A simple SDS-PAGE immunoblotting technique using an enhanced chemiluminescence detection system to identify polyclonal antibody responses to complex cryptosporidial antigen preparations following a monoclonal antibody retest and image overlay technique.
SDS-PAGE immunoblotting is a standard immunochemical technique. We describe a modification of this technique to facilitate the identification of polyclonal antibody responses to complex antigen preparations. The technique utilises an enhanced chemiluminescence (ECL) detection technique where an immunoblot is initially tested with polyclonal antibodies. The polyclonal antibodies are 'stripped' from the nitrocellulose support and the immunoblot is retested with a monoclonal antibody. Polyclonal antibody responses to individual components also recognised by the monoclonal antibody are identified by superimposing the two photographic-film images. This technique has the advantage that antigens can be identified across an individual blot despite distortions which may have occurred. An example of the use of this technique is described in which antibody responses in human sera against antigens from the coccidian parasite Cryptosporidium are identified.
Patel
S
S
Public Health Laboratory Service (PHLS) Food Hygiene Laboratory, Central Public Health Laboratory, London, UK.
McLauchlin
J
J
Casemore
D P
DP
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan; D003457:Cryptosporidiosis / Q000276:immunology; D003458:Cryptosporidium / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005069:Evaluation Studies as Topic; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008163:Luminescent Measurements; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D012967:Sodium Dodecyl Sulfate