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.

Summary report on the ISOBM TD-6 workshop: analysis of 20 monoclonal antibodies against Sialyl Lewisa and related antigens. Montreux, Switzerland, September 19-24, 1997.

The ISOBM TD-6 Workshop is the first international workshop on monoclonal antibodies against the Sialyl Lewisa (SLea) antigen. Eight research groups participated in a blind study to characterize the epitope binding, relative affinity and performance in immunoradiometric assays, of a panel of 20 monoclonal antibodies. The antibodies were tested against a diverse panel of neoglycoconjugates, purified antigens and human serum pools from gastrointestinal malignancies. Epitope specificities were determined for the majority of antibodies in the panel. Cross-reactivity with related saccharide structures was noted in several antibodies. Overall, the results of the TD-6 Workshop show further development of SLea immunoassays may yield yet more specific assays for the detection and management of gastrointestinal and other malignancies.

Multicenter evaluation of the Bayer Immuno I CA 15-3 assay.

We conducted a multicenter evaluation of the analytical and clinical features of the automated Bayer Immuno 1 CA 15-3 assay and compared assay performance to two manual tests. Results of the 10-day imprecision study of the Bayer Immuno 1 assay pooled across four evaluation sites and three lots of reagent produced total CV < or = 4%. Lot-to-lot reproducibility for 26 different lots of reagents and calibrators manufactured over a 2-year period was demonstrated (CV, 1.1%). Results for the Bayer Immuno 1 assay correlated well with the Biomira TRUQUANT BR 27.29 and Centocor CA 15-3 RIAs (r > or = 0.94). The upper limit of the reference interval for the Bayer Immuno 1 assay was 35.9 kilounits/L (35.9 units/mL); values were similar for all methods. Longitudinal monitoring of healthy women yielded assay values with an average CV of 11% and 21% for the Bayer Immuno 1 and Biomira assays, respectively. The Bayer Immuno 1 assay demonstrated the analytical features, intermethod correlation, and long-term performance characteristics that are essential for longitudinal monitoring of breast cancer patients.

Bayer Immuno 1 PSA Assay: an automated, ultrasensitive method to quantitate total PSA in serum.

The Bayer Immuno 1 PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV < or = 3.4% and a biological detection limit of 0.03 microgram/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 micrograms/L for males 50-59 years and 3.3 micrograms/L for males 60-69 years, to 4.6 micrograms/L for males > or = 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5-100%. Specificity of 60-70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx PSA Assay over the range 0.0-6,238 micrograms/L (Y = 1.10 x + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum.

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 approximately 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.

Lack of connectivity between the induced and autoimmune repertoires of lpr/lpr mice.

It has been proposed that the autoantibody-secreting cells active during autoimmune diseases are derived from B cells initially responding to environmental antigens. In order to test the relationship between the antigen-induced and autoimmune repertoires, we monitored the fate of antigen-activated idiotypically defined B cells present in mice that developed the systemic lupus erythematosus (SLE)-like syndrome associated with the lpr mutation. Mice homozygous for both the A/J-derived Igh and Ig kappa region haplotypes and the lpr mutation were bred. Immunization of these mice with p-azophenylarsonate (Ars)-protein conjugates elicited the idiotypic components (IdCR) characteristic of the A/J anti-Ars response and did not interfere with the spontaneous development of the lpr-mediated autoimmune disease. These Id/lpr mice provided an ideal system for studying the relationship between the exogenously and endogenously induced responses because: (1) VHIdCR antibodies have been shown to bind autoantigens in vitro; and (2) serological and molecular reagents exist which can identify and monitor VHIdCR antibody production as disease progresses. Serum samples and hybridoma cell lines derived from non-immune as well as Ars-keyhole limpet haemocyanin (KLH)-immunized Id/lpr mice were monitored for idiotype expression as well as Ars and ssDNA reactivity at various stages of disease progression. We found that antibodies utilizing the VHIdCR gene segment did not preferentially contribute to the autoantibody pool. Moreover, even when IdCR B-cell clones were expanded by deliberate immunization with Ars-KLH, Ars non-binding variants were only rarely detected among the activated B-cell populations of diseased mice. These results indicate that there is only minimal overlap between the VHIdCR conventional and autoimmune repertoires.

Defining the structural correlates responsible for loss of arsonate affinity in an IDCR antibody isolated from an autoimmune mouse.

Immunization of the autoimmune mouse strain (M x A) Id/lpr with Ars-KLH, has been shown to elicit a prolonged anti-Ars IdCR response similar to that found in A/J mice. Cell fusion of splenocytes from a diseased mouse previously immunized with Ars-KLH resulted in a monoclonal antibody, 1-52.30, that was found to express the strain A major cross-reactive idiotype, but failed to bind Ars. Nucleotide sequence analysis demonstrated that 1-52.30: (a) used the "canonical" combination of gene segments associated with this idiotype, and (b) exhibited a pattern of somatic mutation consistent with selection for high affinity Ars binding. Two amino acids, VL 91 and 93, were mutated in 36-65, the germline equivalent of the IdCR antibodies, to 1-52.30-like residues (91G-->D, 93T-->M). The results of the mutagenesis showed that changing a single light chain residue, VL 91, from glycine to aspartic acid, resulted in a dramatic loss of Ars binding activity.

Monoclonal antibody to SER immune suppressor detects polymeric forms of haptoglobin.

A series of monoclonal antibodies has been developed which is directed to a serum immunosuppressive factor, known as suppressive E-receptor factor (SER). SER, purified from the body fluids of cancer patients, is a polymeric form of haptoglobin, which is 100-1,000 times more potent an immunosuppressor than normal plasma haptoglobin and is immunochemically analogous to the neonatal form. Unlike the neonatal haptoglobin found in cord blood, SER, however, does not contain bound-hemoglobin. One group of monoclonal antibodies described in this study detects the polymeric forms of haptoglobin (SER) under non-denaturing conditions, but fails to recognize SER under the denaturing conditions of SDS-PAGE. A second group of monoclonal antibodies reacts only with the alpha subunit of haptoglobin but not with the beta subunit; in contrast, the commercially prepared polyclonal antisera to haptoglobin react with both the alpha and beta subunit. The average level of SER in normal human plasma (n = 19) was 1.0 micrograms/ml, regardless of age or sex. Since macrophages appear to secrete SER but do not synthesize haptoglobin, SER may represent an oxidized form of plasma haptoglobin generated from macrophages activated during an inflammatory response. These studies suggest that SER may be a negative feed-back regulator of immune response produced by activated macrophages.

An analogy between fetal haptoglobin and a potent immunosuppressant in cancer.

In spite of the numerous reports indicating the presence of humoral immunosuppressive factors in cancer patients, only a few of these factors have been biochemically identified. Furthermore, their role as effective immunosuppressors in vivo remains to be established. Our laboratory has attempted to isolate and identify the major immunosuppressive factor in the malignant effusions derived from ovarian and lung cancer patients. We have previously demonstrated that the Mr 52,000 immunosuppressive factor isolated from the ascites fluid of an ovarian cancer patient inhibited T-dependent immune responses in vivo and in vitro including the inhibition of E-rosetting. Thus, this immunosuppressive factor was named "suppressive E-receptor" (SER). Our current study demonstrates that this SER factor purified from malignant effusions derived from ovarian, lung, or head and neck cancer patients had a common component which dissociated equally into Mr 38,000-42,000 and 17,000-19,000 moieties on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under vigorous reducing conditions. Electroelution of these two components followed by a limited amino acid sequence determination revealed these two components to have N-terminal amino acid sequences identical to the beta and alpha 2 subunits of normal adult haptoglobin. Immunoelectrophoresis of SER using a polyclonal antiserum to neonatal cord blood demonstrated that SER, unlike normal haptoglobin, has slower electrophoretic mobility than the normal adult haptoglobin. Western blotting analysis of SER separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions failed to recognize a monoclonal antibody directed specifically to SER. However, this monoclonal antibody exclusively reacted with the SER separated by an analytical polyacrylamide gel electrophoresis gel under nondenaturing conditions while normal adult haptoglobins or purified but denatured haptoglobin obtained from the same malignant fluid as SER all failed to react with this antibody. Thus, SER appears to bear an additional epitope(s) that is absent in normal adult haptoglobin. Since the SER as well as the neonatal haptoglobin have at least 100 to 1000-fold more potent immunosuppressive activity than the normal adult haptoglobin, this additional epitope(s) present in SER may be responsible for the potent immunosuppressive property of SER.

Reactivity of the B6.C-H-2bm-1 mutant to the wild-type tumor EL4: characterization of the serological response.

Hyperimmunization of B6.C-H-2bm-1 (H-2bm-1), a congenic mutant of C57Bl/6J (B6), with the C57Bl lymphoma EL4 resulted in the induction of antibodies with apparent EL4 specificity. EL4 reactivity was demonstrable in H-2bm-1 anti-EL4 sera by complement-mediated cytotoxicity, absorption, and enzyme-linked immunosorbent assay. By these same serological tests, H-2bm-1 anti-EL4 serum was found to be nonreactive with B6 normal lymphoid cells, embryonic fibroblasts, and two fibrosarcomas previously induced in B6 mice by methylcholanthrene. These data suggest that the serological response of H-2bm-1 to EL4 is directed against tumor-associated antigens on EL4. These findings indicate that congenic mutants which differ from the wild-type strain at MHC Class I subloci, but which do not evoke serological responses to MHC components, may provide convenient sources for preparing serological reagents directed against tumor-specific antigens.