Apolipoprotein A1 ELISA Assay Kit

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 A1 ELISA Assay Kit employs the sandwich enzyme immunoassay technique for the detection of Human Apolipoprotein A1 in human plasma and serum samples. Human Apolipoprotein A1 will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, anti-Human Apolipoprotein A1 primary antibody binds to the captured protein. Following a washing to remove unbound substances, secondary antibody 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 Apolipoprotein A1 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 Apolipoprotein A1 in the sample is then determined by comparing the O.D of samples to the standard curve.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 100 µl of standards, samples and zero controls into appropriate wells. Shake plate at 300 rpm for 30 minutes at RT.
3. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1X wash buffer (300 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 buffer by aspirating, decanting or blotting against clean paper towels.
4. Add 100 µl of working Primary Antibody into each well. Shake plate at 300 rpm for 30 minutes at RT.
5. Wash as according to step 3.
6. Add 100 µl of working HRP-conjugated secondary Antibody into each well. Shake plate at 300 rpm for 30 minutes at RT.
7. Wash as according to step 3.
8. Add 100 μl of TMB substrate to each well. Incubate for 1-5 minutes at RT in dark. Substrate will change from colorless to different strengths of blue.
9. Add 50 μl of 1N H2SO4 or HCl to each well. The color of the solution should change from blue to yellow. Mix thoroughly by gently shaking the plate.
10. Read the OD with a microplate reader at 450 nm immediately.