Determination of monoclonal antibody specificity by immunoadsorption and western blotting.
A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).
Schuh
R
R
GSF-Institut für Immunologie, Munich, Germany.
Kremmer
E
E
Ego
E
E
Wasiliu
M
M
Thierfelder
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D015703:Antigens, CD / Q000032:analysis / Q000276:immunology; D019011:Antigens, CD7; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D001666:Binding Sites, Antibody; D015153:Blotting, Western / Q000379:methods; D018956:CD5 Antigens; D015922:Complement C1q / Q000276:immunology; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D051381:Rats; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D019011:Antigens, CD7; D000945:Antigens, Differentiation, T-Lymphocyte; D018956:CD5 Antigens; D015922:Complement C1q