A method for blocking antigen-independent binding of human IgM to frozen tissue sections when screening human hybridoma antibodies.
Using an indirect immunoperoxidase technique antigen independent binding of both human monoclonal and polyclonal IgM was found to a wide range of frozen sections of normal and malignant human glandular epithelia. Identical binding was found using dimeric human IgA (dIgA), whereas no binding was found with monomeric human IgA or human IgG. Secretory component (SC) was found to be the component in these tissues mediating antigen-independent binding of human IgM and dIgA antibodies. A method for the blocking of this antigen-independent binding of human IgM and dIgA was evaluated. Frozen sections of tissues containing SC were blocked with antibody to endogenous immunoglobulin and preincubated with rabbit anti-secretory component antibody (anti-SC) before applying the human monoclonal antibody. This treatment blocked the binding of control polyclonal and monoclonal human IgM to sections of SC-containing tissues such as respiratory and colonic epithelia. The influence of anti-SC on the binding of dIgA could not be established due to interactions between the anti-SC antibody and the IgA preparation. Using this method human hybridoma supernatants containing IgM could be readily screened for reactivity with frozen tissue sections from patients with colo-rectal cancer. This approach is recommended for the screening of human IgM monoclonal antibodies on frozen human tissue sections.
Ditzel
H
H
Department of Medical Microbiology, University of Odense, Denmark.
Erb
K
K
Nielsen
B
B
Borup-Christensen
P
P
Jensenius
J C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D004848:Epithelium / Q000276:immunology; D005615:Freezing; D006801:Humans; D006825:Hybridomas; D007124:Immunoenzyme Techniques; D007070:Immunoglobulin A / Q000276:immunology; D007075:Immunoglobulin M / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D011817:Rabbits; D012635:Secretory Component / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007075:Immunoglobulin M; D012635:Secretory Component