Human IFN-γ ELISA Kit

Need to bring your own test equipment

1. Microplate reader (can measure the absorption value of 450nm detection wavelength and the absorption value of 540nm or 570nm correction wavelength)
2. High-precision liquid dispenser and disposable tip
3. Distilled water or deionized water
4. Bottle washing (spray bottle), multi-channel plate washer or automatic plate washer
5. 500mL measuring cylinder

1. Preparation before the experiment

1. Sample collection and storage

① Cell culture supernatant: particulate matter should be removed by centrifugation; Test the sample immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator at-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×).

② Serum: Use a serum separation tube (SST) to collect samples, and place the samples at room temperature for 30 minutes. Centrifuge for 15 minutes at a rotation speed of 1000 g. The serum was removed immediately and tested immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator ≤-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×).

③ Plasma: Plasma was collected using EDTA, heparin or citric acid as anticoagulant, centrifuged for 15 minutes within 30 minutes after collection, rotated at 1000g, and detected immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator ≤-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×).

1. Reagent preparation (Please place all reagents and samples at room temperature before use and let them stand 15 Minutes. All experimental samples and standards are recommended Do repeat hole detection）

① Preparation of 1 × washing liquid: The concentrated washing liquid in the kit is 20 × mother liquid, which needs to be diluted into 1 × working liquid with distilled water before use.Example: Take 10mL of concentrated washing solution + 190mL of distilled water and make the volume to 200mL. In actual operation, the amount used can be calculated first, and then prepared.

② Preparation of 1 × dilution buffer: The concentration and dilution buffer in the kit is 10 × mother liquor, which should be diluted to 1 × working solution with distilled water before use.Example: Make up to 30 mL with 3 mL of concentration and dilution buffer + 27 mL of distilled water. In actual operation, the required amount of dilution buffer solution can be calculated according to the sample dilution factor, and then prepared.

③ Antibody detection: centrifuge the dry powder to the bottom of the tube, dissolve it with 110uL of dilution buffer (1 ×), and let it stand at room temperature for 5 minutes to obtain 100 × mother liquor; Dilute to 1 × working solution before use. Calculate the required volume according to the dosage of 100uL per well.Example: After 10 wells were used, 10 uL of the detection antibody having a working concentration of 100 times was taken, and the volume was diluted to 1 mL using a dilution buffer (1 ×) to obtain 1 mL of the detection antibody having a working concentration of 1 ×.

④ SA-HRP: SA-HRP is 40 × mother liquor, which needs to be diluted with dilution buffer (1 ×) before use to prepare 1 × working solution, and the required amount per well is 100uL.Example: After 10 wells were used, 25 uL of 40 × mother liquor + 975 uL of dilution buffer (1 ×) was diluted to 1 mL to obtain 1 mL of detection antibody having a 1 × working concentration.

⑤ Developer: According to 100uL per hole, calculate the dosage required for the current test, take out the corresponding volume of developer, and protect it from light; The developer removed is for the same day use only.

⑥ Standard: The freeze-dried standard is re-dissolved with dilution buffer (1 ×), and the re-dissolving volume is 1000uL to obtain the standard mother liquor with a concentration of 600pg/mL. Gently shake for at least 5 minutes and it dissolves well. 300 uL of dilution buffer (1 ×) was added to each dilution tube. Make serial dilutions of the standard mother liquor according to the figure below, and each tube must be thoroughly mixed before pipetting to the next tube. The standard mother solution without dilution can be used as the highest point of the standard curve (600 pg/mL), and the dilution buffer (1 ×) can be used as the zero point of the standard curve (0 pg/mL).

2. Operation steps

1. Prepare all required reagents and standards;
2. Take out the microplate from the sealed bag that has been balanced to room temperature. Please put the unused slats back into the aluminum foil bag and reseal them;
3. Add 300uL of washing liquid to the microplate, let it stand and soak for 30 seconds, discard the washing liquid and pat the microplate dry on absorbent paper. Please use it immediately and do not let the microplate dry;
4. Add different concentration standards, experimental samples or quality control products to the corresponding wells, 100uL per well. Sealing the reaction hole with plate sealing adhesive paper and incubating at room temperature for 2 hours;
5. Suck off the liquid in the plate and wash the plate with a bottle washer, a multi-channel plate washer or an automatic plate washer. 300 uL of washing solution was added to each well, and then the washing solution in the plate was aspirated off. Repeat the operation 3 times. Trying to absorb the residual liquid as much as possible every time you wash the plate will help to get good experimental results. At the end of the last plate washing, please suck all the liquid in the plate or turn the plate upside down, and pat all the residual liquid dry on absorbent paper;
6. Add 100 uL of detection antibody to each well. Seal the reaction wells with plate sealing tape and incubate at room temperature for 2 hours;
7. Repeat the plate washing operation in step 5;
8. Add 100uLSA-HRP to each well and incubate at room temperature for 20 minutes. Be careful to avoid light;
9. Repeat the plate washing operation in step 5;
10. Add 100uL of chromogenic solution to each microwell, incubate at room temperature for 5-30 minutes, and avoid light;
11. Add 50uL of stop solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution changes to green or the color changes are inconsistent, pat the microplate gently to mix the solution evenly;
12. Within 30 minutes after adding the stop solution, measure the absorbance value of 450nm using a microplate reader, and set 540nm or 570nm as the calibration wavelength. If dual-wavelength correction is not used, the accuracy of the results may be affected;
13. Calculation Results: Average the corrected absorbance values (OD450-OD540 or OD570), multiple well readings for each standard and sample, and then subtract the average zero standard OD value. Four-parameter logic (4-PL) curve fitting was performed using computer software to create the standard curve. Alternatively, a curve can be generated by plotting the logarithm of the standard concentration versus the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process can generate a data fit that is sufficiently useful but less accurate. If the sample is diluted, the concentration should be calculated by multiplying the dilution factor.

Note

The standard curve data provided is for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.

3. Kit parameters

1. Recovery rate: Different levels of human IFN-γ were spiked into cell culture medium samples, and the recovery rate was determined. The recoveries ranged from 90 to 116%, with an average recovery of 103%.
2. Sensitivity: The lowest measurable dose (MDD) of human IFN-γ is generally less than 7.8 pg/mL. The lowest measurable value is the corresponding concentration calculated from the mean of the zero-point absorbance values of 20 standard curves plus two standard deviations.
3. Calibration: This ELISA kit was calibrated by high-purity recombinant human IFN-γ protein expressed by E. coli.
4. Linearity: 4 different samples were spiked with high concentrations of human IFN-γ, and then the samples were diluted to the detection range with diluent (1 ×) to determine their linearity.

1. Specificity: This ELISA method can detect natural and recombinant human IFN-γ protein. The following factors were formulated with diluent (1 ×) at a concentration of 50 ng/mL to detect cross-reactivity with human IFN-γ. Interference with human IFN-γ was detected by incorporating 50 ng/mL of the interfering factor into the mid-range recombinant human IFN-γ control. No significant cross-reactivity or interference was observed. There was approximately 20% cross-reactivity with recombinant rhesus IFN-γ.

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4. Analysis of frequently asked questions

1. Whiteboard (after the color development is completed, no color appears)

1. Flower plate (blank and negative positive controls are normal, but the OD value of specimen wells is obviously higher)

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Please use it within the validity period of the kit (new and old products are shipped randomly)

Gamma interferon (also known as type II interferon) is an important cytokine that can exercise immunomodulatory function. It was discovered for its antiviral activity. Interferon gamma plays a key role in host defense through antiviral, antiproliferative, and immunomodulatory functions. In many types of cells, gamma interferon induces cytokine production and upregulates the expression of different membrane proteins, including type I/II major histocompatibility complex (MHC-I, MHC-II), Fc receptors, leukocyte adhesion molecules, B7 family antigens. Gamma interferon can effectively activate macrophages and guide the synthesis, type transformation and secretion of B cell immunoglobulin. Gamma interferon can also affect the development of T helper cell subtypes by inhibiting the differentiation of Th2 and stimulating the growth of Th1. Gamma interferon plays an important role in some inflammatory disease processes such as autoimmunity and atherosclerosis.

Biologically active gamma interferon is a non-covalently bound dimer with a molecular weight of about 20-25KD and varying degrees of glycosylation. Mature human interferon gamma has close to 90% homology with the amino acid sequence of interferon gamma from rhesus monkey, 59-64% homology with interferon gamma from cattle, dog, horse, cat and pig, and 37-43% homology with interferon gamma from cotton rat, mouse and rat. The interferon dimer first binds to the transmembrane receptor IFN-γRI (α subunit), and then binds to IFN-γRII (β subunit) to form a functional complex containing two α subunits and two β subunits; Wherein the IFN-γRII in the complex receptor can increase the affinity of the ligand as well as the signal transduction efficiency. Although the γ subunit is constitutively expressed in many types of cells, the β subunit cell expression regulates the influence of the response state of the receptor interferon γ and is strictly regulated.

Interferon gamma can be secreted by many cells under a series of inflammatory conditions, including dendritic epidermal/γδ T cells, keratinocytes, peripheral blood γδ T cells, mast cells, neurons, CD8 + T cells, macrophages, B cells, neutrophils, natural killer cells, CD4 + T cells and testicular sperm cells, etc.