Zeqi Zhou

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

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.

Technicon Immuno 1 PSA assay measures both free and alpha-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis.

In this study, we investigated the immunoreactivity of the Technicon Immuno 1® PSA assay, a monoclonal‐polyclonal sandwich immunoassay, with free and ACT‐complexed PSA. Assay calibrators prepared with free PSA (standard immuno 1 calibrators) and calibrators consisting of 90% ACT‐complexed and 10% free PSA (90:10 calibrators) yielded virtually identical PSA recoveries at all concentrations tested. Concentrations of total PSA at ∼4 and 10 ng/mL, prepared in varying ratios of free PSA to PSA‐ACT complex, recovered from 92–104% at 4 ng/mL and from 98–102% at 10 ng/mL. Additionally, an excellent correlation of serum total PSA values from a panel of 40 prostatic cancer patient samples was obtained using calibration curves generated with the standard Immuno 1 calibrators or the 90:10 calibrators. These results demonstrate that the Technicon Immuno 1® PSA assay measures free and ACT‐complexed PSA on an equal molar basis.

Equivalent recognition of free and act-complexed PSA in a monoclonal-polyclonal sandwich assay is conferred by binding specificity of the monoclonal antibody

The Bayer Immuno 1TM PSA Assay measures free and ACT‐complexed PSA on an equimolar basis, although it uses a monoclonal antibody (MM1) for capture and polyclonal antibodies for detection. Competitive inhibition studies using antibodies directed at various epitopes on PSA and PSA‐ACT demonstrated that the capture antibody, MM1, does not bind to free PSA simultaneously with antibodies against Epitope E which is exposed only in free PSA. Affinity studies showed that the affinity constants of MM1 for both free PSA and PSA‐ACT are similar. One explanation for the properties of MM1 is that it precludes the binding of antibodies to Epitope E due to steric hindrance. Alternatively, the binding of MM1 causes a conformation change within the free PSA molecule, so that Epitope E is altered in a way that causes a loss of binding affinity. The unusual properties of MM1 are responsible for the equimolar response of this monoclonal‐polyclonal sandwich assay for free and ACT‐complexed PSA. J. Clin. Lab. Anal. 12:242–249, 1998.

Analysis of Monoclonal Antibodies to Prostate-Specific Antigen: Reactivity with Native and Recombinant Prostate-Specific Antigen

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.