A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma

J Immunol Methods. 1998 Oct 1;219(1-2):45-55. doi: 10.1016/s0022-1759(98)00131-8.

Abstract

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.

MeSH terms

  • Antibodies
  • Antibodies, Monoclonal
  • Biotinylation
  • Cross Reactions
  • Dimerization
  • Edetic Acid
  • Endothelial Growth Factors / blood*
  • Endothelial Growth Factors / immunology
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Female
  • Heparin / pharmacology
  • Humans
  • Lymphokines / blood*
  • Lymphokines / immunology
  • Male
  • Neoplasms / blood
  • Neoplasms / diagnosis
  • Protein Isoforms / blood
  • Protein Isoforms / immunology
  • Recombinant Proteins / immunology
  • Reference Values
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factors

Substances

  • Antibodies
  • Antibodies, Monoclonal
  • Endothelial Growth Factors
  • Lymphokines
  • Protein Isoforms
  • Recombinant Proteins
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factors
  • Heparin
  • Edetic Acid