Human IFN-γ(Interferon Gamma) ELISA Kit

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Properties

Protocol PDF Click for Protocol
Catalog Number TK02099
Species Human
Design Sandwich
Standards 7 standards, serially diluted from 1 prepared lyophilized standard.
Controls
Sample Types Serum, plasma and other biological fluids
Sample Volume 100 μL
Assay Desc.
Standard Range 15.63—1000 pg/mL
Sensitivity 9.38 pg/mL
Radioactivity
Other Names IFNG, IFG, IFI, Type II Interferon
Storage 2 - 8°C
Postscript For research use only, not for use in diagnostic procedures.

Intended use

This ELISA kit applies to the in vitro quantitative determination of Human IFN-γ(Interferon Gamma) concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Human IFN-γ(Interferon Gamma). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Human IFN-γ(Interferon Gamma) and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human IFN-γ(Interferon Gamma), biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human IFN-γ(Interferon Gamma). You can calculate the concentration of Human IFN-γ(Interferon Gamma) in the samples by comparing the OD of the samples to the standard curve.

Typical data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration(pg/ml) O.D Average
1000 2.485
2.527
2.506
500 1.558
1.608
1.583
250 0.967
0.957
0.962
125 0.465
0.489
0.477
62.5 0.252
0.242
0.247
31.25 0.172
0.146
0.159
15.63 0.107
0.113
0.11
0 0.048
0.068
0.058

Precision

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of Human IFN-γ(Interferon Gamma). And the sample of Human IFN-γ(Interferon Gamma) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Human IFN-γ(Interferon Gamma). And the sample of Human IFN-γ(Interferon Gamma) were tested on 3 different plates, 30 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample 1 2 3 1 2 3
n 30 30 30 30 30 30
Mean (pg/ml) 49.71 139.76 393.02 48.78 149.14 450.24
Standard deviation 2.58 6.31 16.06 2.6 6.72 18.13
C V (%) 5.19 4.51 4.09 5.33 4.51 4.03

Recovery

Matrices listed below were spiked with certain level of Human IFN-γ(Interferon Gamma) and the recovery rates were calculated by comparing the measured value to the expected amount of Human IFN-γ(Interferon Gamma) in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 92-107 96
EDTA plasma (n=5) 91-101 93
heparin plasma (n=5) 90-97 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IFN-γ(Interferon Gamma) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

    Serum (n=5) EDTA plasma (n=5) heparin plasma (n=5)
1:2 Range (%) 91-104 94-104 92-100
Average (%) 95 94 93
1:4 Range (%) 93-108 89-104 87-103
Average (%) 94 95 94
1:8 Range (%) 89-106 91-99 90-102
Average (%) 94 93 95
1:16 Range (%) 94-99 87-102 92-105
Average (%) 93 93 94

Stability

The stability of Human IFN-γ(Interferon Gamma) ELISA Kit is determined by the loss rate of activity. The loss rate of Human IFN-γ(Interferon Gamma) ELISA Kit is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 4℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Other supplies required

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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