A sandwich ELISA for properdin in clinical specimens

Abstract

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when > 2.5 μg/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions <1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD 405=0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 μg. Day-to-day variation for a group of nine normal sera showed a mean difference of −0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclued that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.