Apolipoprotein AI ELISA Assay Kit

$950.00

The Eagle Biosciences Apolipoprotein AI ELISA Assay Kit is intended ffor the quantification of human Apolipoprotein AI (ApoAI) in human plasma, serum and biological fluids  The Eagle Biosciences Apolipoprotein AI ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.

Apolipoprotein AI ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 50 pg/ml
Dynamic Range: 31.3-2000 pg/ml
Incubation Time: 4.5 hours
Sample Type: Serum, Plasma, or other biological fluids
Sample Size: 100 µL

Additional Information

Assay Background

Apolipoprotein A-I is the major protein component of high density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion, and it is a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. This gene is closely linked with two other apolipoprotein genes on chromosome 11. Defects in this gene are associated with HDL deficiencies, including Tangier disease, and with systemic non-neuropathic amyloidosis. Apolipoprotein A1 participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase (LCAT). As part of the SPAP complex, activates spermatozoa motility.

Assay Principle

The Eagle Biosciences Apolipoprotein AI ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for ApoAI has been pre-coated onto a microtiter plate. Standards or samples are pipetted into the wells and any ApoAI present is bound by the immobilized antibody. After washing away any unbound substances, a biotin-conjugated antibody specific for ApoAI 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 ApoAI 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 ApoAI 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 2 hours at 37°C or incubate overnight at 4°C on 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-ApoAI antibody working solution to each well, incubate for 1 hour at room temperature on 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 room temperature on microplate shaker.

7. Aspirate each well and wash as step 3.

8. Warm TMB substrate solution to room temperature. 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 (optional: read at 620 nm as reference wave length).

Manual

Product Manual