Thermo Fisher VEGF ELISA User Manual

PRODUCT INFORMATION SHEET

Human VEGF ELISA Kit

Catalog Number KHG0111 (96 tests), KHG0112 (2 × 96 tests)

Pub. No. MAN0003960 Rev. 7.0 (33)

CAUTION! This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to

form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and
contact with eyes, skin and mucous membranes. In case of contact, rinse aected area with plenty of water. Observe all federal, state, and
local regulations for disposal.

Note: For safety and biohazard guidelines, see the “Safety” appendix in the ELISA Technical Guide (Pub. no. MAN0006706). Read the Safety Data
Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Product description

The Invitrogen™ Human VEGF ELISA Kit is a solid‑phase sandwich Enzyme‑Linked Immunosorbent Assay (ELISA). This assay is designed to
detect and quantify the level of human VEGF in human serum, plasma, buered solution, or cell culture medium. The assay recognizes both
natural and recombinant human VEGF.

Vascular Endothelial Growth Factor (VEGF), originally named vascular permeability factor (VPF), is an important regulator of angiogenesis and
vasculogenesis. Six identified isoforms of 121, 145, 165, 183, 189, and 206 amino acids are derived from alternative splicing in humans.
These variants dier with respect to two independent heparin binding domains, and the isoforms exhibit altered anity for interaction with
heparan-sulfate proteoglycans.

Contents and storage

Upon receipt, store the kit at 2°C to 8°C.

Contents

Hu VEGF Standard, lyophilized; contains 0.1% sodium azide. 2 vials
Standard Diluent Buer; contains 0.1% sodium azide 25 mL
Incubation Buer 12 mL
Antibody Coated Plate, 96-well plate 1 plate
Hu VEGF Biotin Conjugate; contains 0.1% sodium azide 11 mL
Streptavidin-HRP (100X) 0.15 mL
Streptavidin HRP Diluent; contains 3.3 mM thymol 25 mL
Wash Buer Concentrate (25X) 100 mL
Stabilized Chromogen, Tetramethylbenzidine (TMB) 25 mL
Stop Solution 25 mL
Adhesive Plate Covers 3

Cat. No. KHG0111

(96 tests)

Materials required but not supplied

• Distilled or deionized water

• Calibrated adjustable precision pipettes and glass or plastic tubes
for diluting solutions; beakers, flask and cylinders for preparation
of reagents

• Microtiter plate reader with software capable of measurement at
or near 450 nm

• Plate washer–automated or manual (squirt bottle, manifold
dispenser, or equivalent)

Before you begin

IMPORTANT! Reagents are lot-specific. Do not mix or interchange

dierent reagent lots from various kit lots.

• Review the Procedural guidelines and Plate washing directions

• Allow reagents to reach room temperature before use. Mix to

Prepare 1X Wash Buer

1. Dilute 16 mL of Wash Buer Concentrate (25X) with 384 mL of

2. Store the concentrate and 1X Wash Buer in the refrigerator. Use

For Research Use Only. Not for use in diagnostic procedures.

in the ELISA Technical Guide available at thermofisher.com.

redissolve any precipitated salts.

deionized or distilled water. Label as 1X Wash Buer.

the diluted buer within 14 days.

Sample preparation guidelines

Reconstituted

Standard

Diluent
Volume

Std5Std4Std3Std2Std1 Std6 Std7

Std0

90 μL 300 μL 300 μL 300 μL300 μL 300 μL 300 μL

375 pg/mL1,500 pg/mL 750 pg/mL 23.4 pg/mL46.9 pg/mL93.8 pg/mL188 pg/mL10,000 pg/mL

510 μL 300 μL 300 μL 300 μL 300 μL 300 μL 300 μL 300 μL

0 pg/mL

• Refer to the ELISA Technical Guide at thermofisher.com for detailed sample preparation procedures.

• Collect samples in pyrogen/endotoxin-free tubes.

• Freeze samples after collection if samples will not be tested immediately. Avoid multiple freeze-thaw cycles of frozen samples. Thaw
completely and mix well (do not vortex) prior to analysis.

• Avoid the use of hemolyzed or lipemic sera. If large amounts of particulate matter are present in the sample, centrifuge or filter sample prior to
analysis.

Pre-dilute samples

Sample concentrations should be within the range of the standard curve. Because conditions may vary, each investigator should determine the
optimal dilution for each application.

• Perform sample dilutions with Standard Diluent Buer.

Dilute standards

Note: Use glass or plastic tubes for diluting standards.

1. Reconstitute Hu VEGF Standard to 10,000 pg/mL with Standard Diluent Buer. Refer to the standard vial label for instructions. Swirl or mix

gently and allow the contents to sit for 10 minutes to ensure complete reconstitution. Label as 10,000 pg/mL human VEGF. Use the standard
within 1 hour of reconstitution.

2. Add 90 µL Reconstituted Standard to one tube containing 510 µL Standard Diluent Buer and mix. Label as 1,500 pg/mL human VEGF.

3. Add 300 µL Standard Diluent Buer to each of 7 tubes labeled as follows: 750, 375, 188, 93.8, 46.9, 23.4, and 0 pg/mL human VEGF.

4. Make serial dilutions of the standard as shown in the following dilution diagram. Mix thoroughly between steps.

5. Discard any remaining reconstituted standard. Return the Standard Diluent Buer to the refrigerator.

Prepare 1X Streptavidin‑HRP solution

Note: Prepare 1X Streptavidin‑HRP within 15 minutes of usage.

1. For each 8-well strip used in the assay, pipet 10 μL Streptavidin‑HRP (100X) solution, and dispense the solution into a tube containing 1 mL

of 1X Streptavidin-HRP Diluent. Mix thoroughly.

2. Return the unused Streptavidin‑HRP (100X) solution to the refrigerator.

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Human VEGF ELISA Kit Product Information Sheet