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.

2

Human VEGF ELISA Kit Product Information Sheet

Perform ELISA (Total assay time: 4 hours)

Standards

Sample + Standard

Diluent Buffer

IMPORTANT! Perform a standard curve with each assay.

• Allow all components to reach room temperature before use. Mix all liquid reagents prior to use.

• Determine the number of 8-well strips required for the assay. Insert the strips in the frames for use. Re-bag any unused strips and frames,
and store at 2°C to 8°C for future use.

Bind antigen

1

Add Biotin Conjugate

2

Add Streptavidin‑HRP

3

Add Stabilized

4

Chromogen

Add Stop Solution

5

1.1. Add 50 µL of the Incubation Buer to all wells except the chromogen blanks.

1.2. Add 100 µL of standards to the appropriate wells. For samples and controls, add 50 µL of Standard

Diluent Buer followed by 50 µL of sample (see “Pre-dilute samples” on page 2) to the appropriate
wells. Leave the wells for chromogen blanks empty.

1.3. Tap the side of the plate to mix. Cover the plate with a plate cover and incubate for 2 hours at room
temperature.

1.4. Thoroughly aspirate the solution and wash wells 4 times with 1X Wash Buer.

2.1. Add 100 µL Hu VEGF Biotin Conjugate solution into each well except the chromogen blanks.

2.2. Cover the plate with plate cover and incubate for 1 hour at room temperature .

2.3. Thoroughly aspirate the solution and wash wells 4 times with 1X Wash Buer.

3.1. Add 100 μL 1X Streptavidin‑HRP solution (see page 2) into each well except the chromogen blanks.

3.2. Cover the plate with a plate cover and incubate for 30 minutes at room temperature.

3.3. Thoroughly aspirate the solution from the wells and wash wells 4 times with 1X Wash Buer.

4.1. Add 100 μL Stabilized Chromogen to each well. The substrate solution begins to turn blue.

4.2. Incubate for 30 minutes at room temperature in the dark.

Note: TMB should not touch aluminum foil or other metals.

Add 100 μL Stop Solution to each well. Tap the side of the plate to mix. The solution in the wells changes
from blue to yellow.

Read the plate and generate the standard curve

1. Read the absorbance at 450 nm. Read the plate within 2 hours after adding the Stop Solution.

2. Use curve-fitting software to generate the standard curve. A 4 parameter algorithm provides the best standard curve fit. Optimally, the

background absorbance may be subtracted from all data points, including standards, unknowns and controls, prior to plotting.

3. Read the concentrations for unknown samples and controls from the standard curve. Multiply value(s) obtained for sample(s) by the
appropriate factor to correct for the sample dilution.

Note: Dilute samples producing signals greater than the upper limit of the standard curve in Standard Diluent Buer and reanalyze. Multiply
the concentration by the appropriate dilution factor.

Performance characteristics

Standard curve example

The following data were obtained for the various standards over the
range of 0 to 1,500 pg/mL human VEGF.

Standard Human VEGF (pg/mL) Optical Density (450 nm)

1,500 2.63

750 1.62
375 0.86
188 0.48

93.8 0.25

46.9 0.15

23.4 0.11
0 0.06

Inter-assay precision

Samples were assayed 42 times in multiple assays to determine
precision between assays.

Parameters Sample 1 Sample 2 Sample 3

Mean (pg/mL) 88.6 319 984
Standard Deviation 8.2 27.1 64.4
% Coecient of Variation 9.3 8.5 6.5

Intra-assay precision

Samples with known human VEGF concentration were assayed in
replicates of 14 to determine precision within an assay.

Parameters Sample 1 Sample 2 Sample 3

Mean (pg/mL) 87.4 345 938
Standard Deviation 4.8 12.7 45.8
% Coecient of Variation 5.5 3.7 4.9

Human VEGF ELISA Kit Product Information Sheet 3

Linearity of dilution

Human serum and tissue culture medium containing 10% fetal bovine
serum were spiked with human VEGF and serially diluted in Standard
Diluent Buer over the range of the assay. Linear regression analysis
of samples versus the expected concentration yielded a correlation
coecient of 0.99 in both cases.

Serum Cell Lysate

Dilution

Neat 1,834 — — 1497 — —

1/2 893 917 97 745 749 99
1/4 493 459 107 386 374 103

1/8 245 229 107 196 187 105
1/16 118 115 103 93 94 99
1/32 58 57 102 47 47 100

Measured

(pg/mL)

Expected

(pg/mL) % (pg/mL) %

Measured

(pg/mL)

Expected

Cross-reactivity

Mouse and rat VEGF
respectively. Human VEGF
complete parallelism with human VEGF

showed 0.25% and 0.11% cross‑reactivity,

165

showed 100% cross‑reactivity and

121

165

.

Expected values

Human bronchial smooth muscle cells were cultured for 144 hours
with and without Hu TNF‑α (10 ng/mL). The values were 790 pg/mL
and 630 pg/mL, respectively.

Fifteen fresh matched sera from healthy individuals were evaluated
in this assay. The values for sera ranged from 40– 600 pg/mL (mean
270 pg/mL).

Thirteen previously frozen citrate plasma samples were evaluated in
this assay. All samples fell below the sensitivity of the assay

Recovery

The following table shows the average recovery when adding human
VEGF to the listed sample types.

Sample

Serum 95
Citrate plasma 99
Culture medium containing 1% fetal bovine serum 90
Culture medium containing 10% fetal bovine serum 88

Average %

Recovery

Parallelism

Natural human VEGF was serially diluted in Standard Diluent Buer.
The optical density of each dilution was plotted against the standard
curve. The standard accurately reflects natural human VEGF content
in samples.

Sensitivity

The analytical sensitivity of the assay is <5 pg/mL human VEGF. This
was determined by adding two standard deviations to the mean O.D.
obtained when the zero standard was assayed 30 times.

Specificity

Buered solutions of a panel of substances at 10,000 pg/mL were

assayed with the Human VEGF ELISA Kit. The following substances
were tested and found to have no cross‑reactivity: human IL‑1β, IL‑2,
IL‑6, IL‑8, IL‑10, IL‑13, IL‑15, EGF, FGF basic, FGF acidic, G‑CSF,
GM‑CSF, IFN‑γ, RANTES, SCF, TGF‑α, and TNF‑α; mouse IL‑1β,
IL‑6, IL‑10, G‑CSF, GM‑CSF, IFN‑γ, and TNF‑α; rat IL‑1β, IL‑6, IL‑10,
GM‑CSF, IFN‑γ, and TNF‑α.

4

Human VEGF ELISA Kit Product Information Sheet

Limited product warranty

Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of
Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at

www.thermofisher.com/support.

Product label explanation of symbols and warnings

Catalog

Number

Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

applicable Limited Use Label Licenses.
©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

Batch code

Temperature

limitation

Use by Manufacturer

Consult

instructions for

use

Caution, consult

accompanying

documents

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

2 April 2021