Major Histocompatibility Complex (MHC) ELISA Kit

Antigen: Major Histocompatibility Complex (MHC)

Synonyms: D18.5, MGC134453

Reactivity: Cow (Bovine)

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN364636

Additional Information

Alternative name: Major histocompatibility complex (MHC/BoLA)

Sample Volume: 50 - 100 µL

Assay Time: 1 - 4.5 h

Plate: Pre-coated 8 x 12 strip plate

Components: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120µl HRP-avidin 1 x 120µl Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml 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.

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of bovine MHC concentrations in cell culture supernates, serum, plasma and other biological fluids.

Sensitivity: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Minimum Detection Limit: 0.025 ng/mL

Detection Range: 0.04 ng/mL - 10 ng/mL

Application Details

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. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 50 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (50 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml).
3. Biotin-antibody Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively.
4. HRP-avidin Dilute to the working concentration using HRP-avidin 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. Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. 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: Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the MHC concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. 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. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, 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: Detection wavelength: 450 nm

Handling Advice: 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. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, 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.

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. An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.

Storage: 2-8°C

Storage Comment: Unopened: Store at 2 - 8° C. Do not use past kit expiration date.
Opened: May be stored for up to 1 month at 2 - 8° C. Try to keep coated assay plate in a sealed aluminium foil bag, and avoid the damp.

Expiry Date: Refer to the package label for the expiration date

Restrictions: For Research Use only

Publications

Product: Foucras, Corbière, Tasca et al.: "Alloantibodies against MHC class I: a novel mechanism of neonatal pancytopenia linked to vaccination." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 187, Issue 12, pp. 6564-70, 2011 (PubMed).