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Quantitative differentiation of the haptoglobin-related gene product from haptoglobin in human plasma: a possible
1Department of Microbiology, Boston University School of Medicine, MA 02118.
The haptoglobin-related gene product (HGRP) having greater than 90% sequence homology with plasma haptoglobin, has recently been implicated as a tumor maker of recurrent breast cancer. Therefore, availability of a monoclonal antibody which is specific to the HGRP and ensuing ELISAs to quantitate the HGRP without cross-reactivity with plasma haptoglobin will allow determination of the clinical value of the HGRP in human plasma under various pathophysiological conditions. A peptide representing the first 34 residues of the N-terminal portion of the HRGP was synthesized and conjugated to keyhole limpet hemocyanin (KLH) to immunize Balb/c mice. Hybrid clones that produced specific antibodies to HRGP were screened with the same peptide but conjugated to bovine serum albumin (HRGP-BSA). From 12 hybridoma clones that recognized HGRP-peptide, we obtained one IgM producing clone (27.5) by limiting dilution technique that selectively reacted with HGRP-peptide with minimal cross-reactivity with haptoglobin. Using a separate monoclonal antibody, clone 21.7, which is directed to the alpha subunit of haptoglobin, we developed an enzyme-linked immunosorbent assay (ELISA) to determine the quantities of the HGRP in human plasma, which will allow assessment of HGRP as a clinical marker of malignancy.
The haptoglobin-related gene product (HGRP) having greater than 90% sequence homology with plasma haptoglobin, has recently been implicated as a tumor maker of recurrent breast cancer. Therefore, availability of a monoclonal antibody which is specific to the HGRP and ensuing ELISAs to quantitate the HGRP without cross-reactivity with plasma haptoglobin will allow determination of the clinical value of the HGRP in human plasma under various pathophysiological conditions. A peptide representing the first 34 residues of the N-terminal portion of the HRGP was synthesized and conjugated to keyhole limpet hemocyanin (KLH) to immunize Balb/c mice. Hybrid clones that produced specific antibodies to HRGP were screened with the same peptide but conjugated to bovine serum albumin (HRGP-BSA). From 12 hybridoma clones that recognized HGRP-peptide, we obtained one IgM producing clone (27.5) by limiting dilution technique that selectively reacted with HGRP-peptide with minimal cross-reactivity with haptoglobin. Using a separate monoclonal antibody, clone 21.7, which is directed to the alpha subunit of haptoglobin, we developed an enzyme-linked immunosorbent assay (ELISA) to determine the quantities of the HGRP in human plasma, which will allow assessment of HGRP as a clinical marker of malignancy.