Anti-alpha 1-6 epitope, specificity of a T-cell hybrid-secreted factor: affinity- and ion-exchange chromatographic separation.
We have studied the specificity of the products of a "T-cell" hybridoma, Th 1, a fusion product of AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized mice, which has been shown to affect the anti-Dx but also the anti-SRC response from culture supernatants and ascitic fluids. The anti-Dx-affecting material was separated from unspecific effector molecules by Sephadex affinity chromatography combined with HPLC DEAE chromatography and gel filtration. The activities of fractions were tested for their effects on anti-Dx, anti-SRC, and anti-SSS-III IgM responses. We show that the anti-Dx response-affecting material binds to Sephadex. Its Ig contamination can be reduced by two DEAE chromatographies at pH 6 and 8.1. At pH 8.1 it starts eluting with 0.13 M NaCl, but is still contaminated with materials that affect the anti-SRC and to a smaller extent the anti-SSS-III response. On gel filtration it localizes in the area of 100-40 kDa. The effects of the active material on anti-Dx IgM varied from suppression to enhancement. The details of that effect are largely unknown but three other findings further confirm the Dx specificity of Th 1 products. The growth of Th 1 in mice induces the production of anti-Dx IgA, detectable in their sera with ELISA. The priming of mice with Th 1 products affects the magnitudes of anti-Dx IgM PFC responses to the subsequent immunization with Dx with or without the product. The binding to Dx of material from in vivo active fractions can be verified in the ELISA with an antiserum produced against Th 1.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003911:Dextrans / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006462:Hemolytic Plaque Technique; D006825:Hybridomas / Q000032:analysis / Q000378:metabolism*; D007126:Immunoglobulin Allotypes / Q000096:biosynthesis; D007075:Immunoglobulin M / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D011135:Polysaccharides, Bacterial / Q000276:immunology; D012756:Sheep; D013601:T-Lymphocytes / Q000032:analysis / Q000378:metabolism*
D003911:Dextrans; D000939:Epitopes; D007126:Immunoglobulin Allotypes; D007075:Immunoglobulin M; D011135:Polysaccharides, Bacterial; C041999:alpha-1,6-dextran; C026604:pneumococcal polysaccharide, type III