Serological studies of actionomyces israelii by crossed immunoelectrophoresis: standard antigen-antibody system for A. israelii

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

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confidence: 99%

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Smyth¹,

Friedman‐Kien²,

Saltón³

1976

“…The standard system based on StAgSL and StAbI comprised six reference precipitins, coded SL1 to SL6, the system based on StAgHCl comprised five reference precipitins, coded HCll to HCl5, the system based on StAgurea comprised five reference precipitins, coded ureal to urea5, and the system based on StAgTCA comprised one reference precipitin, coded TCA1. The reference precipitins coded SL1, SL2, SL4, SL5, HCl1, HCl2, HCl3, ureal, urea2, and urea-4 were specific for their respective systems, whereas the other reference precipitins of the systems showed reactions of partial or total identity to each other in CLE analysis (17). The system based on StAgSL and StAbII comprised 10 reference precipitates, coded 1 to 10, of which six were identical to those revealed in the system based on StAgSL and StAbI.…”

Section: Methodsmentioning

confidence: 94%

“…Reference antigen-antibody systems for A. israelii, were prepared from antigen/water extracts on standard preparations of sonic lysates, StAgSL, and chemical extracts from whole cells by 0.2 M hydrochloric acid (StAgHCl), 4 M urea (StAgurea), and 10% (vol/vol) trichloroacetic acid (StAgTCA), and standard rabbit antisera to formalin-treated whole cells (StAbI) and cell lysates (StAbII) of A. israelii (ATCC 12103 and WVU 307). The procedure for preparing standard antigens, standard antisera, and the immunochemical characteristics of the systems have been described elsewhere (17).…”

Section: Methodsmentioning

confidence: 99%

“…Crossed immunoelectrophoresis (CIE) with intermediate gel (1) proved to be a powerful method in combination with a reference precipitin system for characterization of antibodies in mucocutaneous candidiasis (3), Pseudomonas aeruginosa infections (18), and Mycobacterium leprae infections (2). The objectives of this study were to apply CIE with intermediate gel technique to the serological diagnosis of human actinomycosis and classification of A. israelii, using recently developed standard antigen-antibody systems as reference (17).…”

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confidence: 99%

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Serological studies of Actinomyces israelii by crossed immunoelectrophoresis: taxonomic and diagnostic applications

Holmberg¹,

Nord²,

Wadström³

1975

Self Cite

Crossed immunoelectrophoresis (CIE) with intermediate gel was applied to the serological analysis of Actinomyces israelii to develop a test with high efficiency in the laboratory diagnosis of human actinomycosis and classification of A. israelii. Recently developed standard antigen-antibody systems for A. israelii by CIE were used as reference. The reference systems were based on standard preparations of cytoplasmic and whole cell-associated antigens of A. israelii and a standard immunoglobulin G pool purified from rabbit antisera to formalintreated whole cells and cell lysates of A. israelii. The specificity of the standard antigens for A. israelii was evaluated in CIE studies by screening for antibodies to components of the antigens in rabbit antisera raised against related bacteria. The standard system for A. israelii based on cytoplasmic antigens formed speciesspecif'ic precipitins, whereas antisera raised against A. naeslundii and/or Propionibacterium acnes precipitated components of' the other standard antigens. As a result of these analyses, the standard system for A. israelii based on 10 cytoplasmic antigens was used as reference for CIE studies to detect humoral antibodies to A. israelii in sera from nine patients with actinomycosis. All the sera from the patients formed at the time of diagnosis one or more precipitins in terms of the 10 reference precipitins. Up to five precipitins were found in single sera. Follow-up studies covering a period of one-half year after treatment showed a gradually decreased precipitin response in the course of' time. In control sera from patients with newly diagnosed tuberculosis, nocardiosis, deep Candida infection, and aspergillosis, and in sera from healthy blood donors, no antibodies were detected with specificity for the reference antigens.

“…Several previous studies have been conducted on the antigenic composition of A. israelii (3-5, 8,12,13,20). Most investigations have emphasized the importance of wall carbohydrate antigens as possible serotype-specific markers.…”

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confidence: 99%

Identification and preliminary characterization of a major heat-stable surface antigen of Actinomyces israelii by two-dimensional (crossed) immunoelectrophoresis

Ayakawa¹,

Williams²,

Kenny³

1983

We have identified, employing two-dimensional (crossed) immunoelectrophoresis, a major heat-stable surface antigen in Actinomyces israelii with an electrophoretic mobility of 0.86 compared to bovine albumin. The surface component was protease sensitive and non-dialyzable and was found in relatively large amounts in two serotype 2 strains of A. israelii (WVU 307 and ATCC 23860). Serotype 1 strains either lacked antigen 0.86 (ATCC 12103 and CDC X522) or contained small quantities of a cross-reacting antigen which was not identical to antigen 0.86 (CDC W855). Thus, antigen 0.86 may be useful in the serotaxonomy of A. israelii. Our data also suggest the possibility that a greater degree of antigenic heterogeneity exists within the species than can be accommodated within the two currently recognized serotypes.serotypes does not accurately reflect the degree of antigenic heterogeneity within the species. 11 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from