This assay employs a two-site sandwich ELISA to quantitate IFN-γ in samples. An antibody specific for IFN-γ has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IFN-γ present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for IFN-γ 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-γ bound in the initial step. The color development is stopped and the intensity of the color is measured.
Product Overview:
Components:
Reagents
Quantity
Reagents
Quantity
Assay plate (96 Wells)
1
Instruction manual
1
Standard (lyophilized)
2
Sample Diluent
1 x 20 mL
Biotin-Conjugate (concentrate 100 x)
1 x 120 μL
Biotin-Conjugate Diluent
1 x 20 mL
Streptavidin-HRP (concentrate 100 x)
1 x 120 μL
Streptavidin-HRP Diluent
1 x 20 mL
Wash Buffer (concentrate 25 x)
1 x 20 mL
Substrate Solution
1 x 12 mL
Stop Solution
1 x 10 mL
Adhesive Films
4
Specificity:
This assay has high sensitivity and excellent specificity for detection of Cat IFN-γ. No significant cross-reactivity or interference between Cat IFN-γ and analogues was observed.
Recovery:
Matrices listed below were spiked with certain level of recombinant Cat IFN-γ and the recovery rates were calculated by comparing the measured value to the expected amount of Cat IFN-γ in samples.
Sample Type
Number
Recovery range (%)
Average(%)
Serum
10
90-101
96
EDTA plasma
10
89-97
93
Heparin plasma
10
91-99
95
Precision:
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%
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Cat IFN-γ and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample Type
1:2
1:4
1:8
1:16
Serum
78-89%
81-99%
92-103%
95-105%
EDTA plasma
91-101%
90-98%
93-101%
91-98%
Heparin plasma
92-103%
93-102%
92-99%
91-101%
Stability:
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).
Sample collection and storage:
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.
Kits storage instructions:
Store at 2-8°C. Please refer to Instruction Manual.