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Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Epitope mapping analysis was performed on 53 antibodies submitted to the ISOBM TD-3 PSA Workshop. Western blotting and N-terminal amino acid sequencing, using both native and recombinant human prostate-specific antigen (rPSA), identified four different epitope groups for native PSA. Under reducing conditions native PSA was not recognized by 18/53 antibodies suggesting they reacted with conformation-dependent epitopes. Nine other antibodies reacted with a rPSA polypeptide doublet of 34-35 kD corresponding to different rPSA glycoforms. From sequence mapping studies 22/53 antibodies bound epitopes within amino acid residues 25-85, 3/53 antibodies bound to epitopes within residues 86-220, while 10/53 antibodies bound epitopes within residues 221-261. These results indicate that there are multiple immunogenic epitopes localized on the PSA molecule.

We evaluated 56 monoclonal antibodies (MAbs), submitted to the ISOBM TD-4 Workshop, for changes in binding following desialylation of the MUC1 molecule and for epitope specificity. Antibody binding of MAbs was assayed by an ELISA method using microtiter plates coated with the MUC1 mucin obtained from supernatants of the ZR75-1 cell line. The MUC1 mucin was desialylated directly on the plate by treatment with neuraminidase. For each MAb, binding to untreated mucin was compared over a range of antibody concentrations. The concentration at which binding was half-maximal (K50) was determined for all antibodies whose binding reached saturation in the assay. Results showed that K50 values for MAb binding to untreated MUC1 mucin varied from 10(-10) to 10(-6) M. These data suggest that MAbs to MUC1 mucin bind with a broad range of intrinsic affinities. Desialylation was found to have variable effects on antibody binding, in that binding was either increased, decreased, or unchanged. No relationship was found between the apparent affinities for untreated mucin and changes in binding following desialylation. Among the 56 Workshop MAbs, 33 were found reactive with synthetic peptides which mimic the MUC1 tandem repeat. We determined the epitope specificity of the 33 MAbs by competitive binding using 10 amino acid peptides corresponding to various regions of the 20-amino acid tandem repeat domain of MUC1. All antibodies which recognized epitopes in the 1-10 amino acid region of the tandem repeat showed increased binding to desialylated mucin. Antibodies to other peptide epitopes showed no consistent pattern of change in binding following desialylation. Our results suggest that sialic acid residues on the MUC1 mucin may contribute either positively or negatively to antibody binding. In addition, our results suggest that improved antibody selection methods could provide MAbs with improved selectivity for cancer-derived mucin compared with mucin from normal tissues. This could form the basis of improved biomarker assays for breast cancer.

At maximally active concentrations with 20-min exposure, insulin and phorbol myristate acetate (PMA) stimulated hexose transport in 3T3-L1 adipocytes by 11- and 2-fold, respectively. The potential role of phosphorylation of the glucose transporter (GT) in these stimulations was investigated by the isolation of GT through immunoprecipitation from ortho[32P]phosphate-labeled 3T3-L1 adipocytes. It was found that there was no significant 32P incorporation into GT from basal adipocytes after 2- or 18 h-labeling in the presence of 0.5 mCi of 32Pi/ml. Furthermore, under these labeling conditions, insulin treatment for 1, 4, or 30 min failed to stimulate the phosphorylation of GT. Also, there was no detectable phosphate incorporation into GT upon reversal of insulin-stimulated hexose transport by the removal of insulin (half-time for reversal approximately 8 min). In contrast to these results, exposure of adipocytes to PMA (1 microM) for 20 min elicited a phosphorylation of GT to the extent of about 0.1 phosphate/GT molecule. Exposure of cells to both insulin and PMA resulted in a 3-fold increase in the level of phosphate in GT compared to that seen with PMA alone. Possibly this increase is due to the translocation of GT to the plasma membrane where it is a better substrate for activated protein kinase C. Stimulation of hexose transport was the same with the combined treatment of insulin and PMA compared to that seen with insulin alone. These results indicate that neither a change in the phosphorylation state of the GT nor activation of protein kinase C is involved in the mechanism by which the insulin receptor stimulates glucose transport.