificity when tested against other bacterial by coagglutination and was further used for the detection of pili antigen by ELISA. Only one pili out of 4 heterologous pili preparation and 4 out of 35 piliated gonococci were reactive with this anti pili anti ody by ELLISA system, indicating extreme pili heterogeneity among strains. It is also indicative of high specificity of rabbit antibody against a variable epitope on the immunized pilin. The high specificity of anti-pili antibody and pili heterogeneity presents problems in developing an ELISA system for pili antigen determination. Therefore, the use of rabbit antipili against a single local strain of gonococcal pili could not be feasible for the diagnosis of gonococcal infection. The employing of polyvalent anti-pili antibodies from pools of antisera against different strains and/or immunization of purified common fragment of gonococcal pili may be solutions to increase the capacity for pili antigen detection. Nevertheless, antipili antibodies could be invaluable in the analysis of gonococci for taxonomic and epidemiological purposes.