Biomedical subjects

R L Vessella

Publications and source records attributed to R L Vessella.

At least 37 records · Page 2Linked to original sources

PubMed2000

Probability of prostate cancer detection based on results of a multicenter study using the AxSYM free PSA and total PSA assays.

OBJECTIVES: The determination of the percentage of free prostate-specific antigen (%fPSA) enhances the specificity of prostate cancer (CaP) detection. This study was undertaken to assess the performance of %fPSA in differentiating benign prostate disease from CaP and to determine the CaP probability estimates using the AxSYM Free PSA and AxSYM Total PSA assays. METHODS: In this prospective study, 297 men, 50 years old or older, with a total PSA level between 4 and 10 ng/mL and a nonsuspicious digital rectal examination were enrolled at 10 clinical sites. All subjects underwent at least sextant prostate biopsies to establish the diagnosis. fPSA and total PSA (tPSA) levels were determined using the AxSYM Free PSA and AxSYM Total PSA assays. Percent fPSA values were compared with tPSA values to determine the appropriate cutoffs for prostate biopsy and to calculate the CaP probability estimates. RESULTS: The strongest predictor of CaP in a logistic regression model was %fPSA (odds ratio 2.29), which contributed significantly more than age or tPSA to the predictive model. In this study population, a %fPSA cutoff of 26.4% would have detected 96% of subjects with CaP (sensitivity) and would have eliminated 27.4% of unnecessary biopsies (specificity). CaP probability estimates ranged from 9% to 69% and increased as the %fPSA value decreased. Men with a %fPSA level of 10% or lower had a 69% probability of CaP, and men with a %fPSA level of greater than 26% had a 9% probability of CaP. CONCLUSIONS: Percent fPSA values can help differentiate CaP from benign prostate disease and reduce unnecessary biopsies in 27% of men 50 years old or older whose digital rectal examination was normal and whose tPSA level was between 4 and 10 ng/mL. A %fPSA result can assist the physician and patient in determining the probability of CaP and assessing the need for prostate biopsy.

Biopsy↗

PubMed2000

Differential expression of osteonectin/SPARC during human prostate cancer progression.

The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP.

Antibodies, Monoclonal↗

PubMed1999

Development of new biotin/streptavidin reagents for pretargeting.

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.

Affinity Labels↗

PubMed1999

Analysis and sorting of prostate cancer cell types by flow cytometry.

BACKGROUND: Prostate tumor heterogeneity as manifested by differential expression of markers can be attributed to multiple types of cancer cells populating a tumor. Does the composition differ between primary tumor and metastasis? How can one isolate the different cancer cell types to study? What is the relationship among cancer cell types? METHODS: Flow cytometry keying on the prostate epithelial cell surface markers CD57 and CD44 was applied to analyze and sort single cells prepared from tumor tissue samples by collagenase digestion. In normal tissue, CD57 is found on luminal cells and CD44 on basal cells. RESULTS: CD57(+) and CD44(+) cells were sorted from various prostate tumor tissue specimens. The CD57(+) cancer cell type was found to predominate in primary tumors, while the CD44(+) cancer cell type was found to predominate in two visceral metastases. All tumors could be characterized by a ratio of CD57(+) and CD44(+) cancer cells. CONCLUSIONS: Two types of prostate cancer cells, CD57(+) and CD44(+), were identified. The finding that most primary tumors contain a predominantly CD57(+) cancer cell population agrees with the argument that cancer cells arise from the transformation of CD57(+) luminal cells. However, CD44(+) cancer cells are also present in some primary tumors; and in some metastases, they, and not CD57(+) cells, constitute a predominant population.

Abdominal Neoplasms↗

PubMed1999

Interleukin-8 serum levels in patients with benign prostatic hyperplasia and prostate cancer.

OBJECTIVES: Using arbitrarily primed polymerase chain reaction (AP-PCR) ribonucleic acid (RNA) fingerprinting, we discovered a messenger RNA (mRNA) that encoded the cytokine interleukin-8 (IL-8) that was up-regulated in the peripheral blood leukocytes (PBLs) of patients with metastatic prostate cancer (CaP) compared with similar cells from healthy individuals. We compared the total prostate-specific antigen (PSA) levels, the free/total (f/t) PSA ratios, and the immunoreactive IL-8 serum concentrations in patients with either biopsy-confirmed benign prostatic hyperplasia (BPH) or CaP. METHODS: The sera from 35 apparently healthy normal volunteers and 146 patients with biopsy-confirmed BPH and CaP obtained from two academic centers were retrospectively examined to determine the serum levels of IL-8, total PSA (tPSA), and the f/t PSA ratio. Logistic regression and trend analysis statistical methods were used to assess the results. RESULTS: Normals (n = 35), BPH patients (n = 53), patients with clinical Stages A to C CaP (n = 81), and patients with metastatic CaP (n = 1 2) had mean levels of IL-8 of 6.8, 6.5, 15.6, and 27.8 pg/mL, respectively. The IL-8 serum concentrations correlated with increasing CaP stage and also differentiated BPH from clinical Stages A, B, C, or D CaP better than tPSA and performed similarly to the f/t PSA ratio. The combination of the IL-8 levels and f/t PSA ratios using multivariate logistic regression analysis distinguished BPH from Stages A, B, C, or D CaP or only Stages A and B with a receiver operating characteristic area under the curve of 89.8% and 87.5%, respectively (P <0.0001). CONCLUSIONS: The IL-8 serum concentration in our clinically well-defined patient sample was independent of the f/t PSA ratio as a predictor of CaP. When test samples are controlled for extraneous clinical origin of inflammation or infection, the combination of the IL-8 and f/t PSA assay results may offer an improved approach for distinguishing BPH from CaP.

Adult↗

PubMed1999

Effects of phenylbutyrate on proliferation and apoptosis in human prostate cancer cells in vitro and in vivo.

Phenylbutyrate (PB) is a potent differentiating agent and currently under investigation for the treatment of prostate cancer (CaP) and other malignancies. We have studied the impact of PB in vitro and in vivo on differentiation, proliferation and apoptosis in the LNCaP and LuCaP 23.1 prostate cancer xenograft models. In vitro we found that i) PB increased PSA secretion/cell, ii) inhibited cell proliferation in a time- and dose-dependent manner resulting in a cell cycle arrest in G1-phase and iii) induced apoptosis at concentrations of 2.5 mM after 3 days of treatment. In PB treated animals tumor growth stabilized or regressed. Combination of castration and PB treatment had a synergistic antiproliferative effect. The growth-inhibitory and differentiating properties and a low toxicity profile of PB provide rationale for further clinical studies in patients with CaP.

Androgens↗

PubMed1999

High prevalence of antibodies against HERV-K10 in patients with testicular cancer but not with AIDS.

Human endogenous retrovirus K10 (HERV-K10) env and gag expression has been detected in placenta, embryonic tissue, and cell lines. By transfection, these sequences have been expressed in insect cells and developed into serological assays, revealing HERV-K10 antibodies in patients with testicular cancer. Patients with AIDS are at an increased risk for testicular cancer and frequently reactivate latent infections. We postulated that HERV-K10 seroprevalence might be increased with HIV infection or AIDS. Stored, frozen serum samples from 52 patients with testicular cancer (8 patients with HIV and 30 patients with samples near the time of diagnosis) and 84 controls (40 patients with HIV) were diluted 1:40 and tested by immunofluorescence against SF158 cells transfected with HERV-K10 env [ENV1.9(+)] or gag (pACGAG). Seroprevalence rates were compared cross-sectionally in cases and controls, excluding those with indeterminate results (3 of 30 cases and 7 of 84 controls), and also were examined longitudinally in the cases before or after diagnosis of testicular cancer. Seroprevalence to HERV-K10 Env or Gag was 17 of 27 testicular cancer patients (63%) around the time of diagnosis, compared to 4 of 77 controls (5%; P < 0.0001). Seroprevalence was similar (50% to 60%) with seminoma, teratocarcinoma, or embryonal carcinoma, and it was not increased with HIV infection in either cases (33%) or controls (3%). HERV-K10 antibodies were detected in 12 of 19 cases (63%) more than 6 months before seminoma diagnosis, as well as in four cases with residual or recurrent malignancy more than 1 month after initial diagnosis. Thus, HERV-K10 antibodies are detected frequently with testicular cancer and seem to resolve rapidly with effective therapy of the malignancy. Antibody reactivity also occurs in approximately 5% of controls, perhaps because of nonspecific or cross-reactive epitopes. HIV and AIDS were not associated with HERV-K10 antibodies, thus, leaving their higher risk of testicular cancer unexplained.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Study of free and complexed prostate-specific antigen in mice bearing human prostate cancer xenografts.

BACKGROUND: Our objective was to evaluate five preclinical prostate cancer (CaP) xenograft models to determine whether (1) prostate-specific antigen (PSA) formed complexes in murine serum, (2) the percentage of free PSA (f-PSA) was characteristic of a given xenograft line, and (3) the percentage of f-PSA was similar to that in the patient at time of tumor harvest. Our fourth objective was to identify which murine serpin(s) bind(s) to PSA in vivo. METHODS: Xenografts were established from metastatic foci. The percentage of f-PSA, and total PSA (t-PSA) in serum of animals bearing CaP xenografts was determined by immunoassay. Size exclusion high-performance liquid chromatography and Western blots were used to evaluate the presence of PSA complexes in murine serum. Edman degradation was used to determine the N-terminal sequence of complexed proteins. RESULTS: PSA was detected as both free and complexed forms in murine serum from all mice bearing the CaP xenografts. Three xenografts (related sublines) produced PSA that resulted in low mean percentages of f-PSA (1.9-6.4%). In sera from the other two xenografts, the mean percentages of f-PSA were high (>25%); patient sera, where available at time of tumor acquisition, were in agreement. Western blots showed that murine protease inhibitors formed complexes with PSA. Edman degradation yielded a sequence with 80% homology over 15 amino acids with that of murine alpha1-protease inhibitor (alpha1-PI). CONCLUSIONS: Our data have shown that the majority of PSA secreted by these CaP xenografts complexes in murine serum with a protease inhibitor with high homology to murine alpha1-PI and that the percentage of f-PSA is a characteristic of each xenograft line tested, which is in agreement with patient values at time of tumor harvest. These CaP xenografts offer opportunities for study of human PSA biology and phenomenology.

Animals↗

PubMed1998

Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression.

The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription-PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.

Azacitidine↗

PubMed1998

LNCaP produces both putative zymogen and inactive, free form of prostate-specific antigen.

BACKGROUND: Our objective was to investigate the presence of prostate specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) mRNA and protein in prostate cancer cell lines, and the complexing characteristics of expressed PSA. METHODS: RT-PCR, immunohistochemistry, and Western blots were employed. Trypsin treatment of PSA was performed to establish the possible presence of an activatable form of PSA. RESULTS: ACT mRNA and protein were detected in LNCaP, PC-3, and DU 145 by RT-PCR and by immunohistochemistry, respectively. Only LNCaP cells were positive for PSA mRNA and protein. LNCaP expressed approximately 30% active PSA, approximately 40% putative zymogen form of PSA, and approximately 30% stably inactive PSA. CONCLUSIONS: We have shown that the majority of PSA expressed by LNCaP cells is present in free, noncomplexed forms in the conditioned media. A portion (40%) can be activated by trypsin, while the rest is stably inactive PSA. LNCaP cells may serve as a source of the "unreactive" PSA present in prostate cancer patients' serum.

Biomarkers, Tumor↗

PubMed1998

Serum percent free prostate-specific antigen in metastatic prostate cancer.

OBJECTIVES: To define the serum prostate-specific antigen (PSA) isoform profile in patients who have prostate cancer but do not have a prostate gland, that is, men who have had a previous radical prostatectomy (RP) and subsequently persistent disease as evidenced by elevated PSA. PSA can be reliably measured in the serum in two major isoforms: PSA complexed to alpha1-antichymotrypsin and uncomplexed free PSA (fPSA). Multiple investigations have illustrated the usefulness of the free/total PSA proportion (percent fPSA) in differentiating prostate cancer from benign prostate disease in patients who still have their prostate gland in situ. METHODS: Sera were evaluated from 52 men who underwent RP and postoperatively had increased PSA. fPSA and total PSA (tPSA) concentrations were determined using the Abbott AxSYM PSA assays. Percent fPSA was calculated for all patients. RESULTS: Median tPSA was 5.45 ng/mL (range 0.93 to 214.99). Median fPSA was 0.69 ng/mL (range 0.11 to 54.93); the median percent fPSA was 13.3% (range 3.9% to 62.9%). There were 27 (52%) patients with percent fPSA less than 15%, 25 (48%) patients with greater than 15%, and 7 (13%) with greater than 30%. No significant relationship was found between percent fPSA and grade, stage, and severity of disease. Percent fPSA was significantly increased in patients who received hormonal, radiation, or combination treatment versus those who received no treatment (P = 0.02 to 0.0007). CONCLUSIONS: Serum percent fPSA in men after RP with persistent prostate cancer encompasses a wide range of values with no clear stratifying factor or factors. These observations and further serial studies in patients with progressive metastatic disease may be important in determining the mechanism(s) for lower percent fPSA in men with newly diagnosed prostate cancer.

Aged↗

PubMed1998

Engineering the isoelectric point of a renal cell carcinoma targeting antibody greatly enhances scFv solubility.

BACKGROUND: The murine A6H monoclonal antibody targets a cell surface antigen associated with renal cell carcinoma with high specificity and excellent biodistribution properties. Tumor to blood ratios of > 40:1 have been achieved in clinical studies. OBJECTIVES: In order to generate an antibody engineering system that would allow the construction of improved derivatives for diagnostics and therapeutics, a single-chain Fv antibody (scFv) derived from A6H was constructed. The initial single-chain Fv, constructed with a cysteine residue and hexa-histidine sequence at the C-terminus, displayed a limited solubility of 100 microg/ml at pH 7.4. The low solubility and refolding yield of the original single-chain Fv required that a more soluble variant be designed and constructed. STUDY DESIGN: We hypothesized that lowering the pI of the scFv antibody away from the physiological range would yield a more soluble antibody. A derivative was thus subsequently engineered with five glutamic acid residues followed by the cysteine and hexa-histidine residues. The cysteine was included to provide a conjugation site for future radiolabeling studies. RESULTS: The redesigned A6H single-chain Fv has a predicted pI of 6.1, relative to 7.5 for the native scFv. The redesigned A6H scFv displayed a greatly enhanced solubility of > 15 mg/ml at pH 7.4. Both the original scFv and the redesigned single-chain Fv exhibited a strong tendency to form dimers and soluble high molecular weight aggregates. The monomer and disulfide bonded dimer were separated from the aggregates and complete cell binding isotherms were obtained, demonstrating that the purified A6H scFv retains much of the activity of the parent monoclonal. CONCLUSION: The addition of glutamic acid to the C-terminus of poorly soluble scFv antibodies could provide a straightforward avenue for improving their solubility properties. The increased solubility of the A6H scFv allowed the purification of the monomeric and dimeric species from the soluble aggregated species.

Amino Acid Sequence↗

PubMed1998

A novel monoclonal antibody 107-1A4 with high prostate specificity: generation, characterization of antigen expression, and targeting of human prostate cancer xenografts.

Monoclonal antibodies with high specificity for prostate tissue are of interest for prostate cancer research and treatment. Reactivity and specificity of a new murine monoclonal antibody, 107-1A4, was assessed by immunohistochemistry, ELISA and indirect immunofluorescence (IDIF). 107-1A4 stained all normal and malignant prostate tissue specimens while reactivity to non-prostate tissue was limited to the tubules of the normal kidney and renal cell carcinoma. Twenty two human cell lines were included in the reactivity survey; only the immunogen prostate cancer line LNCaP reacted with 107-1A4. Seminal plasma proteins PSA, PAP, PSMA, and PSP-94 were determined not to be the 107-1A4 antigen.

Journal Article↗

PubMed1998

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.

Aged↗

PubMed1998

The value of a reverse transcriptase polymerase chain reaction assay in preoperative staging and followup of patients with prostate cancer.

PURPOSE: The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an extremely sensitive technique of detecting cells expressing prostate specific antigen (PSA). Controversy exists regarding the ability of peripheral blood PSA RT-PCR testing to reflect pathological staging or treatment outcome. We examine the phenomenology of RT-PCR results in patients with prostate cancer, with particular emphasis on the RT-PCR test before and after radical prostatectomy, and correlations with pathological staging and treatment outcome. MATERIALS AND METHODS: Peripheral blood was obtained from a wide variety of patients with and without prostate cancer, including before and after radical prostatectomy. After ribonucleic acid isolation, complementary deoxyribonucleic acid was generated and amplified with a hot-start technique. RT-PCR results were compared with pathological stage, Gleason score, tumor volume and disease-free status. Correlations between preoperative and postoperative RT-PCR tests were also made. RESULTS: The RT-PCR test was positive in 1 of 56 controls (1.8%) without suspicion of prostate cancer. A positive test was obtained in 12 of 65 men (18.5%) with a suspicion of prostate cancer but a negative biopsy. Before radical prostatectomy a positive test was obtained in 13 of 75 men (17.3%) with pT2 disease versus 10 of 46 (21.7%) with pT3 disease. There was no significant difference in serum PSA, Gleason score or tumor volume between the men with positive or negative results. With repetitive testing an increasing percentage of men had at least 1 positive test preoperatively. With a median followup of 8 months 6 of the 7 patients in whom radical prostatectomy failed had had negative RT-PCR before treatment. Of patients with known metastatic disease or failed primary treatment a positive test was obtained in 32 to 75%. Radical prostatectomy and prostate needle biopsy appeared to have a negligible effect on RT-PCR tests immediately following these procedures. Following radical prostatectomy results were variable but many men who are RT-PCR positive preoperatively become RT-PCR negative postoperatively. CONCLUSIONS: The PSA RT-PCR test in our laboratory cannot be used preoperatively to predict pathological stage of prostate cancer or treatment failure. Most cases that are positive preoperatively become negative postoperatively. While increasing tumor burden increases the likelihood of positive tests, there appears to be significant sampling error associated with the use of this test in the peripheral blood.

Adult↗

PubMed1997

Cell-cell interaction in prostate gene regulation and cytodifferentiation.

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57+ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57+ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57+ cells are reconstituted with stromal cells. The CD44+ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44+ cells are cocultured with stromal cells. Our studies show that cell-cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.

Androgens↗

PubMed1997

A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis.

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.

Adenosine Triphosphate↗

PubMed1997

Characterization of 10 new monoclonal antibodies against prostate-specific antigen by analysis of affinity, specificity and function in sandwich assays.

While prostate-specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti-PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f-PSA) and the PSA-alpha-1-antichymotrypsin complex (PSA-ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti-PSA monoclonal antibodies (MAbs). Apparent dissociation constants (Kd) of MAbs were determined by direct ELISA yielding Kd-0.2-164.0 nM. Western blots suggested that 3 of the MAbs (60-1A2, 60-8A2 and 17-1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f-PSA and PSA-ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f-PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants.

Animals↗