Hepatitis B Virus Surface Antigen (HBSAg) ELISA Kit

Antigen: Hepatitis B Virus Surface Antigen (HBSAg)

Epitope: Surface

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN366121

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human HBsAg concentrations in serum, plasma and other biological fluids.

Sample Type: Serum, Plasma

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with HBsAb. Samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated HBsAb and incubated. Then substrate solutions are added to each well. Only those wells that contain HBsAg and enzyme-conjugated HBsAb will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

Protocol: Reagent Preparation:
Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 600 ml of Wash Buffer. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. 4.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, and controls be assayed in duplicate. 1. Set a Blank well without any solution. Add 5 0μl of Negative Control, Positive Control or Sample per well. 2. Add 50 μl of HRP-conjugate to each well (not to Blank!). Cover the microtiter plate with a adhesive strip. Incubate for 30 minutes at 37°C. 3. Aspirate each well and wash, repeating the process five times for a total of five washes. Wash by filling each well with Wash Buffer (200 μl ) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 50 μl of Substrate A and 50μl of Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6 5. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 6. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
For calculation the valence of human HBsAg, compare the sample well with control. When OD negative ＜ 0.05, calculate it as 0.05. While OD sample / OD negative ≥2.1: Positive While OD sample / OD negative ＜ 2.1: Negative.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. 7This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), HRP-conjugate (1x7ml), Positive Control (1x1ml), Negative Control (1x1ml), Substrate A (1x7ml), Substrate B (1x7ml), Wash Buffer 1x30ml (20×concentrate) Stop Solution (1x7ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Research Area: Virology

Restrictions: For Research Use only