Anti-nucleolus antibody (ANA) ELISA Kit

Antigen: Anti-Nucleolus Antibody (ANA)

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN366040

Additional Information

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

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

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with purified nuclear 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-nucleolus antibody IgG in the samples.

Protocol: Preparation of Reagents: 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. Sample Dilute 2µl sample using 198µl Sample Diluent (1:100), respectively. 3. HRP-anti-human IgG Dilute to the working concentration using HRP-anti-human IgG Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. 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. Assay Procedure: Calculation of Results: For calculation the valence of human anti-nucleolus antibody IgG antibody, compare the sample well with control. The well appears significant blue color is determined as Positive. The well not contains significant blue is determined as Negative. 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. 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.

Application Notes: When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Components: Reagent Quantity Assay plate 1 Sample Diluent 1 x 20 ml HRP-anti-human IgG Diluent 1 x 10 ml HRP-anti-human IgG 1 x 120µl (1:100) Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml Negative Control 1 x 800 µl 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.

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. Expiration date: six months from the date of manufacture.

Restrictions: For Research Use only