Purification of an IgM Antibody Response‐Enhancing Factor to the Alpha 1–6 Epitope of Native Dextran from Serum and Supernatants from T‐Cell Hybridomas

Abstract

We have found that some hybrids produced by fusing AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized CBA mice produce factors that affect the primary CBA IgM PFC response. By the use of HPLC-DEAE chromatography, sometimes combined with affinity chromatography, we have purified from hyperimmune anti-Dx serum, from ascites-growing T hybrids, and from normal CBA serum a family of factors that specifically enhance the anti-Dx IgM response. They show specificity for either Dx or anti-Dx, they may contain an Iak determinant, and they are genetically restricted in their function. The factors from all three sources are found in the same fractions after separation and move in fractions without Ig contamination. The affinity-purified factor was eluted at an NaCl concentration of 0.15-0.20 M. On the basis of these characteristics we believe that we have separated a class of T cell-derived helper factors from serum. The concentration of the factor in serum increases after hyperimmunization with Dx. As a specificity control we show that the same sera contain a natural anti-SRBC IgM response-enhancing factor. It co-separates with the anti-Dx-enhancing factor, but they can be distinguished by their preferential binding to their own antigens.

ELISA Assay for the Detection of a Dextran‐Binding Product Secreted by a T‐Cell Hybrid Th 1

“…We therefore sought with ELISA for la*" determinants in the Dx-bound Th 1 material with an anti-la'' serum but without significant binding. This result was not unexpected, since we have previously demonstrated that the Th 1-derived factor could be absorbed onto CBA but not onto C57B1 spleen cells [5], which suggests that the specificity of the factor is directed to recognizing la determinants.…”

“…The same anti-Th 1-positive fractions of anti-Dx CBA sera or of Th I material have been shown to affect the magnitude of the primary anti-Dx response in CBA but not in C57BI/6 mice [5], indicating that they contain a Dx binding, Ia/IgH chain loci-restricted. anti-Dx response-regulating factor.…”

“…Further we have previously separated from hyperimmune CBA anti-Dx scrum a Dx-binding (Sephadex affinity chromatography), non-Ig factor that regulated the magnitude of the IgM anti-Dx primary response, if given simultaneously with or before the immunization. A similar factor material from normal CBA serum could also be enriched with Sephadex affinity chromatography, but there was a clear concentration gradient of the material in the normal and immune sera [5). This was taken as evidence that the immunization with Dx resulted not only in the increased numbers of the regulatory cells eoncerned but also in the increased production and recovery of the cell-produced factors in the serum.…”

“…Furthermore, the effects on the anti-Dx responses could vary from suppression to enhancement. This lack of reproducibly dear specific effects of the Th 1 products on the anti-Dx response, despite our efforts to separate the Dx-specific component from the nonspecific ones, interfered with our studies on the Th I product [5].…”