Solid-phase antigen binding by purified immunoproteins from antigen-specific monoclonal T cell hybridomas.
We have developed two distinct solid-phase immunoassays for the detection of antigen binding activity by products of antigen binding T cell hybridomas in the absence of MHC. Two suppressor T cell hybridomas studied (34s-18 and 34s-704) are specific for keyhole limpet hemocyanin, a protein antigen, and the other suppressor T cell hybridoma (51H7D) binds specifically to the arsonate hapten. We have adapted these hybridomas to growth in serum-free medium and have isolated molecules with antigen binding activity both from the cell membranes and from the culture fluid in which the cells had been grown. The antigen binding molecules (ABM) produced by the KLH-specific hybridomas bound best to native hemocyanin; binding was decreased when KLH was denatured by reduction and alkylation and no binding was found to an arthropod (Limulus) hemocyanin. The arsonate binding hybridoma, on the other hand, produced molecules specific for this hapten; they showed no capacity to bind KLH. The antigen binding molecules affinity-purified from all three T hybridomas have intact masses of either 145,000, 67,000 or 48,000 when run in SDS-PAGE under non-reducing conditions. Following reduction, ABM resolve in SDS-PAGE into a complex of polypeptide chains having apparent masses of 65,000, 56,000 and 49,000, with either a pair of bands at 26,000 and 22,000, or with a single band at 32,000, which is consistent with the size of translation products of mRNA previously isolated from these hybridomas. Two of the hybridomas, 34s-18 and 34s-704, used for isolation of antigen binding products in this study, were previously reported to lack detectable rearranged gamma or beta genes and therefore to lack expression of the alpha/beta or gamma/delta heterodimers. The antigen binding molecules react in solid-phase immunoassay with some antibodies specific for variable (first framework) region and joining (J) region peptide sequences predicted from T cell receptor gene sequence. Furthermore, the affinity-purified antigen binding molecules from mouse T cell hybridomas cross-react in ELISA with goat anti-rabbit IgG and not with protein G, thus allowing the use of these commercially available reagents in standard laboratory assays. Interestingly, ABM anchored in intact cell membranes, which could be shown to specifically bind antigen, did not cross-react with goat anti-rabbit IgG, indicating that the cross-reactive moiety is not detectable when the ABM are in this situation.(ABSTRACT TRUNCATED AT 400 WORDS)
Bristow
C L
CL
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Marchalonis
J J
JJ
D000818:Animals; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D011863:Radioimmunoassay; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*
D000939:Epitopes; D011948:Receptors, Antigen, T-Cell