Insulin Autoantibodies (IAA) ELISA Kit

Antigen: Insulin Autoantibodies (IAA)

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365971

Additional Information

Characteristics: This immunoassay kit allows for the in vitro semi-quantitative determination of human insulin autoantibodies concentrations in serum and other biological fluids.

Sample Type: Serum, Plasma

Specificity: This assay recognizes human insulin autoantibodies. No significant cross-reactivity or interference was observed.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with purified insulin antigen. Samples are then added to the appropriate microtiter plate wells and incubated. Then add Horseradish Peroxidase (HRP)-conjugated goat-anti-human antibody to each well and incubate. Finally, substrate solutions are 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 insulin autoantibodies 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 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. 2. Sample Dilute 10 μl sample using 1 00μl Sample Diluent (1:11), 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:
Bring all reagents and samples to room temperature before use. It is recommended that all samples and controls be assayed in duplicate. 1. Add 100 μl of Positive Control and 100μl of Negative Control, respectively. Add 10 0μl of diluted Sample per well. Cover with the adhesive strip. Incubate for 60 minutes at 37° C. 5 2. Aspirate each well and wash, repeating the process for a total of three or five 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. 3. Add 100μl of HRP-conjugate to each well. Incubate for 30 minutes at 37°C. HRP-conjugate may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 4. Wash plate three or five times as before. 5. Add 5 0μl 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. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. 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 insulin autoantibodies, compare the sample well with control. 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.
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. 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 (1x12ml), HRP-conjugate (1x12ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml), Positive Control (1x1.0ml), Negative Control (1x1.0ml)

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