An enzyme-linked immunosorbent assay for quantifying adherence of Candida to human vascular endothelium.

Success in elucidating the pathogenesis of certain bacterial infections through studies of bacterial adherence to host cells has stimulated interest in parallel investigations of fungal adherence. Fungal adherence differs from bacterial adherence, especially when fungal coadherence (adherence of fungal cells to each other) is a factor. Using human umbilical vein endothelial cells cultured in a living monolayer in microtiter plates, we developed an ELISA to study adherence of Candida albicans to endothelial cells in the absence of yeast coadherence. A rabbit antibody to Candida detected the adherent Candida, and an alkaline phosphatase-conjugated antibody to rabbit IgG was the developing antibody. A linear relationship between the log of the optical density and the log of the number of adherent organisms was seen for wells containing 3 X 10(4)-1 X 10(6) organisms (r = .923- .965). In addition to measuring adherence of living Candida to living target cells and avoiding Candida coadherence, this assay makes it possible to investigate adherence limited to lumenal surfaces, conserves reagents, and facilitates the testing of large numbers of potential adherence modifiers.

A rat anti-mouse kappa chain specific monoclonal antibody, 187.1.10: purification, immunochemical properties and its utility as a general second-antibody reagent.

A rat IgG1 monoclonal antibody, produced by hybridoma 187.1.10, exhibits specificity for mouse immunoglobulins containing kappa light chains (Yelton et al., 1981). The 187.1.10 hybridoma cell line secreted upwards of 200 micrograms/ml of monoclonal antibody in tissue culture and the secreted product was purified in a single step by antigen-immunoadsorbent affinity chromatography. The homogeneity of the purified 187.1.10 protein was determined by isoelectrofocusing and SDS gel electrophoresis. Equilibrium binding analyses of the radioiodinated 187.1.10 antibody indicated a strong interaction with its antigen of KA = 2 X 10(9) l/mole. The 187.1.10 antibody did not readily bind to Staph. aureus protein A unless it was complexed with antigen. The binding of immune complexes of 187.1.10 to protein A was shown to be dependent on the Fc region of the antigen. The utility of the 187.1.10 monoclonal antibody as a general second antibody reagent for studying mouse immunoglobulins was demonstrated in a rapid solid phase immunoprecipitation assay to detect and analyze radioiodinated membrane proteins of a human cytotoxic T cell line.