Enzyme-linked immunosorbent assay for the detection of HBsAg in serum or plasma

The HBsAg EIA is a solid-phase simultaneous sandwich immunoassay, which employs monoclonal antibodies and polyclonal antibodies specific for HBsAg. Microtiter well are coated with monoclonal antibodies specific for HBsAg. A serum specimen is added to the antibody coated Microtiter wells together with enzyme conjugated polyclonal antibodies. HBsAg, if present, will form an antibody-HBsAg-antibody-enzyme complex. The plate is then washed to remove unbound material. Finally, a solution of substrate is added to the wells and incubated. A blue color will develop in proportion to the amount of HBsAg present in the specimen. The enzyme-substrate reaction can be stopped and the result is visualized by naked eye or read by EIA plate reader for absorbance at the wavelength of 450 nm(450 nm/630 nm) and is proportional to the amount of antibodies present in the specimen..

Materials provided with the kits:

ASSAY PROCEDURE:

It is strongly advised to analyze each specimen and controls in duplicate. All the reagents should equilibrate to room temperature before use.

1. Dispense one drop (50μl) of Positive Control as well as Negative Control in duplicate into respective wells. Set one blank well as background control, and 50ul of serum or plasma samples into respective test wells.
2. Add one drop (50μl) of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 minutes. Do not add Enzyme Conjugate to the blank well.
3. Place the microtiter plates into a humidified box, and incubate at 37°C for 30 minutes.
4. Wash each well 5 times by filling each well with diluted wash buffer, then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.
5. Add one drop (50μl) of Substrate Solution A (HRP-substrate) to each well, then add one drop (50μl) of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for 15 minutes.
6. Add 1 drop (50μl) of Stop Solution to each well to stop the color reaction. Read the OD value at 450 nm/630 nm with dual filter plate reader. It is option to read the OD value at 450 nm with single filter plate reader. (using the OD value of the blank well to correct all the OD reading from all wells)