Human VEGF IQELISA™ Kit

RayBio® Human Immunoquantitative (PCR-Based) VEGF ELISA Kit for cell culture supernatants, plasma, and serum samples.

Specifications

Introduction

The RayBio^® Immuno Qunatitative Enzyme Linked ImumunoSorbent Assay (IQELISA™) is an innovative assay that combines the specificity and ease of use of an ELISA with the sensitivity of real-time PCR. This results in an assay that is simultaneously familiar and cutting edge and enables the use of lower sample volumes while also providing more sensitivity. The RayBio^® Human VEGF IQELISA™ kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of human VEGF in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for human VEGF coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and VEGF present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection affinity molecule is added to the plates. After washing away unbound detection affinity molecule, primers and a PCR master mix are added to the wells and data is collected using qPCR. Ct values obtained from the qPCR are then used to calculate the amount of antigen contained in each sample, where lower Ct values indicate a higher concentration of antigen.

Standard Curves

Typical Data

Spiking & Recovery Results

Serum spike tests show recovery is 93.3% with a range of 89.7 to 98.0%

Application Notes

Kit Components

• - VEGF Microplate (Item A)**: 96 well PCR plate coated with anti-human VEGF

• - Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution

• - Standards (Item C): 2 vials of recombinant human VEGF

• - Assay Diluent B (Item E): 15 ml of 5x concentrated buffer

• - Detection Affinity Reagent for VEGF (Item F): 2 vials of a 4x concentrated solution of anti-human VEGF affinity reagent

• - Primer Solution (Item G): 1.7 ml vial

• - PCR Master Mix (Item H): 1.2 ml vial

• - PCR Preparation buffer (Item I): 1 ml vial of 10x concentrated buffer

• - Final Wash Buffer (Item J): 10 ml vial of 10x concentrated buffer

Other Materials Required

• - Real-time PCR instrument, Bio-Rad recommended

• - Precision pipettes to deliver 2 µl to 1 ml volumes

• - Adjustable 1-25 ml pipettes for reagent preparation

• - 100 ml and 1 liter graduated cylinders

• - Absorbent paper

• - Distilled or deionized water

• - Log-log graph paper or computer and software for data analysis

• - Tubes to prepare standard or sample dilutions

• - Heating block or water bath capable of 80°C

Protocol Outline

• 1. Prepare all reagents, samples and standards as instructed

• 2. Add 25 µl standard or sample to each well. Incubate for 1.5 - 2.5 hours at room temperature

• 3. Add 25 µl detection antibody to each well. Incubate 1 hour at room temperature

• 4. Add 15µL Primer solution to each well

• 5. Add 10µL of PCR Master Mix to each well

• 6. Run real-time PCR

Storage/Stability

May be stored for up to 6 months at 2° to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C) after reconstitution. Opened PCR plate or reagents may be stored for up to 1 month at 2° to 8°C. Note: the kit can be used within one year if the whole kit is stored at -20°C. Avoid repeated freeze-thaw cycles.

Catalog #: IQH-VEGF-1 -- 1 Plate Kit

Catalog #: IQH-VEGF-2 -- 2 Plate Kit

Catalog #: IQH-VEGF-5 -- 5 Plate Kit

Please email us at cs@medikonia.com for any enquiry. To place an order, please include the catalog number(s) of the product(s) in the email.

Manual

Safety Data Sheet