Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Abstract

Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimize and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic system. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fast-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic contamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzyme markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of potential use in screening immune responses to N. gonorrhoeae. 1273 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from 1274 SMYTH, FRIEDMAN-KIEN, AND SALTON

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis

“…Detergent extraction of membrane fractions. The nonionic detergent Triton X-100 has proved to be extremely useful in the extraction of bacterial membrane antigens and in their subsequent analysis by crossed immunoelectrophoresis (36,50). However, optimal conditions for membrane protein extraction often require the use of fairly dilute membrane suspensions (45).…”

“…Since the maximum volume of detergent extract that can be conveniently analyzed by crossed immunoelectrophoresis in our system is about 10 pl and since it is necessary to analyze about 50 ,ug of membrane protein for optimum resolution, it is apparent that detergent extracts of complex antigen mixtures should possess a protein concentration in excess of 5 mg/ml. Consequently, membrane fractions often have to be at an initial protein concentration as high as 20 mg/ml (36,50) and must be treated repeatedly with detergent if full extraction is desired.…”

“…Agarose gels were then cast and cut to give a final plate consisting of four parallel gels (width, 50 mm), 10, 8, 2, and 30 mm in length, containing membrane antigens that had been subjected to electrophoresis, lectin, agarose alone, and antienvelope immunoglobulins, respectively. In control runs, the affinity gel dehydrogenase (EC 1.1.1.49), glutamate dehydrogenase (EC 1.4.1.4), and chymotrypsin-like protease (EC 3.4.21.1) were identified by the methods of Uriel (52), and those possessing D-lactate dehydrogenase (EC 1.1.1.27) activity were identified by the method described by Smyth et al (50). Other enzyme activities were detected by incubating pressed immunoplates individually in the following incubation mixtures (final volumes, 20 Analytical procedures.…”

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides

“…Additional chromatophore polypeptide components have been observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6,15, 19), but the function of these components has remained largely unknown because this procedure results in the loss of any biological activity. Recently, crossed immunoelectrophoresis (CIE) has been applied to the study of bacterial membranes (7,14,17,18). Since nondenaturing detergents are used for membrane disruption before CIE, both enzymatic and antigenic activities are usually retained, thus permitting a functional analysis of membrane components.…”

Neisseria gonorrhoeae possesses two nicotinamide adenine dinucleotide-independent lactate dehydrogenases

“…It has remained unclear whether separate, membrane-bound iLDH enzymes or a single iLDH species capable of utilizing both isomers is expressed. The presence of two iLDH isoenzymes was supported by the observation that two membrane-associated, enzymatically active iLDH components were detected following crossed immunoelectrophoresis [8]. In contrast, Bhatnagar et al [9] and Hendry et al [10] obtained results interpreted to indicate the presence of a single iLDH species.…”