The Apolipoprotein B ELISA Assay Kit is intended for for the quantification of Apolipoprotein B in Human plasma, serum and urine samples. The Eagle Biosciences Apolipoprotein B ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.
Apolipoprotein B is the main apolipoprotein of chylomicrons and low density lipoproteins. It occurs in plasma as two main isoforms, apoB-48 and apoB-100: the former is synthesized exclusively in the gut and the latter in the liver. The intestinal and the hepatic forms of apoB are encoded by a single gene from a single, very long mRNA. The two isoforms share a common N-terminal sequence. The shorter apoB-48 protein is produced after RNA editing of the apoB-100 transcript at residue 2180 (CAA->UAA), resulting in the creation of a stop codon, and early translation termination. Mutations in this gene or its regulatory region cause hypobetalipoproteinemia, normotriglyceridemic hypobetalipoproteinemia, and hypercholesterolemia due to ligand-defective apoB, diseases affecting plasma cholesterol and apoB levels. Apolipoprotein B is a major protein constituent of chylomicrons (apo B-48), LDL (apo B-100) and VLDL (apo B-100). Apo B-100 functions as a recognition signal for the cellular binding and internalization of LDL particles by the apoB/E receptor.
The Eagle Biosciences Apolipoprotein B ELISA Assay Kit employs the sandwhich enzyme immunoassay technique for the detection of Human Apolipoprotein B in human plasma, serum, and urine samples. Human Apolipoprotein B will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, anti-Human Apolipoprotein B primary antibody binds to the captured protein. Following a washing to remove unbound substances, secondary antibody conjugated to Horseradise Peroxidase (HRP) is added to each microplate well and incubated. After washing away any unbound antibody-enyme reagent, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of Apolipoprotein B 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 B 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.
Package Inserts
Instructions for Use
SDS
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