Competitive particle concentration fluorescence immunoassay for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol) in serum.

A competitive particle concentration fluorescence immunoassay (PCFIA) is described for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol; Lilly) in human serum. b-Phycoerythrin-labeled lometrexol competes with free lometrexol for binding to a limiting concentration of lometrexol-specific antibodies immobilized by a second antibody to submicrometer-diameter polystyrene particles in specially designed 96-well plates. Reaction particles are washed and concentrated onto filter membranes in the wells of the plates and the fluorescence is measured at 575 nm. The method, including sample preparation and data reduction, is automated and can be completed in less than 2 h. The assay has a standard curve maximum measurable concentration of 1000 micrograms/L and a minimum detectable concentration of 0.1 microgram/L. Analytical recovery of lometrexol in serum is quantitative at concentrations greater than 1 micrograms/L. Intra- and interassay coefficients of variation at 50 micrograms/L in serum are 7.1% (n = 9) and 7.5% (n = 33), respectively. The cross-reactivity of naturally occurring folates, folic acid analogs, and the anti-cancer agent methotrexate is minimal. We report the use of the PCFIA during Phase I clinical studies designed to evaluate the pharmacokinetics of lomextrexol after intravenous administration to cancer patients.

Molecular cloning and characterization of a human adenocarcinoma/epithelial cell surface antigen complementary DNA.

A human adenocarcinoma-associated antigen (KSA) defined by the monoclonal antibody KS1/4 has become the focus of several site-directed strategies for tumor therapy. KSA, a 40,000 Da cell surface glycoprotein antigen, is found at a high density in all adenocarcinomas examined to date and in corresponding normal epithelial tissues. Here we describe the cloning and sequencing of overlapping complementary DNA clones which encode the entire KSA as expressed in UCLA-P3, a human lung adenocarcinoma cell line. We have deduced the 314-amino acid sequence and have compared it to the N-terminal amino acid sequence data of the affinity-purified antigen. The KSA is synthesized as a 314-residue-long preproprotein that is then processed to a 232-residue-long antigen. KSA appears to have a single transmembrane domain of 23 residues that separates the highly charged 26-residue cytoplasmic domain from the extracellular domain. The N-terminal region of the propeptide is rich in cysteines and contains three potential N-glycosylation sites. Computer-assisted analyses at both the DNA and protein levels have found no significant similarities of this protein to known sequences, but a GC-rich 5' terminus is evident. Northern blot analysis shows that transcription of KSA can be detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver.

Isolation and characterization of the human adenocarcinoma-associated glycoprotein gp40.

The human adenocarcinoma-associated antigen gp40 is a cell surface glycoprotein recognized by murine monoclonal antibody KS1/4. A KS1/4-Sepharose affinity matrix was utilized to purify gp40 from detergent lysates of either tissue culture cells or nude mouse xenograft tumors of the human lung adenocarcinoma cell line P3-UCLA. This single immunoaffinity chromatography step yielded an antigen preparation of approximately 95% purity which was further characterized by immunochemical and enzymatic techniques. The gp40 molecule was shown to have both complex and high-mannose oligosaccharides comprising some 16% of the apparent molecular weight. The antigen preparation was suitable for gas-phase N-terminal amino acid sequencing and the first 16 residues of the N-terminus were determined. Despite considerable molecular heterogeneity, gp40 shows a single N-terminal sequence.

Enzyme-linked immunosorbent assay for LY163502, a potent dopamine agonist.

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for the potent selective dopaminergic receptor (D2) agonist, LY163502, is described. The ELISA is a competitive assay in which peroxidase-labeled LY163502 competes with unlabeled LY163502 for binding to solid-phase antibodies specific for LY163502. The limit of detection is 8 pg/mL; intra- and interassay coefficients of variation are 7.2 and 12.3% respectively, for human plasma determinations. Recovery of LY163502 from plasma and urine is quantitative. We found two potential metabolites of LY163502, despropyl-LY163502 and N-oxide LY163502, to be 44 and 0.5% cross-reactive at the ED50 level, respectively. However, LY163502 levels measured by the ELISA and by a specific gas-liquid chromatography (GLC) assay are highly correlated [Pearson's correlation coefficient, r = 0.964]. This suggests that the ELISA assay of biological fluids is not affected by cross-reactivity due to LY163502 metabolites. Application of the ELISA to the measurement of LY163502 in biological fluids is demonstrated.