Disc-crossed immunoelectrophoresis. A simple ‘laying-on’ technique permitting the use of commercially available agarose

Ekwall, K.; Söderholm, J.; Wadström, Torkel

doi:10.1016/0022-1759(76)90100-9

Cited by 13 publications

(3 citation statements)

References 27 publications

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“…Neither prolonged electrophoresis in the first dimension nor reduction of the Triton X-100 concentration in the extract improved separation. Using dextran, Mr 20,000, as a marker, these batches of Miles agarose were shown to possess rather high electroendoosmotic flow (38), confirming data recently obtained by Ekwall et al on other batches of Miles agarose (16).…”

Section: Resultssupporting

confidence: 85%

“…Electroendoosmotic flow characteristics of agarose preparations (5,16,38,47) were a major determining factor in achieving resolution of membrane-associated antigens extracted with Triton X-100 from gonococcal envelopes. Such problems were not reported with crossed immunoelectrophoresis of membrane antigens from Acholeplasma laidlawii (Bio-Rad agarose), Micrococcus lysodeikticus (Miles agarose), and rat liver microsomes (Behring agarose) (28, 37, terized by clustering of immunoprecipitates around the antigen well in the initially antibody-free portion of the gel (51).…”

Section: Discussionmentioning

confidence: 99%

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Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Smyth¹,

Friedman‐Kien²,

Saltón³

1976

Infect Immun

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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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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Smyth¹,

Friedman‐Kien²,

Saltón³

1976

Infect Immun

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“…Alternatively, antigens were separated in Davis or discontinuous SDS-PAGE disc gels (diameter, 6 mm), and then the gels were sliced into four strips with a Longitudinal Gel Slicer II (Miles Research Products). The Davis gel strips were then sliced in half with a 15cm razor blade (Bio-Rad Laboratories), soaked in water for 60 min, and laid on a standard rocket immunoelectrophoresis plate for the second-dimension electrophoresis (12). The SDS-PAGE strips were soaked for 60 min in 1.5% Triton X-100 in water to remove SDS and renature the antigen and then electrophoresed on a rocket plate which contained 1.5% Triton X-100 in the cathodal agarose strip.…”

Section: Methodsmentioning

confidence: 99%

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis

Dasch¹,

Samms²,

Williams³

1981

Infect Immun

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Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 560C for 30 min but not by 50°C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-x-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide pattems on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.

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Precipitation and Related Immunoassay Techniques

Nilsson¹

1981

Immunoassays for the 80s

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Disc-crossed immunoelectrophoresis. A simple ‘laying-on’ technique permitting the use of commercially available agarose

Cited by 13 publications

References 27 publications

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis

Precipitation and Related Immunoassay Techniques

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