Free PSA forms in prostatic tissue and sera of prostate cancer patients: analysis by 2-DE and western blotting of immunopurified samples.

OBJECTIVE: The isoform pattern of free prostate-specific antigen (fPSA) from sera of patients with prostate cancer (PCa) and no evidence of prostate cancer (NPCa), cancerous and non-cancerous tissues and seminal plasma, have been compared, above all regarding the degree of sialylation, with the aim to show a better discrimination of PCa and NPCa. DESIGN AND METHODS: The samples have been immunopurified and analyzed by two dimensional gel electrophoresis and western blotting. It was investigated which patterns were obtained when looking for the fPSA and the (-5/-7)proPSA (precursor form) before and after desialylation. RESULTS: The fPSA sialylation and the proPSA pattern in cancerous and non-cancerous prostate tissues were similar to each other and only slightly different from PCa and NPCa sera. The different fPSA isoforms observed could not be due solely to differences in the degree of sialylation, because different fPSA and (-5/-7)proPSA precursor isoforms were still present after complete desialylation. CONCLUSIONS: Although slight differences in the fPSA and (-5/-7) proPSA glycosylation and isoform pattern were observed, they were not large enough to be considered to improve PCa diagnosis.

Detection of anti-EPO antibodies in human sera by a bridging ELISA is much more sensitive when coating biotinylated rhEPO to streptavidin rather than using direct coating of rhEPO.

Sensitive and efficient methods for detecting anti-erythropoietin (anti-EPO) antibodies are needed for analysis and, above all, for large scale screening of human serum samples. ELISA is an attractive alternative to labor-intensive radioimmunoprecipitation assays but apparently conflicting reports question its sensitivity. We sought to resolve this issue by directly comparing different reported ELISA approaches to determine whether rhEPO-coating methods affect detection of anti-EPO antibodies. Investigators reporting low sensitivity had used ELISAs in which rhEPO was directly coated to microtiter plates while the high sensitivity ELISA used plate-bound streptavidin to bind biotinylated rhEPO. Using anti-EPO positive human sera, our results confirmed a large (100- to 300-fold) difference in sensitivity between the ELISAs and suggested that the inferiority of the low sensitivity ELISA was caused by the direct coating of rhEPO which may disrupt epitopes by masking recognition sites or introducing conformational changes. Thus, a bridging ELISA can be an appropriate and effective system for antibody analysis and screening of human sera with high sensitivity and specificity but only if performed with streptavidin binding of biotinylated antigen. This finding may also be more generally applicable to the detection of antibodies against other protein antigens.

Electrophoretic subforms of free prostate-specific antigen in serum as promising diagnostic tool in prostate cancer diagnostics.

OBJECTIVES: To assess the diagnostic performance of the major electrophoretic subforms of free prostate-specific antigen (PSA), named F2 and F3, for differentiating between benign and malignant prostatic disease in men with total PSA (tPSA) concentrations up to 10 microg/L. METHODS: In sera from 50 patients with prostate cancer (PCa) and 44 men without evidence of malignancy (NPCa), F2 and F3 were quantified by two-dimensional electrophoresis and Western blotting. The F2/F3 ratios were compared with the conventional parameter tPSA and percentage fPSA/tPSA ratio (%fPSA) in univariate and multivariate analyses using receiver operating characteristic analysis. RESULTS: F2 was lower in the NPCa group (median 17%) than in the PCa group (55%), and F3 was greater in the NPCa group (62%) than in the PCa group (45%), resulting in a significantly lower F2/F3 ratio in the NPCa group than in the PCa group (0.32 versus 1.21). The F2/F3 ratio correlated with the %fPSA and prostate volume but not Gleason score, tumor stage, age, or tPSA. The F2/F3 ratio and F2-F3/%fPSA ratio had greater areas under the receiver operating characteristic curves than did tPSA or %fPSA, especially in the subgroup of %fPSA greater than 15%. Models of binary logistic regression confirmed the improvement of diagnostic accuracy using the F2/F3 ratio as an independent variable. CONCLUSIONS: Compared with tPSA and %fPSA, the fPSA subforms F2 and F3, assessed as F2/F3 or F2-F3/%fPSA ratios, enhanced the differentiation between men with and without PCa for tPSA levels up to 10 microg/L. Additional characterization of these forms should be performed to develop a feasible assay.

Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA.

Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states.

Analysis of subforms of free prostate-specific antigen in serum by two-dimensional gel electrophoresis: potential to improve diagnosis of prostate cancer.

BACKGROUND: The aim of this study was to develop a method to separate and quantify subforms of free prostate-specific antigen (fPSA) in serum by two-dimensional electrophoresis and to assess the diagnostic accuracy of these subforms for prostate cancer (PCa) diagnosis in comparison with total PSA (tPSA) and the ratio of fPSA to tPSA (%fPSA). METHODS: Sera from 50 patients with and without PCa, respectively, were studied. PSA was isolated by immunoadsorption on streptavidin-coated magnetic beads with biotinylated anti-PSA antibodies and separated by two-dimensional electrophoresis. After semidry blotting, the intensities of the fPSA spots were quantified by chemiluminescence using an imager analyzer. RESULTS: The method detected subforms to a concentration of 0.1 mug/L fPSA with an imprecision (CV) <16%. We detected 15 immunoreactive fPSA spots of different intensities. Spots F2 and F3 were present in all samples. F2 was lower in samples from non-PCa patients (median, 23%) than in samples from PCa patients (49%), whereas F3 behaved inversely (non-PCa, 73%; PCa, 45%). Ratios of F2 to F3 and F2/F3 to %fPSA, respectively, showed improved diagnostic accuracy compared with tPSA and %fPSA. Better differentiation by F2/F3 or by F2/F3 to %fPSA was particularly evident in patients with %fPSA values >15%. There were no associations between the PCa grading scale and fPSA subforms. CONCLUSIONS: fPSA subforms separated by two-dimensional electrophoresis may improve both sensitivity and specificity in prostate cancer diagnostics compared with tPSA and %fPSA. The development of a practicable assay based on the immunologic properties of these different fPSA subforms seems to be promising.