BioTechniquesVol. 45, No. 5 Application Forum - Sponsored PaperOpen AccessChemiluminescent VEGF ELISA Using the SpectraMax® L Microplate LuminometerCathy OlsenCathy OlsenMDS Analytical Technologies, 1311 Orleans Drive, Sunnyvale, CA, 94089Search for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000113044AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionVascular endothelial growth factors (VEGFs) are a family of secreted polypeptides implicated in mammalian vascular development and in disease processes involving abnormal blood vessel growth.(1) The QuantiGlo Chemiluminescent VEGF Immunoassay is a solid phase ELISA that uses the quantitative sandwich enzyme immunoassay technique to measure VEGF165 levels in cell culture supernatants, serum, plasma, saliva, and urine.(2) Luminescent signal is produced in proportion to the amount of VEGF present in samples and is measured using a microplate luminometer. Here, assay results were detected using the SpectraMax® L microplate luminometer, which performs flash and glow applications in 96- and 384-well microplate formats. Its dedicated luminescence optical design yields an extremely high signal-to-noise ratio, low crosstalk, and a dynamic range of over eight orders of magnitude. Data analysis was performed using a preconfigured protocol in the SoftMax® Pro software.MaterialsQuantiGlo Human VEGF Immunoassay (R&D Systems P/N QVEOOB).SpectraMax L microplate luminometer.MethodsPreparation of reagents and standardsStep 1. All reagents were brought to room temperature.Step 2. Wash Buffer Concentrate was diluted 10-fold with deionized water.Step 3. Working Glo Reagent was prepared as directed in the product insert.Step 4. The VEGF standard was reconstituted as directed. Working standards ranging from 20,000 to 1.3 pg/mL were prepared by serially diluting the stock standard in Calibrator Diluent RD5L.Assay procedureStep 1. 150 µL of Assay Diluent RD1-8 was pipetted into each well.Step 2. 50 µL of working standard or diluent was pipetted into triplicate wells of the supplied microplate. The microplate was incubated for 2 hours at room temperature with shaking.Step 3. Wells were washed 4 times with 400 µL Wash Buffer.Step 4. 200 µL of VEGF Conjugate was added to each well, and the microplate was incubated for 3 hours at RT with shaking.Step 5. The plate was washed as in step 3.Step 6. 100 µL Working Glo Reagent was added to each well. The microplate was covered and incubated for 5-20 minutes at RT.Step 7. The microplate was read on the SpectraMax L microplate luminometer using 1-second integration, AutoRange PMT, and 470 nm target calibration wavelength.ResultsFigure 1 shows the VEGF standard curve obtained with the SpectraMax L microplate luminometer. The dynamic range for this assay spanned greater than four orders of magnitude, and the calculated sensitivity of about 1.5 pg/mL correlated closely with R&D Systems’ stated value of 3.3 pg/mL. Data were analyzed and plotted using SoftMax Pro software. Similar results may be obtained using the SpectraMax M5 and FlexStation® 3 multi-mode microplate readers (data not shown).Figure 1. VEGF Standard Curve. Standards ranging from 20,000 to 1.3 pg/mL were assayed as described in the assay procedure. The microplate was read on a SpectraMax L microplate luminometer with a 1-second integration time and target calibration wavelength of 470 nm. Results were plotted using SoftMax Pro software.SummaryThe QuantiGlo Human VEGF Immunoassay is a highly sensitive method for assaying VEGF in a variety of samples. The SpectraMax L microplate luminometer with SoftMax Pro software provides excellent sensitivity and dynamic range for this assay as well as simplified data acquisition and analysis.