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. By decreasing the FetalClone III serum from 10% to 0.5% in Eagles Minimal Essential Medium (EMEM) we were able to increase the number of AChR on RD cells to meet the need of sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Less than 6% modulation was found with the normal serum (n = 42). The CVs of both the intra- and day-to-day precision were less than 20%. When clinical samples (n = 105) were assayed in our laboratory and also at Nichols Institute, a correlation coefficient of 0.816 was obtained. The selection of RD cell line, the success of increasing the expression of the AChR on RD cells and the use of 125I alpha-bungarotoxin of high specific activity allowed the establishment of an assay which can be used in routine clinical laboratory for the measurement of modulating anti-AChR autoantibodies for the management of patients with myasthenia gravis.

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. Elevated serum CgA levels also were found in patients not subject to hormonal therapy. Serial specimens from two out of three prostate cancer patients, randomly selected, contained elevated serum CgA. Serum NSE was not detectable in any of the serial specimens we studied, suggesting that CgA, not NSE, should be used as a marker for neuroendocrine differentiation. We also compared the serum CgA in random serum specimens between patients with BPH (benign prostate hyperplasia) and with prostate cancer in the concentration range of serum tPSA between 3-15 ng/mL. Although serum CgA concentrations in BPH patients overlapped considerably with those levels in patients with prostate cancer, levels > 100 ng/mL should suggest prostate cancer. The early appearance of elevated serum CgA allows an early change of therapy to be made and can lead to the effective prevention of any further development of metastases.

Identification and characterization of c-erbB-2 proteins in serum, breast tumor tissue, and SK-BR-3 cell line.

We have identified and characterized c-erbB-2 protein molecules in sera from patients with carcinomas, in both cytosol and cell membrane extract from breast tumor tissue and in both the culture medium and cell extract of the SK-BR-3 cell line. These proteins were characterized by various chromatographic techniques and identified by the use of two immunoassays; one measures both the c-erbB-2 oncoprotein (p185) and its ectodomain (p120), and the other in-house assay reacts specifically for p185. We found that the majority of the immunoreactivity detected in the serum, tumor tissue cytosol, and conditioned cell medium was derived from the ectodomain molecule (p120) of the c-erbB-2 oncoprotein (p185), whereas only p185 was detected in the extracts from cell membrane of both tumor tissue and the SK-BR-3 cell line. The ectodomain molecules (p120) found in the serum, cytosol, and cell medium were very similar in terms of molecular size and charge property. The molecular weight was determined to be 120 kDa by the size exclusion HPLC method. Both p120 and p185 are glycoproteins and were retained by the ConA Sepharose column. Both molecules are also heterogeneous in charge and multiple peaks could be identified in the elution profiles of anion exchange HPLC and chromatofocusing. This information should not only facilitate the isolation of these molecules, but also improve preparation of specific antibodies, preparation of calibrators, and development of improved assays for these proteins.

PSA immunoreactivity detected in LNCaP cell medium, breast tumor cytosol, and female serum.

We made an effort to identify a reliable source for obtaining large quantities of both free (PSA) and PSA-ACT complex for the preparation of the calibrator for the PSA assay. Using size exclusion chromatography, we found both free PSA and PSA-ACT complex in the conditioned cell medium of the LNCaP cell line, which was derived from a human metastatic adenocarcinoma of the prostate. An assay specific for PSA-ACT reacted only with the PSA-ACT complex from cells grown in serum-free medium, and not with the complex from the cell medium grown in 10% calf serum. We also found both free PSA and PSA-ACT complex in 15% of cytosols prepared from breast tumor tissues; the cytosol PSA concentrations ranged from 0.1 to 110 ng/ml. No correlation was found between cytosol PSA and concentrations of estrogen receptor, progestin receptor, epidermal growth factor receptor, cathepsin D, or the ectodomain of c-erbB-2 protein. Based on chromatographic characterizations and the slope of their dose-response curves, it appears that both free PSA and PSA-ACT complex found in the cytosols are similar to PSA complex from the cell medium and the serum of prostate cancer patients. Ectopic PSA was also detected in pooled sera from patients with breast, ovarian, pancreatic, and colon carcinoma. The PSA concentrations in these serum pools increased with the level of their dominant tumor marker. In any event, the LNCaP cell medium appears to be a reliable source for obtaining both free and ACT-complexed PSA of human tumor origin for the preparation of PSA assay calibrators.

Measurement of c-erbB-2 proteins in sera from patients with carcinomas and in breast tumor tissue cytosols: correlation with serum tumor markers and membrane-bound oncoprotein.

Using a commercial kit with antibodies against the ectodomain of c-erbB-2 protein, we detected c-erbB-2 immunoreactivity in human serum. We found that the percentages of patients with elevated serum c-erbB-2 immunoreactivities were 35, 21, and 9% in breast, prostate, and ovarian carcinoma, respectively. The majority of the elevated immunoreactivities were associated with sera containing highly elevated tumor markers with the highest in breast carcinoma (35%) and lowest in ovarian cancer (9%). Excellent correlations were also observed between the serum levels of c-erbB-2 immunoreactivity and the dominant tumor markers in serial specimens from individual cancer patients. We could also detect the c-erbB-2 immunoreactivity in the cytosols prepared from the breast tumor tissue for estrogen and progesterone receptor (ER & PgR) measurements using the same commercial kit for serum studies, and the intact c-erbB-2 oncoprotein (p185) in the extracts of the tissue membrane fractions with a different kit designed for tissue extract. The level of c-erbB-2 immunoreactivity in the cytosol from 124 human breast tumor specimens had an excellent correlation with the cell membrane concentrations of p185 (gamma = 0.89). Most of the elevated cytosol c-erbB-2 immunoreactivities were also found to associate with breast tumor specimens containing low concentrations of ER & PgR. It appears that measuring the c-erbB-2 immunoreactivity potentially could be used as a prognostic marker without performing tissue biopsies and also as a serum tumor marker for managing cancer patients.

Evaluation of free PSA isoforms, PSA complex formation, and specificity of anti-PSA antibodies by HPLC and PAGE-immunoblotting techniques.

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14-fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.

Purification of PSA-ACT complex: characterization of PSA-ACT complex by various chromatographic procedures.

We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the extracellular domain of c-erbB-2 oncoprotein in sera from patients with various carcinomas: correlation with tumor markers.

Using a serum enzyme immunoassay (EIA) kit from Triton diagnostics we detected c-erbB-2 oncoprotein activity in random sera containing highly elevated tumor markers and also in serial specimens from cancer patients expressing elevated oncoprotein activities. Elevated oncoprotein activity was found not only in sera of breast and ovarian carcinomas but also in sera from colorectal, pancreatic, and prostate carcinomas and even from primary hepatoma. Whenever oncoprotein was overexpressed in an individual patient, there was usually an excellent correlation between the oncoprotein activity and the level of dominant tumor marker in serial serum specimens. Based on the size exclusion S-200 column chromatography, we found only a single molecule containing c-erbB-2 oncoprotein activity in pooled sera from cancer patients whereas two oncoproteins slightly different in size were detected in breast tumor tissue cytosol. Using HPLC on a Superose 12 HR column, the serum portion of the oncoprotein was eluted at a position near IgG, suggesting that the extracellular domain of the oncoprotein exists as a dimer in the serum.

Rhabdomyosarcoma cell line can be used for the isolation of soluble acetylcholine receptor and for assaying blocking and modulating autoantibodies.

We found that the Rhabdomyosarcoma (RD) cell line expresses human acetylcholine receptor (AChR) based on the following evidences: 1. Soluble AChR can be isolated from RD cells following the isolation procedure for AChR from human muscle; 2. Intact RD cells bind to alpha-bungarotoxin (alpha Butx) in a time-dependent and saturable fashion. The apparent dissociation constant (5.3 x 10(-10) M) is very similar to that reported for TE671 cells, which is known to express AChR; 3. Like fresh muscle culture, RD cells not only bind but also internalize 125I-alpha Butx. Soluble AChR from RD cells can be labeled specifically with 125I-alpha Butx and then used to quantify binding autoantibodies in myasthenic patients. We also demonstrate that blocking antibodies can be detected in sera from patients with myasthenia gravis (MG) using RD cells and the ability of RD cells to internalize alpha Butx. Consequently, RD cells can be used as a reliable source for obtaining soluble AChR and as a replacement for rodent or human muscle cultures in measuring blocking and modulating antibodies.

Dual fluorometric/colorimetric detection system for an automated random-access instrument utilizing standard polystyrene test tubes as precision cuvettes.

To attain the optical precision necessary to precisely quantify fluorescent or colorimetric signals, analytical systems have typically included quality-controlled cuvettes, flow cells, or dual-beam reference systems. We describe a system where a fluorescence or transmittance signal is quantified in single, standard, 12-mm-diameter polystyrene test tubes. Tube-to-tube variation is minimized by referencing the primary signal to a second reference signal. The tube is carefully oriented within a positioner that allows for the precise placement of the tube within a light path 7.6 mm in diameter. The detection system allows for use of either four pairs of fluorescence excitation/emission wavelengths or eight transmittance wavelengths, which are selected by using specific interference filters. The impact of temperature, tube imperfections, surface flaws, and distortions is minimized by using a reference ratio. Fluorescence is measured with an orthogonal photomultiplier tube, and transmittance with a photodiode; both are illuminated with an ordinary long-life tungsten-halogen lamp. This system is used with the Becton Dickinson AFFINITY system, an automated random-access analyzer with analyte-specific unit-package reagents. The polystyrene tube of the reagent package, which has an antibody-absorbed surface, serves as both the cuvette and the separation medium. Use of the reference ratio method reduces intertube imprecision of fluorometric or transmittance signals, for more precise quantification of various analytes.

Effect of cephapirin on formation of D-alanine carboxypeptidase in growing Bacillus subtilis cells.

Cephapirin was utilized to examine the interaction of beta-lactam antibiotics with growing Bacillus subtilis cells and the biological effects simultaneously produced. Saturation binding and quantitative cell death were observed at the cephapirin concentration of 0.1 mug/ml. Cephapirin bound to all penicillin-binding proteins except the d-alanine carboxypeptidase. A specific [(14)C]benzylpenicillin-binding assay was developed for the d-alanine carboxypeptidase. At the lowest saturating concentration of antibiotic (0.1 mug/ml), cephapirin inhibited formation of the d-alanine carboxypeptidase. Upon incubation with cephapirin, 18% of the membranous d-alanine carboxypeptidase was released into the media. The data suggest that beta-lactam antibiotics may affect the formation of bacterial cytoplasmic membranes in addition to their effect on cell wall synthesis.