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Derivation of a monoclonal anti-murine IL-4 antibody specific for an epitope expressed on denatured molecules
1Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Monoclonal antibodies to interleukin 4 (IL-4) were generated by immunization with recombinant IL-4 and screening by binding to IL-4 adsorbed to plastic surfaces. Three antibodies were obtained that scored well in this assay and one, 13E1, was studied in detail. It was very effective in detecting IL-4 by Western blotting whereas a neutralizing anti-IL-4 antibody, 11B11, was 50-100-fold less sensitive as a detecting agent. Sequential immunoprecipitation and biosynthetic labelling studies indicated that the 11B11 and 13E1 epitopes are largely expressed on different forms of IL-4. 13E1 was able to detect cytosolic IL-4 both by immunohistochemical and flow cytometric analysis of fixed cells. This was routinely successful in an insect cell line (Sf9) expressing large amounts of IL-4 as a result of infection with a recombinant 'IL-4 baculovirus'. Although stimulated D10 cells could also be shown to express IL-4 in their cytosol, positive results were not obtained in all such studies and we have failed to detect IL-4 production by normal T cells using this method. This antibody may have substantial value in detecting IL-4 by Western blots and as a tool to analyze the biosynthesis of IL-4. With suitable improvement in sensitivity, it also may prove valuable in the detection of IL-4 in the cytosol of individual cells.
Monoclonal antibodies to interleukin 4 (IL-4) were generated by immunization with recombinant IL-4 and screening by binding to IL-4 adsorbed to plastic surfaces. Three antibodies were obtained that scored well in this assay and one, 13E1, was studied in detail. It was very effective in detecting IL-4 by Western blotting whereas a neutralizing anti-IL-4 antibody, 11B11, was 50-100-fold less sensitive as a detecting agent. Sequential immunoprecipitation and biosynthetic labelling studies indicated that the 11B11 and 13E1 epitopes are largely expressed on different forms of IL-4. 13E1 was able to detect cytosolic IL-4 both by immunohistochemical and flow cytometric analysis of fixed cells. This was routinely successful in an insect cell line (Sf9) expressing large amounts of IL-4 as a result of infection with a recombinant 'IL-4 baculovirus'. Although stimulated D10 cells could also be shown to express IL-4 in their cytosol, positive results were not obtained in all such studies and we have failed to detect IL-4 production by normal T cells using this method. This antibody may have substantial value in detecting IL-4 by Western blots and as a tool to analyze the biosynthesis of IL-4. With suitable improvement in sensitivity, it also may prove valuable in the detection of IL-4 in the cytosol of individual cells.