Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.     

Bioassay of prostate-specific antigen (PSA) using microcantilevers

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.     

Fluorine-18 labeling of monoclonal antibodies and fragments with preservation of immunoreactivity

A new method is reported for labeling proteins with the positron-emitting nuclide 18F. Initially, 4-[18F]-fluorobenzylamine was prepared in two steps from aqueous [18F]fluoride in high yield. The 18F acylation agent was formed by reaction of this product with disuccinimidyl suberate. Overall yields for the 4-[18F]fluorobenzylamine succinimidyl ester ([18F]SFBS), decay corrected to the end of cyclotron bombardment, were about 30% in a synthesis time of 60 min. After a 15-min reaction, 30-45% (decay corrected) of the [18F]SFBS could be coupled to intact antibodies and their F(ab')2 and Fab fragments. Coupling yields were dependent on protein concentration but not reaction time. HPLC purification of [18F]SFBS was necessary to obtain optimal coupling efficiency and immunoreactivity. The immunoreactivities of 18F-labeled F(ab')2 and Fab fragments of an antimyosin antibody were 89 +/- 5% and 75 +/- 9%, respectively. Biodistribution studies in normal mice demonstrated similar in vivo behavior of 18F-labeled antibody fragments and those labeled with 125I by using N-succinimidyl 3-[125I]iodobenzoate. These results indicate that this method may be useful for labeling monoclonal antibodies and other proteins and peptides with 18F.     

Serum level of prostate-specific antigen (PSA) in women with breast cancer

ObjectiveTo identify the diagnostic role of total and free prostate-specific antigen (TPSA and FPSA) in breast cancer in women.MethodsBlood samples of 55 women with breast cancer were prospectively analyzed for PSA before and after breast surgery, with a control group of 82 healthy women.ResultsTotal and free PSA levels were significantly higher in women with breast cancer (preoperatively) than in healthy women (P < 0.001). Both serum TPSA and FPSA showed a significant decline in their pre-surgical values after surgical removal of the tumor (P < 0.001). A significant proportion of breast cancer patients (83.6%) had free PSA as the predominant molecular form in serum as compared to 0% of controls and 1.8% of postoperative groups (P < 0.001). TPSA and FPSA levels were significantly associated with younger age and earlier cancer stage, whereas no significant association was found between these two variables and FPSA as a predominant molecular form.ConclusionsThis study indicated a clinical significance of preoperative measurement of serum TPSA and FPSA in the diagnosis of women with breast cancer, and may be a useful marker for monitoring the response to treatment.     

Further characterization of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Two monoclonal antibodies (24.14, 61A12) to Echinococcus granulosus Antigen 5 and two (31.15 and 39B3) to Antigen B were further characterized using modified sheep hydatid cyst fluid antigens (SHCF) in ELISA. None of these four monoclonals were directed against carbohydrate or lipid epitopes of SHCF antigens since they all reacted strongly with periodate or lipase-treated SHCF. On the other hand, they appeared to recognize SHCF determinants of protein nature as protease treatment of SHCF destroyed binding with the monoclonals. Anti-Antigen B monoclonals 31.15 and 39B3 showed strong reaction with boiled SHCF and anti-Antigen 5 monoclonal 24.14 did not. However, the second anti-Antigen 5 monoclonal 61A12 also reacted with boiled SHCF suggesting that some epitopes of Antigen 5 are heat stable. 24.14 and 61A12 may recognize a similar epitope of Antigen 5 whereas 39B3 may be against an epitope of Antigen B different from that recognized by 31.15.     

The molecular and biochemical characterization of mutant monoclonal antibodies with increased antigen binding.

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.     

Lymphoma associated antigen (LAA)--a unique biomarker for lymphomas.

A lymphoma associated antigen (LAA) isolated from pooled lymph nodes of confirmed Hodgkin's and non-Hodgkin's lymphomas has been purified and characterized. Using a xenogenic rabbit anti-serum, enzyme-linked immunosorbent assay (ELISA) and RIA were developed for LAA. LAA was detected in the sera of all confirmed lymphomas, the test being negative for normals, for patients with benign lymphadenitis and various other types of cancers. Except for a very few false positive results, no false negative was observed. LAA was identified in urine, CSF, saliva and gastric juice of a few lymphoma patients, and the test proved to be of diagnostic potential, as for a few patients it had a lead time of a few months over the histological diagnosis. In order to render the LAA test more precise and specific, monoclonal antibodies were generated by both in vitro and in vivo immunization procedures. Seven monoclonals were generated, viz. 7D6, 7D2, 7G2, 7C5, 6G2, 23B7 and 23G11, which exhibited cytoplasmic staining of frozen sections of malignant lymphoid tissues of B cell derived non-Hodgkin's lymphomas. Two of these monoclonal antibodies, 7D6 and 23B7, revealed strong cytoplasmic staining of frozen sections, impression smears and cytospin specimens of B cell non-Hodgkin's lymphomas. The reactivity was very weak or negative for T cell lymphomas. The test was negative for Hodgkin's disease and controls. These results were confirmed by dot blotting, immunoprecipitation and immunofluorescence study. By ELISA with a sensitivity of 15 ng/ml, serum LAA levels for lymphomas were in the range 72-1250 ng/ml. LAA could not be detected in the sera of normals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution

Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors. In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells. Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells. The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A. Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats. The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14. The binding was always higher with 16F12 than with 2G4F8 supernatants. No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species. Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage. It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance.     

Characterization of a human granulocyte differentiation antigen (CDw15) commonly recognized by monoclonal antibodies

Many different anti-human granulocyte monoclonal antibodies recognize the same carbohydrate antigen which contains the trisaccharide 3-fucosyl-N-acetyllactosamine. The antigen is expressed mainly on two cell surface glycoproteins of molecular weights around 105 K and 160 K which are apparently not members of the LFA-1 family of proteins. Although specific for granulocytes in blood, the antigen is expressed on a wide range of non-haemopoietic cell types.     

Monoclonal anti-idiotopic antibodies against myasthenia-inducing anti-acetylcholine receptor monoclonal antibodies. Preponderance of nonparatope-directed antibodies affecting antigen binding

To analyze components of the idiotypic network in experimental autoimmune disease, we produced 17 isogeneic anti-idiotopic monoclonal antibodies (anti-Id) against two experimental autoimmune myasthenia gravis-producing anti-acetylcholine receptor (anti-AChR) monoclonal antibodies. We studied the binding of five of the anti-Id to the anti-AChR monoclonal antibodies bearing the complementary idiotopes (Id-mAb). They bound with Kd values ranging from 0.06 to 0.86 nM, values comparable to those of Id-mAb:AChR complexes (0.26 and 0.34 nM). All of the anti-Id tested moderately inhibited the binding of AChR to Id-mAb, whereas for each anti-Id, AChR either strongly inhibited anti-Id binding or had no effect on anti-Id binding. Hence, the inhibition of Id-mAb:AChR binding by anti-Id was not reciprocal with the inhibition of anti-Id:Id-mAb binding by AChR. For each anti-Id, the relative affinities of anti-Id and AChR for Id-mAb together with the lack of symmetry of inhibition by anti-Id compared to inhibition by AChR indicate that these two "ligands" are not competitive inhibitors. Consequently, anti-Id and AChR do not bind to overlapping sites on the Id-mAb, suggesting that the observed inhibition is mediated allosterically. This may be a common mechanism of anti-Id:Id binding, which would have important implications for the mechanism of anti-Id-induced suppression.     

Human seminal vesicle-specific antigen is a substrate for prostate-specific antigen (or P-30)

Human seminal vesicle and prostatic fluids were obtained separately and reconstituted in vitro to test the hypothesis that proteolytic enzymes of prostatic origin would degrade seminal vesicle-specific antigen (SVSA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis to follow the fate of SVSA over time, we found that upon mixing the two secretions, SVSA was converted to forms of intermediate and low molecular weight identical to transformations seen in normal liquefied ejaculates. Diisoproprylfluorophophate, a serine protease inhibitor, prevented this degradation, indicating serine protease involvement in the proteolysis of SVSA. Prostate-specific antigen (PSA; also known as P-30), recently identified as a serine protease, was examined for its ability to mimic the effects of prostatic fluid on SVSA. Purified PSA catalyzed degradation of SVSA to produce proteolytic fragments that comigrated and were immunologically related to SVSA fragments produced by prostatic fluid. Purified PSA in the presence of serine protease inhibitors was unable to degrade SVSA. These results demonstrate that SVSA is a substrate for PSA during human semen liquefaction.     

Potentiation of anti-carcinoembryonic antigen immunotoxin cytotoxicity by monoclonal antibodies reacting with co-expressed carcinoembryonic antigen epitopes

The initial step in ricin A-chain (RTA)-immunotoxin-mediated cell cytotoxicity involves binding to the target cell Ag through the antibody moiety. One of the factors influencing this is the affinity of the antibody component for the target cell Ag. Multiple epitopes on carcinoembryonic Ag have been mapped providing a range of mAb of known specificity. These have been used to show that the cytotoxicity of an immunotoxin containing RTA conjugated to an anti-carcinoembryonic Ag mAb (228-RTA) is potentiated by mAb recognizing different epitopes. The potentiating antibodies also increased the level of target cell binding of antibody 228. Cross-linking of cell bound antibody was not involved because monovalent fragments of a potentiating antibody were effective. The potentiating antibodies modified the binding affinity of 228 antibody increasing the t1/2 of antibody at the tumor cell surface. This increased the dwell time of cell bound antibody and using conjugates of 228 linked to albumin-tetramethylrhodamine it was shown to enhance conjugate endocytosis. These investigations indicate that enhanced antibody affinity leads to increased endocytosis of bound immunoconjugate and potentiates cytotoxicity.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

The human lymphocyte function-associated (HLFA) antigen and a related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies

The structural and functional relations between the alpha- and beta-subunits of the human lymphocyte function-associated antigen (HLFA) and the human Mac-1 antigen (HMac-1) have been analyzed with the use of five monoclonal antibodies that react with these proteins. The specificities of these antibodies were examined by immunoprecipitation of proteins from 125I-labeled cells and purified HLFA and HMac-1 antigens. Three antibodies reacted with the Mr 95,000 common beta-subunit of the proteins, and also co-precipitated the Mr 175,000 HLFA alpha-subunit, the Mr 165,000 HMac-1 alpha-subunit, and a third polypeptide alpha-subunit of Mr 150,000. The other antibodies were specific to noncross-reactive epitopes present on the alpha-subunits of HLFA or HMac-1. These specificities were confirmed in sequential immunoprecipitation studies. Peptide mapping showed that the beta-subunits of HLFA and HMac-1 were identical, whereas the two alpha-subunits differed considerably. The HLFA alpha-subunit-specific monoclonal antibody inhibited phytohemagglutinin stimulation, the mixed lymphocytes reaction, cytolytic T lymphocyte-mediated killing, and tetanus toxoid stimulation, but did not affect natural killer cell-mediated killing or complement receptor type 3 function. The HMac-1 alpha-subunit-specific monoclonal antibody inhibited complement receptor type 3 function but had no effect on T cell or natural killer cell functions. Three monoclonal antibodies to the beta-subunit inhibited all functions tested, including T cell, natural killer cell, and complement receptor type 3 activities. The results suggest that the functions of the HLFA and HMac-1 molecules may be determined by the alpha-subunit, and that the common beta-subunit also bears functionally important epitopes.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

Species-specific monoclonal antibodies for a membrane antigen(s) in all developmental forms of Trypanosoma cruzi

Two monoclonal antibodies (designated as TCF48 and TCF87 were raised against Trypanosoma cruzi, strain Tulahuen, Both antibodies reacted with all developmental forms of several different strains of Trypanosoma cruzi. The antibodies showed no detectable cross-reactivity with other species of Trypanosomatidae, so far examined. TCF48 and TCF87 were classified as immunoglobulin subclasses IgG1 and IgG2b, respectively. Apparent molecular weight of the corresponding antigen(s) to these monoclonal antibodies was 25,000 in amastigotes and epimastigotes, and 25,000 and 24,000 in trypomastigotes, as determined by the Western immunoblotting analysis. This antigen appeared to be located at the plasma membrane and the flagellum ofT. cruzi. However, no evidence supported the localization of the epitope(s) at the external surface of the live cell. Since this antigen reacted with the sera from the chronically infected mice, these monoclonal antibodies may be useful in the study of Chagas' disease.