Accurate estimation of vascular endothelial growth factor levels in microsamples with a low-cost bead-based assay.
OBJECTIVE
The purpose of this study was to evaluate the accuracy of measurement of a known concentration of vascular endothelial growth factor (VEGF) using cytometric bead-based assay in microsamples (50 microL) and compare it with that obtained by enzyme-linked immunosorbent assay.


METHODS
A known concentration of VEGF samples was prepared with addition of VEGF (100 pg/mL) to 1 mL of Hank balanced salt solution. This preparation was further diluted serially to 50, 25, and 12.5 pg/mL; and VEGF estimation was done on each of these samples using Luminex, a commercially available cytometric bead-based assay and enzyme-linked immunosorbent assay. The assay was replicated three times at each dilution.


RESULTS
The mean concentrations of VEGF measured using Luminex were 97.7 +/- 6.2, 47.3 +/- 8.1, 24.9 +/- 2.2, and 14.2 +/- 1.8 pg/mL at 100, 50, 25, and 12.5 pg/mL dilutions, respectively. Similarly, the mean concentrations of VEGF measured using enzyme-linked immunosorbent assay for equivalent dilutions were 100.6 +/- 24.4, 53.8 +/- 16.8, 44.8 +/- 35.5, and 14.19 +/- 0.7 pg/mL, respectively.


CONCLUSIONS
Bead-based assay accurately measures VEGF concentration in a microsample and has better validity and reliability as compared with enzyme-linked immunosorbent assay.
Chalam
Kakarla V
KV
Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida 32209, USA. kchalam@jax.ufl.edu
Balaiya
Sankarathi
S
Murthy
Ravi K
RK
D001826:Body Fluids / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis*
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A