Biomedical subjects
B W Maidment
Publications and source records attributed to B W Maidment.
Isoelectric focusing analysis of soluble immune complexes bound to protein A-Sepharose.
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Recovery of immunologically reactive antibodies and antigens from breast cancer immune complexes by preparative isoelectric focusing.
A new technique for the recovery of immunologically reactive antibodies and putative antigens from breast cancer-associated immune complexes was described. Soluble immune complexes were isolated from extracellular fluids by 2.5% polyethylene glycol fractionation and affinity chromatography on Protein A:Sepharose CL-4B. The immune complexes bound to immobilized Protein A were then subjected to preparative isoelectric focusing. The dissociated immunoglobulins and putative antigens were recovered from the appropriate pH regions of the preparative isoelectric focusing gel. G-type immunoglobulins recovered from immune complexes isolated from a breast cancer patient bound to the recovered putative antigen(s) and three breast cancer cell culture lines, but these immunoglobulins did not bind to carcinoembryonic antigen or other cancer cell culture lines originating in colon, pancreas, prostate, or lymphoblast. The recovered putative antigens had isoelectric points between pH 3.0 and 5.0 and molecular weights of 20,000 and 42,000.
Isoelectric focusing--a new approach to the study of immune complexes.
Isoelectric focusing was used to examine antigen-antibody complexes. BSA : anti-BSA complexes were dissociated and antigen was separated from antibody based upon differences in pI value by isoelectric focusing (pH gradient 3--10). The recovered proteins were homogeneous as determined by immunodiffusion and polyacrylamide gel electrophoresis analyses. The technique was capable of resolving 6.4 microgram of BSA : anti-BSA complexes. More than 90% of the complexes applied to isoelectric focusing gels were dissociated and entered the gels. It was further demonstrated, by the use of complexes containing enzymes (acid phosphatase or alkaline phosphatase), that the dissociated enzymes retained their native pI as well as enzymatic activities. The isoelectric focusing technique, therefore, represents a new and effective approach to the dissociation of antigen-antibody complexes.