Fig. 8.

An ELISA to detect GPIHBP1 in human plasma. We coated 96-well plates with mAb RF4 (1 μg/well), blocked them with BSA, and incubated them overnight at 4°C with dilutions of human plasma (ranging from 1:2 to 1:256) or dilutions of purified human GPIHBP1. In the case of the purified GPIHBP1, the “1:2 dilution” corresponds to 500 pg/ml of recombinant GPIHBP1, and the “1:256 dilution” corresponds to buffer alone (no recombinant GPIHBP1). After washing the plates, GPIHBP1 captured by mAb RF4 was detected with HRP-labeled RE3 Fab′. Samples 8 and 17 were normal control plasma samples; sample 3 was from a subject homozygous for a deletion of GPIHBP1 (21); samples 11 and 15 were from subjects homozygous for a GPIHBP1 nonsense mutation (C89×). The plasma GPIHBP1 levels in samples 8 and 17 were 1,043 and 1,051 pg/ml, respectively. As expected, the GPIHBP1 levels in the GPIHBP1-deficient subjects were essentially absent. (Note that GPIHBP1-C89X is not bound by mAb RE3 [see supplemental Fig. S6] and therefore cannot be detected by this ELISA.) The plot represents a log-transformation of the data.