Technical Manual for Human Interferon Gamma (IFN - γ) ELISA Detection Kit

BS-0033 Human Interferon Gamma (IFN - γ) ELISA Kit

1、 Detection principle

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Pre coat the anti human IFN - γ monoclonal antibody onto a microplate, and sequentially add the sample, standard, and biotinylated detection antibody to form an antibody antigen enzyme-linked antibody complex. After washing, Streptavidin HRP labeled with horseradish peroxidase was added for binding, and color was developed using TMB substrate. The color depth is positively correlated with the concentration of IFN - γ in the sample, and quantitative analysis is performed by measuring the absorbance (OD value) using an enzyme-linked immunosorbent assay (ELISA).

2、 Composition of reagent kit

Component 96 hole configuration, 48 hole configuration, storage conditions

Pre packaged microporous plate with 12 holes x 8 strips, 12 holes x 4 strips, 2-8 ℃

Freeze dried standard 1 tube 1 tube 2-8 ℃ (-20 ℃ after reconstitution)

Biotin labeled antibody 100 μ L 100 μ L 2-8 ℃ (away from light)

Streptomycin HRP 100 μ L 100 μ L 2-8 ℃ (away from light)

Sample diluent 30mL 15mL 2-8 ℃

20 x Wash buffer 20mL 10mL 2-8 ℃

TMB substrate solution 10mL 5mL 2-8 ℃, avoid light

Termination solution 10mL 5mL 2-8 ℃

4 sheets of sealing film and 2 sheets at room temperature

3、 Sample requirements

1. Sample type

Serum/plasma: Use EDTA or heparin for anticoagulation to avoid hemolysis.

Cell culture supernatant: Centrifuge to remove particles and avoid repeated freezing and thawing.

Other biological fluids, such as cerebrospinal fluid and ascites, need to be clarified before testing.

2. Sample processing

Dilution ratio: Serum/plasma is recommended to be diluted 1:5 (sample: diluent=1:4), and cell supernatant is recommended to be diluted 1:2.

Storage conditions: Short term (≤ 7 days) storage at 2-8 ℃, long-term packaging and freezing at -80 ℃ (to avoid repeated freeze-thaw cycles).

4、 Experimental steps

1. Preparation of reagents

Washing solution: Dilute 20 x concentrated solution with distilled water to 1 x working solution (such as 20mL concentrated solution+380mL water).

Standard solution reconstitution: Add 1mL of standard dilution solution to freeze-dried standard solution, let it stand for 15 minutes, and prepare a high concentration mother liquor.

Gradient dilution: Take 7 EP tubes and add 500 μ L of diluent to each tube. Transfer 500 μ L from the mother liquor to one tube and mix well. Dilute in multiple ratios in sequence (example: 1000 → 500 → 250 → 125 → 62.5 → 31.25 → 15.625 pg/mL).

2. Sample addition and incubation

Add 100 μ L of standard substance or diluted sample to each well (recommended for retesting).

Incubate at 37 ℃ in the dark for 90 minutes and seal with a sealing film.

3. Washing

Shake off the liquid in the hole, wash 3 times with 1 x detergent, soak for 1 minute each time, and pat dry.

4. Color rendering and termination

Add 100 μ L of biotin labeled antibody to each well (diluted 1:100 with antibody diluent in advance), and incubate at 37 ℃ in the dark for 60 minutes.

After washing, add 100 μ L of streptavidin HRP to each well (diluted with ABC diluent 1:100 in advance), and incubate at 37 ℃ in the dark for 30 minutes.

After washing, add 100 μ L of TMB substrate solution to each well and incubate at 37 ℃ in the dark for 15-20 minutes.

Add 50 μ L of termination solution to each well to stop the reaction (the solution turns from blue to yellow).

5. Measurement

Measure the OD value at a wavelength of 450nm (reference wavelength 570-630nm) using an enzyme-linked immunosorbent assay (ELISA) reader within 30 minutes.

5、 Result calculation

Draw a standard curve (four parameter fitting) with standard concentration as the horizontal axis and OD value as the vertical axis.

Based on the OD value of the sample, calculate the actual concentration through regression equation. If the sample is diluted, multiply it by the dilution factor.

6、 Performance indicators

Parameter indicators

Sensitivity ≤ 1.0 pg/mL

Detection range 15.63-1000 pg/mL

Precision in board CV<10%

Recovery rate 83% -113%

There is no cross reactivity between specificity and human IL-2, IL-4, etc

7、 Precautions

Reagent storage: the unused Flat noodles should be sealed and stored at 2-8 ℃ to avoid moisture; TMB substrate solution should be kept away from light.

Sample processing: Avoid using hemolysis and hyperlipidemia samples; High concentration samples require pre experimentation to determine the dilution factor.

Operating standards:

Avoid bubbles when adding samples, and replace the nozzle.

The incubation time should be strictly controlled within the specified range to prevent evaporation.

Quality control: Blank wells (S0) and negative controls must be set up for each batch of experiments.

Safety reminder: If the termination liquid contains acid, gloves should be worn for operation; Waste should be treated according to biological hazards.

8、 Validity period

The validity period of the reagent kit is 6 months at 2-8 ℃. After unpacking, it is recommended to pack and freeze the standard sample at -20 ℃.

Note: The above is for reference only. This product is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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