A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma.
A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF165) in human plasma. The ELISA can be performed 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) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF165 isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the intact VEGF165 homodimer, however, the homodimers VEGF121, VEGF110 and the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy 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 males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml.
Rodriguez
C R
CR
Department of BioAnalytical Technology, Genetech, South San Francisco, CA 94080, USA.
Fei
D T
DT
Keyt
B
B
Baly
D L
DL
D000906:Antibodies; D000911:Antibodies, Monoclonal; D019921:Biotinylation; D003429:Cross Reactions; D019281:Dimerization; D004492:Edetic Acid; D016228:Endothelial Growth Factors / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008222:Lymphokines / Q000097:blood* / Q000276:immunology; D008297:Male; D009369:Neoplasms / Q000097:blood / Q000175:diagnosis; D020033:Protein Isoforms / Q000097:blood / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000906:Antibodies; D000911:Antibodies, Monoclonal; D016228:Endothelial Growth Factors; D008222:Lymphokines; D020033:Protein Isoforms; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D006493:Heparin; D004492:Edetic Acid