A SENSITIVE FLUOROMETRIC ENZYME-LINKED-IMMUNOSORBENT-ASSAY THAT MEASURES VASCULAR ENDOTHELIAL GROWTH FACTOR(165) IN HUMAN PLASMA

A specific and sensitive fluorometric enzyme-linked immunosorbent assa y (ELISA) was developed to measure endogenous levels of vascular endot helial growth factor (VEGF(165)) in human plasma. The ELISA can be per formed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF (rhVEGF) a dded to human plasma ranges from 89 to 100%. The capture antibody depl etes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coeffici ents of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18% , respectively. Characterization of the ELISA using plasmin derived VE GF variants suggests the assay is specific for the VEGF(165) isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the inta ct VEGF(165) homodimer, however, the homodimers VEGF(121), VEGF(110) a nd the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal he althy individuals range from 20 to 141 pg/ml, with a mean of 42 +/- 22 pg/ml. There were no significant differences in VEGF levels between m ales and females. Circulating endogenous VEGF levels in cancer patient s ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml. (C) 1998 El sevier Science B.V. All rights reserved.