Canine Interferon alpha-1/2 (IFN-α1/2) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate IFN-α1/2 in Canine serum, plasma, cell culture supernates. An antibody specific for IFN-α1/2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IFN-α1/2 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for IFN-α1/2 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IFN-α1/2 bound in the initial step. The color development is stopped and the intensity of the color is measured.

The interferons (IFNs) are a family of cytokines with potent antiviral, antiproliferative and immunomodulatory properties. IFNs were originally discovered as molecules that could reduce the ability of a normal virus to infect cells, a process called viral 'interference'. IFNs have been classified into two major types of IFNs, type I and type II, based on their interactions to a specific cell surface receptor. In Canines, there are 13 different IFN-alpha genes, designated as IFN-α1, -α2, - α4, - α5, - α6, - α7, - α8, - α10, - α13, - α14, - α16, - α17 and - α21, and one each of the IFN beta (IFNB), IFN-Epsilon, IFN-Kappa and IFN-Omega genes. The Canine IFNA gene family shares 70-80% amino acid sequence homology, and about 35% identity with IFNB. The high degree of amino-acid sequence similarity within the IFNA genes suggests a common ancestor gene.

This assay has high sensitivity and excellent specificity for detection of Canine IFN-α1/2. No significant cross-reactivity or interference between Canine IFN-α1/2 and analogues was observed.

Matrices listed below were spiked with certain level of recombinant Canine IFN-α1/2 and the recovery rates were calculated by comparing the measured value to the expected amount of Canine IFN-α1/2 in samples.

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Canine IFN-α1/2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C 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 EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.