Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-alpha. Implications for antibody screening.
Three enzyme-linked immunosorbent assays (ELISA) were compared in the initial screening of some 400 hybridoma supernatants for antibodies to a recombinant human interferon-alpha subtype, 4a (IFN-alpha 4a). In these assays, (i) the antigen was coated directly to polystyrene microtitre plates (ELISA-PS), (ii) the antigen was coated directly to nitrocellulose (ELISA-NC), or (iii) the antigen and mouse antibody were reacted in solution and the resulting complex immobilized to a solid support precoated with polyclonal rabbit anti-IFN-alpha antibody (ELISA-SW). The ELISA-PS detected eight antibodies, the ELISA-NC 15 and the ELISA-SW 18. The interferon specificity of the MAbs detected by each of the ELISAs was confirmed by neutralization of IFN-alpha antiviral activity and Western immunoblotting analysis. The results suggest that in ELISAs, the presentation of an antigen and its recognition by antibodies is substantially influenced by the method used in the immobilization of antigen and the type of solid support used. The ELISA-SW proved optimal for screening hybridoma supernatants for antibodies to IFN-alpha 4a, and is recommended for screening for antibodies to other soluble antigens.
Overall
M L
ML
Centre for Molecular Biology and Medicine, Monash University, Clayton, Australia.
Marzuki
S
S
Hertzog
P J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000145:classification / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002474:Cell-Free System; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000032:analysis* / Q000145:classification; D007370:Interferon Type I / Q000276:immunology*; D008567:Membranes, Artificial; D051379:Mice; D008807:Mice, Inbred BALB C; D011137:Polystyrenes; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D007370:Interferon Type I; D008567:Membranes, Artificial; D011137:Polystyrenes; D011994:Recombinant Proteins; D003101:Collodion