Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants.
A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D001943:Breast Neoplasms / Q000473:pathology; D002460:Cell Line; D002470:Cell Survival; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas / Q000032:analysis*; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D051379:Mice; D011137:Polystyrenes; D013347:Subcellular Fractions; D013997:Time Factors
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007074:Immunoglobulin G; D011137:Polystyrenes