RAPID, SENSITIVE ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA) FOR SERUM AMYLOID-A (APOSAA) IN HUMAN PLASMA AND TISSUE-CULTURE FLUIDS

Two direct binding enzyme-linked immunosorbent assays (ELISA) for human acute phase serum amyloid A isoforms (apoSAA(1) and apoSAA(2)) are described, a single antibody method for serum and plasma samples and a double antibody method for tissue culture supernatants. Both methods employ polyvalent, monospecific rabbit anti-human apoSAA antiserum that does not react with constitutive apoSAA, in plasma or tissue culture samples. In the presence of 3M KBr, pH 9.6, all apoSAA isoforms are passively adsorbed to microtiter plate wells in quantities proportional to their concentrations. The absolute concentrations of total acute phase apoSAA isoforms in samples can be determined from a standard curve constructed from samples of known apoSAA concentration incubated at the same rime. For clinical evaluation of serum and plasma specimens, apoSAA is bound to microtiter wells at ambient temperature (similar to 25 degrees C) after which ifs concentration is determined directly with horseradish peroxidase (HRP)conjugated anti-apoSAA immunoglobulins. The sensitivity for detection of acute phase apoSAA isoforms in plasma was found to be 1 mu g/ml. In order to measure apoSAA in tissue culture supernatants, apoSAA is bound to wells at 37 degrees C and detected using anti-rabbit apoSAA antiserum and HRP-conjugated goat anti-rabbit immunoglobulins the sensitivity for detection of acute phase isoforms was I mu g/ml.