H1N1 IgG Antibody ELISA Kit

Antigen: H1N1 IgG Antibody

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN367611

Additional Information

Characteristics: This immunoassay kit allows for the in vitro semi-quantitative determination of mouse H1N1 IgG antibody concentrations in serum, plasma and other biological fluids. 

Sample Type: Serum, Plasma

Specificity: This assay recognizes human H1N1 IgG antibody. No significant cross-reactivity or interference was observed.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with purified HA antigen. Samples are then added to the appropriate microtiter plate wells and incubated. Then add Horseradish Peroxidase (HRP)-conjugated anti-human IgG to each well and incubate. Finally, a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. 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. Calculate the valence of anti-H1N1 IgG antibody in the samples.

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 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. HRP-anti-human IgG Dilute to the working concentration using HRP-anti-human IgG Diluent(1:100), respectively. 3. Sample Dilute 1 μl sample using 400μl Sample Diluent (1:401), respectively. 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:
To ensure the accurateness of the results, please control the total time of adding all samples each step (includes add sample, HRP-anti-human IgG working solution, TMB Substrate and Stop Solution) in 3 minutes. We recommend the customers divided the total samples into several batches in order to obtain the most accurate results. 5 1. Bring all reagents and samples to room temperature before use. It is recommended that all samples, and controls be assayed in duplicate. 2. Add 100 μl of Positive Control and Negative Control, respectively. Add 100μl of Sample per well. Cover with the adhesive strip. Incubate for 30 minutes at 37° C. 3. Aspirate each well and wash, repeating the process for a total of three washes. Wash by filling each well with Wash Buffer (200μl) 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 100μl of HRP-anti-human IgG working solution to each well. Incubate for 30 minutes at 37°C. HRP-anti-human IgG working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 5. Wash plate five times as before. 6. Add 90μl of TMB Substrate to each well. Incubate in 20 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 7. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 8. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. 6.
Calculation of Results:
For calculation the valence of human H1N1 IgG antibody, compare the sample well with control. While ODsample ≥1. 4x ODnegative: Positive While ODsample ＜ 1.4x ODnegative: 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.This 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), Sample Diluent (2x20ml), HRP-anti-human IgG Diluent (1x10ml), HRP-anti-human IgG 1x120μl (1:100) Wash Buffer (25×concentrate) (1x20ml), TMB Substrate (1x10ml), Stop Solution (1x10ml), Positive Control (1x800 µl), Negative Control (1x800 µl)

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.

Restrictions: For Research Use only

Publications

Publications: Temiz, Kasifoglu, Kiris et al.: "Immune response after a single vaccination against 2009 influenza A H1N1 in hemodialysis patients." in: Renal failure, Vol. 32, Issue 6, pp. 716-20, 2010 (PubMed).