Test Principle:t|The microtiter plate has been pre-coated with an antigen. Standards or samples are then added to the appropriate microtiter plate with HRP-conjugated secondary antibody. After TMB substrate solution is added, those wells that contain AIRA will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of AIRA in the samples is then determined by comparing the O.D. of the samples to the standard curve.

3.125-200ng/ml

<1.29ng/ml

*152281A: Microtiter Plate, 96 wells, Pre-coated, ready to use.*152281B: Standard, 2x1vial 152281C: Standard Diluent, 1x20ml*152281D: Detection Reagent A, 1x120ul 152281E: Assay Diluent A, 1x12ml152281F: TMB Substrate, 1x9ml152281G: Stop Solution, 1x6ml152281H: Wash Buffer, 30X, 1x20ml

Store *152281A, *152281B and *152281D at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.

1. Microplate reader with 450 ± 10nm filter.2. Precision single or multi-channel pipettes and disposable tips.3. Eppendorf Tubes for diluting samples.4. Deionized or distilled water.5. Absorbent paper for blotting the microtiter plate.6. Container for Wash Solution

Serum

Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination.2. Sample hemolysis will influence the results so hemolyzed specimens should not be used.3. When performing the assay, bring samples to room temperature.

1. Determine the number of wells required for standards, blank and samples. Add 100ul each of standards (see Reagent Preparation), blanks and samples into the appropriate wells. Cover with the plate sealer. Incubate for 2 hours at 37°C.2. Remove the liquid of each well; do not wash.3. Add 100ul of Detection Reagent A working solution to each well. Incubate for 1 hour at 37°C after covering it with the plate sealer.4. Aspirate the solution and wash each well for 1-2 minutes with 350ul of 1x Wash Solution using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Wash for a total of 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.5. Add 90ul Substrate Solution to each well. Cover with a new plate sealer. Incubate for 15-25 minutes at 37°C (Do not exceed 30 minutes). Protect from light. Addition of Substrate Solution will turn the liquid blue.6. Add 50ul of Stop Solution to each well. The liquid will turn yellow on addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there are no bubbles on the surface of the liquid. Read the absorbance at 450nm immediately.

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