CN113702362A - Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof - Google Patents
Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof Download PDFInfo
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Abstract
The invention discloses a method for quantitatively detecting IFN-gamma concentration by using a chemiluminescence method, which is characterized in that based on a ligand receptor binding principle, coating protein, a detection object IFN-gamma and a detection antibody are sequentially bound on a solid medium, and then a chromogenic substrate is added for detection; the detection steps are as follows: fixing a solid medium and specific protein of IFN-gamma, wherein the specific protein is coating protein, adding a detection sample, specifically fixing the IFN-gamma in the sample on the solid medium, then using a second antibody capable of combining the IFN-gamma, namely a detection antibody, fixing the second antibody on the fixed medium, and washing off non-specifically adsorbed impurities along with a washing process; the second antibody is directly or indirectly specially labeled, and finally, a substrate matched with the label is used for color development detection. The invention has the advantages of low cost, high sensitivity, controllable source and strong sustainable service.
Description
Technical Field
The invention relates to the field of biological analysis and detection, in particular to a detection method and a kit for detecting IFN-gamma concentration in human matrix by a double-antibody sandwich method and adopting a chemiluminescence detection mode.
Background
Interferon (IFN), a cytokine first discovered by Isaacs of England scientists in 1957 when studying the interference phenomenon of influenza virus by using chick chorioallantoic membrane, has various effects of inhibiting cell division, regulating immunity, resisting virus, resisting tumor, etc. The essence is protein, and the types of the protein can be divided into alpha, beta, gamma, omega and the like. IFNs induce cell resistance to viral infection by interfering with viral gene transcription or translation of viral protein components, thereby preventing or limiting viral infection, and are currently the leading antiviral and antitumor biologics.
The interferon involved in immune regulation is IFN-gamma, also known as immunomodulatory interferon. The immunoregulation interferon can express Fc receptor of IgG, so as to facilitate phagocytosis of antigen by macrophage, killing target cell by K, NK cell and activation of T, B lymphocyte, and enhance the immune response capability of organism. IFN-gamma can increase the expression of MHC II molecules on the surface of macrophage and enhance the antigen presenting capability. Furthermore, the Fc receptor expressed on the surface of macrophages can be enhanced, and the macrophages can be promoted to phagocytose immune complexes, antibody-coated pathogens and tumor cells. Meanwhile, the compound can stimulate neutrophils, enhance the phagocytic capacity of the neutrophils, activate NK cells, enhance the cytotoxic effect of the NK cells and the like to participate in immune regulation.
Interferon is an innate and adaptive cytokine in the defense against tumor development, IFN-gamma is produced by T lymphocytes stimulated by specific antigens, has a structure different from that of type I interferon, is acid-resistant, is a main macrophage stimulating factor of an organism, and has various regulation effects on immune response of the organism. Can activate effector cells, improve activity of natural killer cells, macrophages and tumor infiltrating lymphocytes, promote monocyte circulation, enhance expression of antigens and antibodies on the surface of immune cells, stimulate production of cytokines such as IL-2, tumor necrosis factor, interferon-alpha and the like, inhibit tumor cell division, induce genes to become antiviral proteins and the like.
The measurement of IFN-gamma concentration in serum and plasma is an important reference index for the safety evaluation of virus infection and immune regulation agent drugs. At present, the bioanalysis method for IFN-gamma concentration adopts an ELISA detection method based on a receptor ligand binding principle, and most of the bioanalysis methods exist in the form of a kit, such as an ELISA method R & D systems Cat. SIF50C or an electrochemiluminescence detection method MSD Cat. K151QOD and the like.
Although the available kits for measuring IFN-. gamma.concentration are available on the market, there is still an unmet need for performance. According to the information of the MSD kit, the concentration range of INF gamma in normal serum is 0.64-14.4pg/mL, and the sensitivity requirement on the method is strict. For example, the detection range of the kit of Abcam (ab48490) is 400-12.5pg/ml, and a sensitivity of about 5pg/ml is not required. The detection interval of an electrochemical luminescence detection method MSD kit (K151QOD-2) is 0.20-938pg/m, the requirement can be met, but the whole kit is very high in cost, the price of a matched instrument is high, compared with an ELISA kit, the kit has multiple choices, the MSD electrochemical luminescence detection method has monopoly status, replaceable products are not available, and once the purchase period is too long or the products are broken, the continuous operation of IFN-gamma concentration determination clinical detection business is not facilitated.
Disclosure of Invention
The present invention aims to provide a detection method and a detection kit thereof for quantitatively detecting IFN-gamma concentration by using a chemiluminescence method, which solves one or more of the above-mentioned problems of the prior art.
According to the method for quantitatively detecting the concentration of IFN-gamma by using the chemiluminescence method, which is provided by the invention, based on the ligand receptor binding principle, the envelope protein, the detection object IFN-gamma and the detection antibody are sequentially bound on a solid medium, and then a chromogenic substrate is added for detection; the method is characterized by comprising the following detection steps: fixing a solid medium and specific protein of IFN-gamma, wherein the specific protein is coating protein, adding a detection sample, specifically fixing the IFN-gamma in the sample on the solid medium, then using a second antibody capable of combining the IFN-gamma, namely a detection antibody, fixing the second antibody on the fixed medium, and washing off non-specifically adsorbed impurities along with a washing process; the second antibody is directly or indirectly specially marked, and finally, a chemiluminescent substrate matched with the marker is used for color development detection.
The detection method and the detection kit for quantitatively detecting the concentration of IFN-gamma by using the chemiluminescence method have high sensitivity and controllable sources, and are suitable for the detection kit for the concentration of IFN-gamma in a human blood matrix.
In some embodiments, the label of the detection antibody is HRP, and the second antibody is labeled directly or indirectly with HRP.
In some embodiments, the specific steps are as follows:
preparation of an ELISA plate containing the coating protein: diluting the coating antibody to a proper concentration, adding 100 mu L/hole into a 96 enzyme label plate, uniformly mixing at 500rpm for 10min, and incubating overnight at 4 ℃ for 16-24 h;
the next day, the solution of the microplate is discarded, washed with washing solution for three times, patted dry, added with 300. mu.L/well of blocking solution, and incubated for 1 hour at room temperature and 500 rpm;
setting up IFN-gamma gradient of detection object: the IFN-gamma standard is subjected to gradient dilution by using a sample analysis solution, the initial dilution concentration is 100pg/mL, and then the dilution is carried out for 9 points by two times of gradient dilution, the concentration range of the standard of a standard curve is 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.125pg/mL, 1.563pg/mL, 0.781pg/mL, 0.391pg/mL and 0.195pg/mL, and the sample analysis solution is used as a blank sample;
detection reaction: taking a sealed 96 enzyme label plate, discarding the solution of the microplate, washing for three times by using a washing solution, patting to dry, adding the diluted standard substance gradient solution into the microplate at 100 mu L/hole, and incubating for 1 hour at room temperature and 500 rpm; discarding the solution of the microplate, washing for three times with a washing solution, patting to dry, diluting the detection antibody by 100 times with a method analysis solution, adding the diluted detection antibody into the microplate at 100 muL/hole, and incubating for 1 hour at room temperature and 500 rpm;
discarding the solution of the microplate, washing three times with a washing solution, patting dry, and using a method analysis solution to convert the HRP-labeled streptavidin 1: 200, adding 100 mu L/hole into a microplate, and incubating for 1 hour at room temperature and 500 rpm;
and (3) color development detection: and (3) balancing the chemiluminescence detection reagent solution A and the chemiluminescence detection reagent solution B to room temperature half an hour in advance, and carrying out a reaction of 1: 1 mix, add 100 μ L/well to microplate and incubate for 3 minutes at room temperature at 500rpm, read using a multifunctional microplate reader using a chemiluminescent detection program.
A detection kit for quantitatively detecting IFN-gamma concentration by using a chemiluminescence method comprises: the kit comprises a 96-well plate, coating protein, a detection antibody and a chemiluminescence detection reagent, and the kit takes the 96-well plate as a solid phase carrier.
In some embodiments, the coating protein of the kit may be a recombinant receptor protein or a recombinant antibody; the kit adopts enzyme to be directly or indirectly combined with a detection antibody, and uses a chemiluminescent substrate with enzyme specificity to carry out chemiluminescent detection, wherein the enzyme is but not limited to HRP and luciferase, and the corresponding substrate is the chemiluminescent substrate of the enzyme.
Luminescence refers to the transition from a low-level ground state to a high-level excited state after energy is absorbed by electrons in a molecule or atom, and the high-level excited state returns to the low-level ground state in a short time to release energy, which is released as photons. Light emission can be classified into photo-stimulation light, bio-light, electro-stimulation light, chemiluminescence, and the like according to the energy source of the excited state formed by molecules or atoms. Chemiluminescence refers to the phenomenon of emission of light that accompanies a chemical reaction process. When some substance molecules in a detection reaction system, such as enzyme, substrate and the like, carry out chemical reaction, the signal molecules are excited to an excited state due to absorption of chemical energy generated during the reaction, and when the excited molecules return to a ground state from the excited state, light with a certain wavelength is emitted. The chemiluminescence detection method is a trace analysis method which is mainly characterized in that chemiluminescence is captured by an instrument according to the principle that the concentration of an object to be detected in a detection system and chemiluminescence intensity are linearly related under a certain condition, an optical signal is converted into an electric signal, and the content of the object to be detected is determined according to the size of the electric signal. The existing chemiluminescence analysis technology is mostly used in the field of western blotting, enzyme-labeled antibodies are used for reaction, and when chemiluminescence substrates (such as luminol, hydrogen peroxide and the like) are added for enzymatic reaction, an optical signal is generated. The signals may be acquired by photographic film or dedicated imaging equipment. Traces of protein down to the femtogram (10-15g) scale can be detected using chemiluminescence analysis techniques with extremely high sensitivity. However, in the western blot experiment, membranes are required to be used as solid carriers, the sample loading amount on one membrane is not high, the requirement of high-flux detection in drug research and development in the pharmaceutical industry cannot be met, the stability of the result is poor due to a complex operation process, and the requirements of methods such as precision, accuracy and stability of clinical detection cannot be met.
Receptor ligand binding enzyme-linked immunosorbent assays were developed in the early 70, 80 s of the 19 th century to replace radiolabelling techniques for studying protein-protein interactions (ligand-receptor, antigen-antibody) and concentration assays, and have been in the past for nearly 50 years. The ELISA technology and the MSD technology are both based on the receptor ligand binding enzyme-linked immunosorbent assay technology, the ELISA technology and the MSD technology take a 96-well plate as a solid carrier, and different chromogenic substrates are used for detection, so that the ELISA technology and the MSD technology have the characteristics of high throughput, and excellent precision, accuracy and stability of the method.
The invention uses the chemiluminescence analysis technology to combine with the receptor ligand and the enzyme-linked immunosorbent assay technology, takes a 96-pore plate as a solid carrier, exerts the high-sensitivity advantage of the chemiluminescence analysis technology, and simultaneously has the characteristics of high flux, excellent precision, accuracy and stability of the ELISA technology and the MSD technology. The biological analysis method for quantitatively detecting IFN-gamma concentration by a chemiluminescence method has the sensitivity as low as 0.5pg/mL, and the precision between plates and the precision in plates are less than 10%.
Drawings
FIG. 1 is a standard curve of IFN-. gamma.for the chemiluminescence method according to one embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1 establishment of IFN-. gamma.Standard Curve by ELISA method
The Abcam kit Human Interferon gamma ELISA Set (without plates) cat No.: the coated antibody IFN gamma Capture antibody in ab48490 was diluted 100-fold with 1 XPBS and 96-well plates (Thermofeisher) were added at 100. mu.L/wellTM439454) and mixing at 500rpm for 10min, and incubating at 4 deg.C overnight for 16-24 h.
The next day, discard the solution of the plate, wash three times with washing solution (washing solution component 1 XPBS + 0.1% Tween 20), pat dry, add 300 uL/hole of blocking solution (blocking solution/method analysis solution component 1 XPBS + 1% BSA + 0.1% Tween 20), incubate 1 hour at room temperature 500 rpm; IFN-gamma standard (standard from Abcam kit Human Interferon gamma ELISA Set (without plates) cat # ab48490) is diluted with sample analysis solution (the sample dilution is 10% rabbit serum added to the method analysis solution) in gradient with initial dilution concentration of 400pg/mL, and then diluted in gradient twice for 8 points, the standard concentration range of the standard curve is 400pg/mL, 200pg/mL, 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.125pg/mL, and the sample analysis solution is used as blank sample; taking a sealed 96 enzyme-linked immunosorbent assay plate, discarding the solution of the microplate, washing for three times by using a washing solution (the washing solution component is 1 multiplied by PBS + 0.1% Tween 20), patting to dry, adding the diluted standard substance gradient solution into the microplate at 100 mu L/hole, and incubating for 1 hour at room temperature and 500 rpm; discarding the solution of the plate, washing with washing solution (washing solution component 1 XPBS + 0.1% Tween 20) for three times, beating to dry, diluting with method analysis solution (blocking solution/method analysis solution component 1 XPBS + 1% BSA + 0.1% Tween 20) for 100 times the Detection biotinylated anti-IFN gamma antibody (Detection antibody from Abcam kit Human Interferon gamma ELISA Set (without out plates) cat # ab48490), adding to the plate at 100. mu.L/well, incubating at 500rpm for 1 hour at room temperature; discarding the solution of the microplate, washing three times with a washing solution (washing solution component 1 × PBS + 0.1% Tween 20), patting dry, and using a method analysis solution (blocking solution/method analysis solution component 1 × PBS + 1% BSA + 0.1% Tween 20) to convert HRP-labeled streptavidin (R & D cat # DY998) 1: 200, adding 100 mu L/hole into a microplate, and incubating for 1 hour at room temperature and 500 rpm; the TMB color developing solution (Thermo, product number 34028) A solution and B solution are balanced to room temperature half an hour in advance, and the reaction is carried out in a ratio of 1: 1 mixing, adding 100 mu L/well into a microplate, incubating for 15 minutes at room temperature in a dark place, adding 50 mu L/well ELISA termination solution for termination, using an enzyme-linked immunosorbent assay (Molecular Device model SpectraMax plus 384) to read by using absorbance and 450nM as detection wavelength and 650nM as reference wavelength, using SoftMax software (Molecular Device) to perform standard curve fitting by using 4 parameters according to the reading result, and selecting a weight factor of 1.
After data analysis, the data analysis result of the ELISA method IFN-gamma standard curve after function fitting is shown in Table 1, relative to the background signal of 0.013, the maximum detection signal is 2.401, the ELISA method generally considers that when the OD value is greater than 0.1, a stable detection result exists, a detection window which is 24 times that of the ELISA method is judged, the point within 25% of the return measurement deviation Bias% of each concentration point of the standard curve is calculated, and the detection range of the ELISA method is 12.5-400 pg/mL.
TABLE 1 data analysis results after function fitting of IFN-gamma standard curve by ELISA method
Example 2 establishment of IFN-gamma Standard Curve by chemiluminescence
The Abcam kit Human Interferon gamma ELISA Set (without plates) cat No.: the coated antibody IFN gamma Capture antibody in ab48490 was diluted 100-fold with 1 XPBS and 100. mu.L/well was added to a 96-well plate (Thermo Fisher)TM436110), mixing at 500rpm for 10min, and incubating at 4 deg.C overnight for 16-24 h.
The next day, discard the solution of the plate, wash three times with washing solution (washing solution component 1 XPBS + 0.1% Tween 20), pat dry, add 300 uL/hole of blocking solution (blocking solution/method analysis solution component 1 XPBS + 1% BSA + 0.1% Tween 20), incubate 1 hour at room temperature 500 rpm; IFN-gamma standard (standard from Abcam kit Human Interferon gamma ELISA Set (without plates) cat # ab48490) is diluted with sample analysis solution (the dilution of the sample is 10% rabbit serum is added to the method analysis solution) in gradient, the initial concentration of dilution is 100pg/mL, then the dilution is carried out for 9 points in double gradient, the concentration range of the standard curve is 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.125pg/mL, 1.563/mL, 0.781pg/mL, 0.391pg/mL, 0.195pg/mL, as blank sample; taking a sealed 96 enzyme-linked immunosorbent assay plate, discarding the solution of the microplate, washing for three times by using a washing solution (the washing solution component is 1 multiplied by PBS + 0.1% Tween 20), patting to dry, adding the diluted standard substance gradient solution into the microplate at 100 mu L/hole, and incubating for 1 hour at room temperature and 500 rpm; discarding the solution of the plate, washing with washing solution (washing solution component 1 XPBS + 0.1% Tween 20) for three times, beating to dry, diluting with method analysis solution (blocking solution/method analysis solution component 1 XPBS + 1% BSA + 0.1% Tween 20) for 100 times the Detection biotinylated anti-IFN gamma antibody (Detection antibody from Abcam kit Human Interferon gamma ELISA Set (without out plates) cat # ab48490), adding to the plate at 100. mu.L/well, incubating at 500rpm for 1 hour at room temperature; discarding the solution of the microplate, washing three times with a washing solution (washing solution component 1 × PBS + 0.1% Tween 20), patting dry, and using a method analysis solution (blocking solution/method analysis solution component 1 × PBS + 1% BSA + 0.1% Tween 20) to convert HRP-labeled streptavidin (R & D cat # DY998) 1: after 200 dilution, 100. mu.L/well was added to the plate and incubated at 500rpm for 1 hour at room temperature.
The chemiluminescence detection reagent (Thermo good No. 37074) solution A and solution B were equilibrated to room temperature half an hour in advance, and 1: 1 mixing, adding 100 mu L/well into a microplate, incubating for 3 minutes at room temperature and 500rpm, reading by using a multifunctional microplate reader (Tecan model Infinite F plex) and a chemiluminescence detection program, and performing standard curve fitting by using SoftMax software (Molecular Device) and 4 parameters according to the reading result, wherein the weight factor is 1/Y ^ 2.
The data analysis shows the IFN-gamma standard curve chart in figure 1.
The data analysis results after the standard curve is subjected to function fitting are shown in the following table 2, the background signal is 96551, the maximum detection signal is 13159275, a 136-time detection window is provided, the back detection deviation Bias% of each concentration point of the standard curve is 25% as a standard, the detection range of the method is expanded to 0.195-100pg/mL, and the method has excellent sensitivity and back detection accuracy.
TABLE 2 parameters of each concentration point of the standard curve after function fitting
And (3) comparing experimental data: the results of the example 1 and the example 2 show that the detection range of the ELISA method is 12.5-400pg/mL, the detection range of the chemiluminescence method is expanded to 0.195-100pg/mL, and the comparison result shows that the chemiluminescence method is more sensitive compared with the ELISA method, and the lowest detection limit can be reduced to 0.195pg/mL from 12.5 pg/mL; the detection window of ELISA is 32 times, the detection window of chemiluminescence method is 512 times, and chemiluminescence method has wider detection window compared with ELISA method.
Example 3 chemiluminescence method parallelism test of serum samples
The experiment was carried out according to the procedure of example 2 with the kit assembled according to example 2, and the preparation of serum samples was carried out using the sample diluent at the same time as the preparation of standard dilution, and the serum samples were diluted 1 and 2 times, respectively.
Standard curve fitting was performed using the same data processing method as in example 2 and the results obtained for the serum samples are shown in table 3 below.
The test results of 4 serum samples show that each sample can measure a concentration value close to each other in two dilution levels, which indicates that the IFN-gamma standard in the method and the samples in the serum have similar dose response curves in the method system, and the method has good parallelism.
The kit can be used for detecting IFN-gamma in a human serum matrix sample.
| Sample | Value | Result | MeanResult | ％CV | Dilution | | Bias％ | 1 | ％CV |
| SM-09-1 | 387710 | 2.076 | 2.065 | 0.8 | 1 | 2.065 | -7.17 | 5.3 | |
| 384506 | 2.054 | ||||||||
| SM-09-2 | 229651 | 0.947 | 0.959 | 1.7 | 2 | 1.917 | |||
| 232801 | 0.97 | ||||||||
| SM-18-1 | 295382 | 1.428 | 1.431 | 0.4 | 1 | 1.431 | -21.80 | 17.3 | |
| 296439 | 1.435 | ||||||||
| SM-18-2 | 175643 | 0.532 | 0.559 | 7 | 2 | 1.119 | |||
| 182696 | 0.587 | ||||||||
| SM-26-1 | 465596 | 2.608 | 2.649 | 2.2 | 1 | 2.649 | -0.60 | 0.4 | |
| 477786 | 2.69 | ||||||||
| SM-26-2 | 282176 | 1.333 | 1.316 | 1.7 | 2 | 2.633 | |||
| 277687 | 1.3 | ||||||||
| SM-30-1 | 396919 | 2.14 | 2.087 | 3.6 | 1 | 2.087 | 2.73 | 1.9 | |
| 381663 | 2.035 | ||||||||
| SM-30-2 | 246281 | 1.07 | 1.072 | 0.2 | 2 | 2.144 | |||
| 246749 | 1.074 |
TABLE 3 serum sample parallelism determination results
Example 4 precision and accuracy testing of the chemiluminescent method
The experiment was carried out according to the procedure of example 2 using the kit assembled according to example 2, and the QC samples were prepared using the standards at theoretical concentrations of 50pg/mL, 6.25pg/mL and 0.781 pg/mL.
The QC sample concentration calculation was performed using the same data processing method as in example 2. The results of the three tests are shown in Table 4 below, and the results of the three tests show that the precision of the bioanalysis method for quantitatively detecting IFN-gamma concentration by the chemiluminescence method is within +/-5%, and the accuracy of the method is within +/-5%, so that the method has excellent precision and accuracy.
| Theoretical concentration pg/ml | First measurement | Second measurement | Third measurement | Accuracy Bias% | Precision% CV |
| 50 | 47.81 | 51.49 | 49.61 | -0.73 | 3.7 |
| 6.25 | 6.41 | 6.04 | 6.15 | -0.80 | 3.1 |
| 0.781 | 0.81 | 0.79 | 0.82 | 3.29 | 1.9 |
TABLE 4 precision and accuracy test results
The invention uses the chemiluminescence analysis technology to combine with the receptor ligand and the enzyme-linked immunosorbent assay technology, takes a 96-pore plate as a solid carrier, exerts the high-sensitivity advantage of the chemiluminescence analysis technology, and simultaneously has the characteristics of high flux, excellent precision, accuracy and stability of the ELISA technology and the MSD technology. The biological analysis method for quantitatively detecting IFN-gamma concentration by a chemiluminescence method has the sensitivity as low as 0.195pg/mL, and the precision between plates and the precision in plates are less than 10 percent.
The foregoing is only a preferred form of the invention and it should be noted that numerous similar variations and modifications could be made by those skilled in the art without departing from the inventive concept herein, which shall be considered to be within the scope of the appended claims.
Claims (6)
1. A method for quantitatively detecting IFN-gamma concentration by using a chemiluminescence method is characterized in that based on a ligand receptor binding principle, coating protein, a detection object IFN-gamma and a detection antibody are sequentially bound on a solid medium, and then a chromogenic substrate is added for detection; the method is characterized by comprising the following detection steps: fixing a solid medium and specific protein of IFN-gamma, wherein the specific protein is coating protein, adding a detection sample, specifically fixing the IFN-gamma in the sample on the solid medium, then using a second antibody capable of combining the IFN-gamma, namely a detection antibody, fixing the second antibody on the fixed medium, and washing off non-specifically adsorbed impurities along with a washing process; the second antibody is directly or indirectly specially marked, and finally, a chemiluminescent substrate matched with the marker is used for color development detection.
2. The method of claim 1, wherein the label of the detection antibody is HRP, and the second antibody is labeled directly or indirectly with HRP.
3. The method for quantitatively determining the concentration of IFN- γ using the chemiluminescence method according to claim 1 or 2, wherein the method comprises the following steps:
preparation of an ELISA plate containing the coating protein: diluting the coating antibody to a proper concentration, adding 100 mu L/hole into a 96 enzyme label plate, uniformly mixing at 500rpm for 10min, and incubating overnight at 4 ℃ for 16-24 h;
the next day, the solution of the microplate is discarded, washed with washing solution for three times, patted dry, added with 300. mu.L/well of blocking solution, and incubated for 1 hour at room temperature and 500 rpm;
setting up IFN-gamma gradient of detection object: the IFN-gamma standard is subjected to gradient dilution by using a sample analysis solution, the initial dilution concentration is 100pg/mL, and then the dilution is carried out for 9 points by two times of gradient dilution, the concentration range of the standard of a standard curve is 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.125pg/mL, 1.563pg/mL, 0.781pg/mL, 0.391pg/mL and 0.195pg/mL, and the sample analysis solution is used as a blank sample;
detection reaction: taking a sealed 96 enzyme label plate, discarding the solution of the microplate, washing for three times by using a washing solution, patting to dry, adding the diluted standard substance gradient solution into the microplate at 100 mu L/hole, and incubating for 1 hour at room temperature and 500 rpm; discarding the solution of the microplate, washing for three times with a washing solution, patting to dry, diluting the detection antibody by 100 times with a method analysis solution, adding the diluted detection antibody into the microplate at 100 muL/hole, and incubating for 1 hour at room temperature and 500 rpm;
discarding the solution of the microplate, washing three times with a washing solution, patting dry, and using a method analysis solution to convert the HRP-labeled streptavidin 1: 200, adding 100 mu L/hole into a microplate, and incubating for 1 hour at room temperature and 500 rpm;
and (3) color development detection: and (3) balancing the chemiluminescence detection reagent solution A and the chemiluminescence detection reagent solution B to room temperature half an hour in advance, and carrying out a reaction of 1: 1 mix, add 100 μ L/well to microplate and incubate for 3 minutes at room temperature at 500rpm, read using a multifunctional microplate reader using a chemiluminescent detection program.
4. A detection kit for quantitatively detecting IFN-gamma concentration by using a chemiluminescence method comprises: the method is characterized in that: the kit comprises a 96-well plate, coating protein, a detection antibody and a chemiluminescence detection reagent, and the kit takes the 96-well plate as a solid phase carrier.
5. The kit for quantitative determination of IFN- γ concentration according to claim 4, wherein the coating protein of the kit is a recombinant receptor protein or a recombinant antibody; the kit adopts enzyme to be directly or indirectly combined with a detection antibody, and uses a chemiluminescence substrate with enzyme specificity to carry out chemiluminescence detection.
6. The detection kit for the quantitative determination of IFN-y concentration by chemiluminescence according to claim 5, wherein the enzyme is but not limited to HRP or luciferase, and the corresponding substrate is a chemiluminescent substrate of the enzyme.
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