Simultaneous isolation of intestinal IgA and IgG from rabbits infected intraduodenally with Vibrio cholerae 01 by combined lectin affinity chromatography involving jacalin and protein A.
Immunoglobulins of IgA and IgG isotypes are formed in host intestines after enteric infections with bacteria such as Vibrio cholerae. A method has been developed for the separation of intestinal immunoglobulins from rabbits immunized intraduodenally with live V. cholerae cells by sequential affinity chromatography on immobilized jacalin and immobilized protein A. The jacalin-Sepharose 4B bound IgA was desorbed with 0.5 M galactose. The protein A-Sepharose 4B bound IgG was desorbed with 0.1 M citrate buffer. The quality of the isolated IgA and IgG was checked by immunodiffusion in agarose gels, enzyme-linked immunosorbent assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The procedure described here is rapid and convenient. It can be used to isolate intestinal IgA and IgG in pure form.
Kabir
S
S
Department of Immunology, Karolinska Institute, Stockholm, Sweden.
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D004386:Duodenum / Q000276:immunology* / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007070:Immunoglobulin A / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose; D013205:Staphylococcal Protein A / Q000378:metabolism*; D014734:Vibrio cholerae / Q000276:immunology*
D000907:Antibodies, Bacterial; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D037102:Lectins; D037121:Plant Lectins; D013205:Staphylococcal Protein A; C043696:jacalin; D012685:Sepharose