Development of an assay for modulating anti-acetylcholine receptor autoantibodies using human rhabdomyosarcoma cell line.

Three types of autoantibodies against the acetylcholine receptors (AChR) of skeletal muscle are detectable in patients with myasthenia gravis including binding, blocking, and modulating anti-AChR antibodies. Modulating autoantibodies correlate best with the severity of the disease, but are also technically most difficult to measure because the assay generally requires fresh human muscle cells. We have developed an assay for the modulation of anti-AChR antibodies using a rhabdomyosarcoma (RD) cell line expressing AChR on the cell surface.

Impact of assay parameters on the accuracy of free PSA test: source and stability of calibrator, calibration curve fitting, and level of total PSA in the serum.

The measurement of PSA is recommended for men over 50 years of age for screening of prostate cancer. However, proper differentiation of prostate cancer from benign prostate hyperplasia (BPH) relies on an accurate measurement of free PSA (fPSA) and a correct calculation of percent fPSA. Because of the extremely low concentration of fPSA in the serum, any slight deviation from its true value may produce large errors in percent fPSA calculated.

Serum chromogranin A: early detection of hormonal resistance in prostate cancer patients.

We monitored both chromogranin A (CgA) and neuron specific enolase (NSE) in serial serum specimens from 14 patients with prostate cancer (CAP patients) showing resistance to hormonal treatment. Elevated serum CgA was detected in 10 out of these 14 patients (71%) during treatment, and an early appearance of elevated serum CgA was found in 6 of 14 (43%) of these patients when serum tPSA levels were still in the normal range. If patients with radical prostatectomy were not included, the percentage of patients showing an early appearance of elevated serum CgA would have been much higher.

Development of an immunoassay specific for the PSA-ACT complex without the problem of high background.

We have developed an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex that effectively diminishes the problem of high assay background commonly reported by other investigators. The assay follows a two-site ELISA format. Polyclonal anti-PSA antibodies were coated on the microplate to capture the PSA complex from the serum, whereas the biotinylated anti-ACT polyclonal antibodies and HRP-conjugated streptavidin were used for detection.

Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: difference between fPSA from LNCaP cell and seminal plasma.

We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation.