VIP ELISA Assay Kit

Additional Information

Assay Background

VIP (vasoactive intestinal peptide) belongs to the glucagon family. It stimulates myocardial contractility, causes vasodilation, increases glycogenolysis, lowers arterial blood pressure and relaxes the smooth muscle of trachea, stomach and gall bladder. The protein also acts as an antimicrobial peptide with antibacterial and antifungal activity. Alternative splicing occurs at this locus and two transcript variants encoding distinct isoforms have been identified. 

VIP causes vasodilation, lowers arterial blood pressure, stimulates myocardial contractility, increases glycogenolysis and relaxes the smooth muscle of the trachea, stomach and gall bladder. PHM and PHV also cause vasodilation. PHM-27 is a potent agonist of the calcitonin receptor CALCR, with similar efficacy as calcitonin. 

Assay Principle

This assay employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for ApoE has been pre-coated onto a microtiter plate. Standards or samples are pipetted into the wells and any ApoE present is bound by the immobilized antibody. After washing away any unbound substances, a biotin-conjugated antibody specific for ApoE is added to each well and incubate. Following a washing to remove unbound substances, streptavidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After washing away any unbound antibody-enzyme reagent, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of ApoE bound in the initial step. The color development is stopped by the addition of acid and the intensity of the color is measured at a wavelength of 450nm ±2nm. The concentration of ApoE in the sample is then determined by comparing the O.D of samples to the standard curve.

Assay Procedure

1. Remove excess microtiter strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it. Standards and samples should be assayed in duplicates.

2. Add 100 μl of the Standards and diluted samples into the appropriate wells. Incubate for at least 2 hours at 37°C or incubate overnight at 4°C on a microplate shaker.

3. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1× Wash Buffer (250 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting

4. Add 100 µl of the 1:1000 diluted Conjugated-ApoE antibody working solution to each well, incubate for 1 hour at RT on a microplate shaker.

5. Aspirate each well and wash as step 3.

6. Add 100 µl of the diluted HRP-Streptavidin working solution to all wells and incubate for 1 hour at RT on a microplate shaker.

7. Warm TMB substrate solution to RT before next wash step. Aspirate each well and wash as step 3. Proceed immediately to the next step.

8. Add 100 μl of TMB substrate solution into each well. Incubate for 2-30 mins at RT on microplate shaker. Avoid exposure to light. Note: Watch plate carefully; if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation. 

9. Add 100 μl of Stop Solution to each well and shake lightly to ensure homogeneous mixing.

10. Read the OD with a microplate reader at 450nm immediately (optional: read at 620 nm as reference wave length).