Kit for detecting tumor necrosis factor alpha and application thereof

Abstract

The invention discloses a kit for detecting a tumor necrosis factor alpha and application of the kit. The kit comprises a reagent R1, a reagent R2 and a reagent R3, the R1 reagent comprises a biotin-labeled anti-tumor necrosis factor alpha antibody 1 solution; the R2 reagent comprises an alkaline phosphatase labeled anti-tumor necrosis factor alpha antibody 2 solution; and the R3 reagent comprises a magnetic particle suspension coated with streptavidin. When the kit is used for detecting the tumor necrosis factor alpha, the kit has the advantages of short reaction time, simplicity and convenience in operation, high sensitivity, strong specificity, reagent stability, good repeatability and accurate test result, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay, and particularly relates to a kit for detecting tumor necrosis factor alpha and application thereof. BACKGROUND

[0002] Tumor necrosis factor (TNF) is a cytokine naturally produced by macrophages in response to bacterial infection or other immune source, is a kind of cytokine that can directly cause tumor cell death, and is one of the strongest biological active factors found so far that can directly kill tumor cells. Tumor necrosis factor and interferon can synergistically kill tumor cells, and are divided into two types according to their sources and structures, namely TNF-α and TNF-β. TNF-α is mainly secreted by monocyte-macrophages; and TNF-β is mainly secreted by activated T lymphocytes.

[0003] Tumor necrosis factor alpha (TNF-α) is an important inflammatory mediator and immune regulator, has a molecular weight of 17 kDa, and the natural form exerting biological effect is a homotrimer, and belongs to pro-inflammatory cytokines. Because TNF-α receptors exist on the surface of almost all cells, TNF-α shows various biological activities, plays a core role in inflammation, apoptosis and immune system development, participates in normal inflammatory response and immune response, and can synergistically regulate the production of other cytokines, cell survival and death to coordinate the homeostasis of tissues.

[0004] TNF-α has cytotoxicity to various tumor cells, and is an important factor for mediating immune response of bacterial infection. Normal level of TNF-α can participate in resisting bacterial, viral and parasitic infections, promote tissue repair, and induce tumor cell apoptosis. However, when TNF-α is produced and released in large quantities in the body, it can destroy the immune balance of the body, cause various pathological damages, such as multiple organ failure caused by cachexia and septic shock, rheumatoid arthritis, multiple sclerosis, various autoimmune diseases, bone marrow hematopoietic disorder syndrome and other diseases. Accurate determination of the content of TNF-α in serum is helpful for the diagnosis and treatment monitoring of these diseases, and can be used as an evaluation index of disease condition, treatment effect and prognosis.

[0005] The main technologies of tumor necrosis factor alpha (TNF-α) detection reagent include ELISA detection technology, chemiluminescence immunoassay, radioimmunoassay, electrochemical immunosensor, lateral flow chromatography and the like. The main defects of the prior art are that the sensitivity is insufficient, it is difficult to meet the early diagnosis demand; the detection time is long, it is difficult to meet the emergency demand; the cross reaction rate is high, the specificity is insufficient; there is radioactive pollution; the stability is poor, the shelf life is short; the operation is time-consuming and complicated, professional technical personnel and multi-step operation are required, the equipment is expensive, and it is difficult to popularize and apply. SUMMARY

[0006] In order to solve the problems of insufficient sensitivity, long detection time, insufficient specificity, poor stability, time-consuming and laborious operation and expensive equipment of the prior art for detecting tumor necrosis factor alpha, the application provides a kit for detecting tumor necrosis factor alpha and application thereof.

[0007] The application solves the above technical problems by adopting the following technical scheme.

[0008] The first aspect of the application provides a kit for detecting tumor necrosis factor alpha, which comprises R1 reagent, R2 reagent and R3 reagent.

[0009] The R1 reagent comprises biotin-labeled anti-tumor necrosis factor alpha antibody 1.

[0010] The R2 reagent comprises alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2.

[0011] The R3 reagent comprises streptavidin-coated magnetic microparticles.

[0012] The anti-tumor necrosis factor alpha antibody 1 and the anti-tumor necrosis factor antibody 2 are monoclonal antibodies that bind to different antigen epitopes; the working concentration of the anti-tumor necrosis factor alpha antibody 1 is 0.3-1.0 μg / mL, and the working concentration of the anti-tumor necrosis factor alpha antibody 2 is 0.1-0.9 μg / mL.

[0013] In some embodiments, the solution of the biotin-labeled anti-tumor necrosis factor alpha antibody 1 is selected from any one of TM023-D of Suzhou Yuanqi Biotechnology Co., Ltd., AB1001 of Guangzhou Maichuan Biotechnology Co., Ltd., and biotin-labeled 4T10-2C8cc of Shanghai Haipai Biotechnology Co., Ltd.

[0014] In some embodiments, the solution of the alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2 is selected from any one of TM022-D of Suzhou Yuanqi Biotechnology Co., Ltd., AB1002 of Guangzhou Maichuan Biotechnology Co., Ltd., and alkaline phosphatase-labeled 4T10-F6C5 of Shanghai Haipai Biotechnology Co., Ltd.

[0015] In some embodiments, the streptavidin-coated magnetic microparticles are selected from TM063-D of Suzhou Yuanqi Biotechnology Co., Ltd. or MS160 / Streptavidin of JSR Life Sciences.

[0016] In some embodiments, the working concentration of the anti-tumor necrosis factor alpha antibody 1 is 0.5-0.8 μg / mL, and the working concentration of the anti-tumor necrosis factor alpha antibody 2 is 0.2-0.8 μg / mL.

[0017] In some embodiments, the biotin-labeled anti-TNFa antibody 1 is TM023-D from Suzhou YuanQi Biotech Co., Ltd., the alkaline phosphatase-labeled anti-TNFa antibody 2 is TM022-D from Suzhou YuanQi Biotech Co., Ltd., and the streptavidin-coated magnetic microparticles are TM063-D from Suzhou YuanQi Biotech Co., Ltd.

[0018] In some embodiments, the R1 reagent, the R2 reagent, and / or the R3 reagent further comprises:

[0019] (1) a buffer: the buffer is selected from one or more of Tris buffer, MES buffer, and HEPES buffer;

[0020] (2) a protective agent: the protective agent is BSA; and

[0021] (3) a preservative: the preservative is selected from one or more of ProClin 300, ProClin 950, gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazole alkyl urea, thiomersal, and 2-methyl-3(2H)-isothiazolone 5-bromo-5-nitro-1,3-dioxolane.

[0022] In some embodiments, the preservative is ProClin 300.

[0023] In some embodiments, the concentration of the magnetic beads is 0.2-0.5 mg / ml.

[0024] In some embodiments, the R1 reagent comprises Tris buffer containing 0.05%-0.15% (m / v) bovine serum albumin, 0.05%-0.15% (v / v) Proclin 300, and 0.4-0.9 μg / ml biotin-labeled anti-TNFa antibody 1; and the pH of the R1 reagent is 7.2-7.6.

[0025] In some embodiments, the R2 reagent comprises MES buffer containing 0.05%-0.15% (m / v) bovine serum albumin, 0.05%-0.15% (v / v) Proclin 300, and 0.1-0.9 μg / ml alkaline phosphatase-labeled anti-TNFa antibody 2; and the pH of the R2 reagent is 5.8-6.2.

[0026] In some embodiments, the R3 reagent comprises a HEPES buffer containing 0.5-1.5% (m / v) sodium caseinate, 0.05-0.15% (v / v) Proclin 300, and 0.1-0.6 mg / ml streptavidin-coated magnetic microparticle suspension; the pH of the R3 reagent is 7.2-7.6.

[0027] In some embodiments, the R1 reagent comprises a Tris buffer containing 0.1% (m / v) bovine serum albumin, 0.1% (v / v) Proclin 300, and 0.5-0.8 μg / ml biotin-labeled anti-tumor necrosis factor alpha antibody 1; the pH of the R1 reagent is 7.4.

[0028] In some embodiments, the R2 reagent comprises a MES buffer containing 0.1% (m / v) bovine serum albumin, 0.1% (v / v) Proclin 300, and 0.2-0.8 μg / ml alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2; the pH of the R2 reagent is 6.0.

[0029] In some embodiments, the R3 reagent comprises a HEPES buffer containing 1% (m / v) sodium caseinate, 0.1% (v / v) Proclin 300, and 0.2-0.5 mg / ml streptavidin-coated magnetic microparticle suspension; the pH of the R3 reagent is 7.4.

[0030] In some embodiments, the kit further comprises an R4 reagent, which comprises a chemiluminescent substrate.

[0031] In some embodiments, the chemiluminescent substrate comprises 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt (AMPPD), luminol, and 3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane.

[0032] In some specific embodiments, the chemiluminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane disodium salt.

[0033] The second aspect of the present application provides a method for detecting tumor necrosis factor alpha, which comprises the step of incubating the reagents in the kit as described in the first aspect of the present application with a sample to be tested.

[0034] In some embodiments, the sample to be tested is a serum sample.

[0035] In some embodiments, the method comprises the steps of:

[0036] S1: adding the R1 reagent and the R2 reagent in the sample to be tested, incubating at 35-40°C for 8-12 min;

[0037] S2: adding the R3 reagent, incubating at 35-40°C for 1-10 min;

[0038] S3: after removing the unbound substances, adding the R4 reagent, incubating at 35-40°C for 1-10 min.

[0039] In some specific embodiments, the method comprises the following steps:

[0040] S1: adding the R1 reagent and the R2 reagent in the sample to be tested, incubating at 37°C for 10 min;

[0041] S2: adding the R3 reagent, incubating at 37°C for 5 min;

[0042] S3: after removing the unbound substances, adding the R4 reagent, incubating at 37°C for 5 min.

[0043] In some embodiments, the method comprises the following steps:

[0044] The volume ratio of the sample to be tested, the R1 reagent and the R2 reagent is 1:1:1.

[0045] In some embodiments, the volume ratio of the sample to be tested and the R3 reagent is 1:1.

[0046] In some embodiments, the sample loading amount of the sample to be tested is 30-70 μl.

[0047] In some embodiments, the sample loading amount of the sample to be tested is 50 μl.

[0048] In some specific embodiments, the method comprises the following steps:

[0049] 1) adding the R1 reagent and the R2 reagent in the sample, reacting under incubation conditions for 10 min to form an antibody-antigen-antibody sandwich complex.

[0050] 2) after incubation, adding the R3 reagent, reacting under incubation conditions at 37°C for 5 min, and the sandwich complex is combined to the magnetic microparticles under the interaction of streptavidin and biotin.

[0051] 3) Detection reading: precipitate by magnetic field, remove supernatant, wash the precipitate complex with washing solution, and aspirate waste liquid to remove substances not combined with magnetic particles, and then send the reaction cup into the measuring chamber. The automatic chemiluminescence immunoassay instrument (Anhui Tongke Biology, model TK-2000) automatically pumps in the substrate solution for automatic immune testing system (substrate solution for automatic immune testing system, AMPPD, Anhui Tongke Biology), and the substrate solution for automatic chemiluminescence immunoassay instrument (Anhui Tongke Biology, model TK-2000) emits photons under the catalysis of alkaline phosphatase, and the number of generated photons is proportional to the concentration of tumor necrosis factor alpha in the sample, and the light intensity is detected by an optical detection system.

[0052] 4) The instrument automatically calculates the detection result through a working curve.

[0053] The third aspect of the present application provides a kit as described in the first aspect of the present application for detecting tumor necrosis factor alpha.

[0054] It is known in the art that alkaline phosphatase and acridinium ester are common reagents for chemiluminescence. Alkaline phosphatase uses enzymatic luminescence, which is indirect luminescence, can sustain luminescence, but needs to be incubated for a certain period of time; acridinium ester uses hydrolysis luminescence, which is direct luminescence, has fast luminescence speed, but short luminescence time, and has high standards for various detection instruments. The present application obtains a reagent reaction system for tumor necrosis factor alpha with high sensitivity, high specificity, and strong stability by combining and screening the conditions for alkaline phosphatase detection, and the corresponding kit has good correlation with commercial reagents, low requirements for instruments, and reduced detection cost.

[0055] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e., to obtain each preferred example of the present application.

[0056] The reagents and raw materials used in the present application are commercially available.

[0057] The positive progress effect of the present application is that:

[0058] The preparation method of the kit described in the present application is simple, the reaction time is relatively short, and the operation is simple; the sensitivity is high, the specificity is strong, the reagent is stable, the repeatability is good, the test result is accurate, and the kit has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The test result is a working calibration.

[0060] Figure 2 The comparison result is a clinical sample. DETAILED DESCRIPTION

[0061] The application will be further described in the following by way of examples without limiting the application to the examples described. The experimental methods in the following examples, for which no specific conditions are indicated, are selected in accordance with the usual methods and conditions, or in accordance with the instructions of the commercial suppliers. All other examples which can be derived by a person skilled in the art without inventive step on the basis of the examples in the present application, are intended to belong to the scope of the present application.

[0062] The kit of the present application is used for detecting the content of tumor necrosis factor alpha, and is calibrated by using a full-automatic chemiluminescence immunoassay analyzer. Then, according to the needs, the clinical sample is tested according to the above-mentioned detection method, and according to the proportional relationship between the light quantum number of the sample and the concentration of tumor necrosis factor alpha, the concentration of tumor necrosis factor alpha is automatically calculated by the instrument. Finally, the performance of the tumor necrosis factor alpha determination kit of the present application is evaluated.

[0063] Example 1 Preparation method of R1, R2, R3 reagent

[0064] 1. Raw material screening

[0065] 1.1 Antibody raw material screening

[0066] According to market research, we selected three companies, MaiChuan (Guangzhou) Biotechnology Co., Ltd., Haipai Biotechnology (Shanghai) Co., Ltd. and Suzhou Yuanqi Biotechnology Co., Ltd., for antibody research. The raw material information is as shown in Table 1:

[0067] Table 1 Antibody raw material information

[0068]

[0069] The antibodies in combination 2 and combination 3 are naked antibodies, which are connected with Biotin on AB1001 and 4T10-2C8cc by a conventional method in the art; and are connected with AP on AB1002 and 4T10-F6C5 by a conventional method in the art.

[0070] The three combinations are prepared into reagents, and the same concentration of antigen sample (TNF-alpha antigen, Suzhou Yuanqi Biotechnology Co., Ltd., TM024-D) is tested to investigate the reactivity. Among them, S0 is a calibrator diluent (0.1% bovine serum albumin in phosphate buffer, pH 6.0); the antigen sample is diluted to 10 pg / mL using the calibrator diluent to obtain antigen sample 1; the antigen sample is diluted to 1000 pg / mL using the calibrator diluent to obtain antigen sample 5; according to the experimental results (Table 2), the reaction ratio, the background luminescence intensity (the mean value of S0, the lower the better), and the overall luminescence intensity range (referring to the mean value of the luminescence value that can be reached by antigen sample 5, the larger the better) of combination 1 are all obviously superior to those of combination 2 and combination 3, so the raw materials of combination 1 are selected.

[0071] Table 2 Test results of different antibody raw materials

[0072]

[0073] 1.2 Magnetic bead screening

[0074] According to the market research, we selected two companies, JSR Life Sciences and Suzhou Yuanqi Biotechnology Co., Ltd., to study magnetic beads. The raw material information is shown in Table 3:

[0075] Table 3 Magnetic bead information

[0076]

[0077] The magnetic beads (coated with streptavidin) from the two companies were diluted to the recommended working concentration by the manufacturer, combined into reagents, and tested with the same concentration of antigen samples to investigate the reactivity. According to the experimental results (Table 4), the reaction ratio, background luminescence intensity, and overall luminescence intensity range of magnetic bead 1 were significantly better than those of magnetic bead 2, so magnetic bead 1 was selected.

[0078] Table 4 Test results of different magnetic beads

[0079]

[0080] Preparation of R1 reagent: TNF-α antibody 1-Biotin was diluted to 0.5-0.8 μg / mL with R1 reagent diluent (Tris buffer containing 0.1% bovine serum albumin, 0.1% Proclin300, pH 7.4).

[0081] Preparation of R2 reagent: TNF-α antibody 2-AP was diluted to 0.2-0.8 μg / mL with R2 reagent diluent (MES buffer containing 0.1% bovine serum albumin, 0.1% Proclin300, pH 6.0).

[0082] Preparation of R3 reagent: Streptavidin magnetic beads (Suzhou Yuanqi Biotechnology Co., Ltd., TM063-D, particle size: 1 μm) were washed 3 times with R3 reagent diluent (HEPES buffer containing 1% sodium casein, 0.1% Proclin300, pH 7.4), and then diluted to a magnetic bead concentration of 0.2-0.5 mg / mL.

[0083] Example 2 Preparation method of calibrators and quality controls

[0084] 1) Preparation of calibrators: TNF-α antigen (Suzhou Yuanqi Biotechnology Co., Ltd., TM024-D) was diluted to two concentration points of 25.00 pg / mL and 250.00 pg / mL with calibrator diluent (0.1% bovine serum albumin in phosphate buffer, pH 6.0), then freeze-dried according to the specification, and after freeze-drying, reconstituted with water, tested together with the working calibrator (traceable to international standard NIBSC 12 / 154), and assigned values.

[0085] 2) Preparation of quality control: TNF-α antigen was diluted to two concentration points of 10.00 pg / mL and 250.00 pg / mL with calibrator diluent, then freeze-dried according to the specification, and after freeze-drying, reconstituted with water, tested together with the working calibrator, and assigned values.

[0086] Assignment method:

[0087] The selected two automatic luminous immune analyzers were used to determine the working calibrator, calibrator and quality control, each sample was determined three times, and each day morning and afternoon were tested once, lasting two days. Record the data, remove outliers, and use four-parameter fitting mode for main calibrator curve fitting. The luminescence values of the calibrators and the quality control were substituted into the main calibration curve to obtain the determination values; the average of the determination values of the calibrators and the quality control was taken as the assignment result.

[0088] The freeze-drying parameters are as follows in Table 5:

[0089] Table 5 Freeze-drying parameters

[0090]

[0091] The test results of the working calibrator are shown in Figure 1 .

[0092] Example 3 A tumor necrosis factor alpha assay kit

[0093] A tumor necrosis factor alpha assay kit comprises: R1 reagent: biotin-labeled tumor necrosis factor alpha antibody 1 solution; R2 reagent: alkaline phosphatase-labeled tumor necrosis factor alpha antibody 2 solution; R3 reagent: streptavidin-coated magnetic particle suspension; and matched calibrators and quality controls. The concentration of R1 reagent is 0.5-0.8 μg / mL, the concentration of R2 reagent is 0.2-0.8 μg / mL, and the R3 reagent contains streptavidin-coated magnetic particle suspension 0.2-0.5 mg / mL. Take 3.7 mL of R1 reagent, 3.7 mL of R2 reagent and 3.7 mL of R3 reagent prepared in Example 1, take two bottles of calibrators and quality controls of different concentrations prepared in Example 2, and package them in a packaging box, and the tumor necrosis factor alpha assay kit is obtained.

[0094] Example 4 Detection method of alpha tumor necrosis factor assay kit

[0095] 1. Reaction system optimization

[0096] 1.1 Reagent dosage

[0097] Three groups of R1, R2, R3 reagent sample amounts were set as follows: ① 40 µL, 40 µL, 40 µL; ② 50 µL, 50 µL, 50 µL; ③ 60 µL, 60 µL, 60 µL, and other detection conditions remained unchanged. Antigen samples were determined to investigate the reactivity. According to the experimental results (Table 6), from the reaction ratio, background luminescence and overall luminescence range, the detection results were best when the R1, R2, R3 reagent sample amounts were set as 50 µL, 50 µL, 50 µL.

[0098] Table 6 Optimization results of reagent dosage

[0099]

[0100] 1.2 Reaction time

[0101] Three reaction times were set as follows in the one-step delay method: ① 5 min+5 min; ② 10 min+5 min; ③ 10 min+10 min, and other detection conditions remained unchanged. Antigen samples were determined to investigate the reactivity. According to the experimental results (Table 7), from the reaction ratio, background luminescence and overall luminescence range, the detection results were best when the reaction time was set as 10 min+5 min.

[0102] Table 7 Detection results of different reaction times

[0103]

[0104] 1.3 Sample loading amount

[0105] Three sample loading amounts were set as follows: ① 30 µL; ② 50 µL; ③ 70 µL, and other detection conditions remained unchanged. Antigen samples were determined to investigate the reactivity. According to the experimental results (Table 8), from the reaction ratio, background luminescence and overall luminescence range, the detection results were best when the sample loading amount was set as 50 µL.

[0106] Table 8 Detection results of different sample loading amounts

[0107]

[0108] 2. Detection method

[0109] 1) Incubate 50 μL of sample, 50 μL of biotin-labeled α-tumor necrosis factor antibody 1 solution and 50 μL of alkaline phosphatase-labeled α-tumor necrosis factor antibody 2 solution at 37°C for 10 min to form an antibody-antigen-antibody sandwich complex;

[0110] 2) Add 50 μL of streptavidin-coated magnetic microparticle suspension and incubate at 37°C for 5 min, and the sandwich complex is bound to the magnetic microparticles under the interaction of streptavidin and biotin;

[0111] 3) Precipitate by applying a magnetic field, remove the supernatant, wash the precipitated complex with a washing solution (Tris buffer containing 0.1% Tween 20, 0.1% BND complexing agent and 0.006% triton X-100, pH 8.4), dry the waste liquid, remove the substances not bound to the magnetic microparticles, and then put the reaction cup into a measuring chamber;

[0112] 4) The instrument automatically pumps in 200 μL of substrate solution (AMPPD) for automatic immune testing system, and the substrate solution for automatic immune testing system emits photons under the catalysis of alkaline phosphatase (catalysis temperature: 37°C, catalysis time: 5 min), the number of generated photons is proportional to the concentration of α-tumor necrosis factor in the sample, the light intensity is detected by an optical detection system, and the detection result is automatically calculated by the instrument through a working curve.

[0113] Example 5 Comparison of clinical samples

[0114] The kit of Example 3 above is used (when the reagent test value is >10.00 pg / mL, the sample to be tested is positive; when the reagent test value is <10.00 pg / mL, the sample to be tested is negative, i.e., the reference range is <10.00 pg / mL), and 100 clinical serum samples are tested according to the detection method of Example 4 above after calibration, and the results are shown in Figure 2 and Table 9, the kit of the present application has good correlation with the Siemens IMMULITE / IMMULITE 1000 TNFα kit (TNF-α / α-tumor necrosis factor assay kit (chemiluminescence method), 01.09.01.LKNF1, Siemens, USA, when the reagent test value is >8.1 pg / mL, the sample to be tested is positive; when the reagent test value is <8.1 pg / mL, the sample to be tested is negative, i.e., the reference range is <8.1 pg / mL), has high coincidence rate of positive and negative, and the correlation coefficient r=0.9913.

[0115] Table 9 Results of detection of 100 clinical serum samples by two kinds of kits

[0116]

[0117]

[0118]

[0119] Example 6 Performance evaluation of tumor necrosis factor alpha assay kit

[0120] 1. Blank limit

[0121] The zero concentration calibrator or sample diluent was used as the sample for detection, and the detection was repeated 20 times to obtain the RLU value (relative luminescence value) of 20 measurement results, the average value (M) and standard deviation (SD) were calculated, the RLU value corresponding to M+2SD was obtained, and a linear equation was obtained by two-point regression fitting according to the concentration-RLU value results between the zero concentration calibrator and the adjacent calibrator: Y=2406.3X+7993.9, the RLU value corresponding to M+2SD was substituted into the above equation, and the corresponding concentration value was obtained, that is, the blank limit. The results are shown in Table 10, and the blank limit is 0.27 pg / mL; the reagent blank limit standard is ≤2.00 pg / ml, and the verified blank limit is 0.27 pg / ml, which meets the requirements.

[0122] Table 10 Detection results of zero concentration calibrator

[0123]

[0124] 2. Accuracy

[0125] The reference material with a concentration of (15.00±3.00) pg / mL and (150.00±30.00) pg / mL was repeatedly detected 3 times, and the relative deviation of the measured concentration value and the target value was calculated. The results are shown in Table 11, and the deviation of the 3 results is <5.00%.

[0126] Table 11 Accuracy test results

[0127]

[0128] 3. Linearity

[0129] The sample with a concentration close to the upper limit of the linear range was prepared into at least 5 concentrations, and the low concentration sample should be close to the lower limit of the linear interval. Each concentration level was repeatedly detected 3 times, and the average value was calculated as the measured value. The measured value and the theoretical value were linearly fitted by the least square method, and the linear correlation coefficient was calculated. The results are shown in Table 12, and the correlation coefficient is r=0.9987 in the range of 4.00-1000.00 pg / mL, and the linearity is good. The correlation coefficient is r=0.9987 in the range of 4.00-1000.00 pg / mL, and the linearity is good.

[0130] Table 12 Linear correlation test results

[0131]

[0132] 4. Reproducibility

[0133] The sample with the concentration level of (15.00 ± 3.00) pg / mL and (150.00 ± 30.00) pg / mL was determined for 10 times in the same experiment, and the average value (M), standard deviation (SD) and coefficient of variation (CV) of the 10 determination values were calculated. The results are shown in Table 13, and the maximum value of the reproducibility CV is 1.32%, which is good reproducibility.

[0134] Table 13 Kit reproducibility test results

[0135]

[0136] 5. Specificity

[0137] The interleukin 1β antigen (≤10000 pg / mL), interleukin 2 antigen (≤10000 pg / mL), interleukin 6 antigen (≤10000 pg / mL), interleukin 8 antigen (≤10000 pg / mL), and interleukin 10 antigen (≤10000 pg / mL) were determined for 1 time, respectively. The results are shown in Table 14, and the results show that there is no cross reaction with the above-mentioned substances.

[0138] Table 14 Specificity test results

[0139]

[0140] 6. Stability

[0141] The kit in Example 3 was stored at 2-8°C in a sealed state for 0, 3, 6, 9, 12, and 13 months, respectively. The sample with the concentration level of (15.00 ± 3.00) pg / mL and (150.00 ± 30.00) pg / mL was determined for 3 times at each time point, and the deviation between the 0th month was calculated. The results are shown in Table 15, and the maximum deviation of the results measured within 13 months is -5.93%, which is good stability of the kit.

[0142] Table 15 Stability test results

[0143]

[0144] 7. Batch difference

[0145] The repeatability reference samples B1 (15.00±3.00) pg / mL and B2 (150.00±30.00) pg / mL of the enterprise were detected for 10 times respectively by using three batches of kits, and the average (M) and standard deviation (SD) of 30 measurement results were calculated, and the coefficient of variation (CV) was calculated. The results are shown in Table 16, and the batch difference CV is less than 5%.

[0146] Table 16 Test results of batch difference

[0147]

[0148] 8. Interference test

[0149] The interference material stock solution (hemoglobin, triglyceride, bilirubin, biotin, rheumatoid factor and human anti-mouse antibody HAMA) was added to the sample (volume ratio 0.5:9.5) to prepare an interference sample containing the interference material with the final concentration shown in the table below. The target concentration of the analyte is 10.00 pg / mL and 100.00 pg / mL. The results are shown in Table 17, and the relative deviation is less than 5%, and the anti-interference ability is strong.

[0150] Table 17 Test results of interference test

[0151]

[0152] From the above detection results, it can be seen that the kit of the present application has the advantages of high sensitivity and accuracy, good linearity, reagent stability, strong specificity, small batch difference, strong anti-interference ability, and good correlation with Siemens reagent.

[0153] The above embodiments of the present application are only examples for clearly illustrating the present application, and do not limit the protection scope of the present application. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A kit for detecting tumor necrosis factor alpha, characterized by, The kit comprises an R1 reagent, an R2 reagent and an R3 reagent; The R1 reagent comprises a biotin-labeled anti-tumor necrosis factor alpha antibody 1; The R2 reagent comprises an alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2; The R3 reagent comprises a streptavidin-coated magnetic microsphere; The anti-tumor necrosis factor alpha antibody 1 and the anti-tumor necrosis factor alpha antibody 2 are monoclonal antibodies that bind to different antigen epitopes; the working concentration of the anti-tumor necrosis factor alpha antibody 1 is 0.3-1.0 μg / mL, and the working concentration of the anti-tumor necrosis factor alpha antibody 2 is 0.1-0.9 μg / mL.

2. The kit of claim 1, wherein The biotin-labeled anti-tumor necrosis factor alpha antibody 1 is selected from any one of TM023-D of Suzhou Yuanqi Biotechnology Co., Ltd., AB1001 of Guangzhou Maichuan Biotechnology Co., Ltd., and 4T10-2C8cc of Shanghai Haipai Biotechnology Co., Ltd.; and / or, the alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2 is selected from any one of TM022-D of Suzhou Yuanqi Biotechnology Co., Ltd., AB1002 of Guangzhou Maichuan Biotechnology Co., Ltd., and 4T10-F6C5 of Shanghai Haipai Biotechnology Co., Ltd.; and / or, the streptavidin-coated magnetic microsphere is selected from TM063-D of Suzhou Yuanqi Biotechnology Co., Ltd. or MS160 / Streptavidin of JSR Life Sciences; and / or, the working concentration of the anti-tumor necrosis factor alpha antibody 1 is 0.5-0.8 μg / mL, and the working concentration of the anti-tumor necrosis factor alpha antibody 2 is 0.2-0.8 μg / mL; Preferably, the biotin-labeled anti-tumor necrosis factor alpha antibody 1 is TM023-D of Suzhou Yuanqi Biotechnology Co., Ltd.; the alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2 is TM022-D of Suzhou Yuanqi Biotechnology Co., Ltd.; and the streptavidin-coated magnetic microsphere is TM063-D of Suzhou Yuanqi Biotechnology Co., Ltd.

3. The kit of claim 1, wherein The R1 reagent, the R2 reagent and / or the R3 reagent further comprise: (1) a buffer: the buffer is selected from one or more of Tris buffer, MES buffer and HEPES buffer; (2) a protective agent: the protective agent is BSA; (3) a preservative: the preservative is selected from one or more of ProClin300, ProClin950, gentamicin sulfate, sodium azide, 2-hydroxy pyridine-N-oxide, chloroacetamide, imidazole alkyl urea, thiomersal and 2-methyl-3(2H)-isothiazolone 5-bromo-5-nitro-1,3-dioxolane; Preferably, the preservative is ProClin300.

4. The kit of any one of claims 1-3, wherein The concentration of the magnetic beads is 0.2-0.5 mg / ml.

5. The kit of any one of claims 1-4, wherein The R1 reagent comprises Tris buffer containing 0.05%-0.15% (m / v) bovine serum albumin, 0.05%-0.15% (v / v) Proclin 300 and 0.4-0.9 μg / ml biotin-labeled anti-tumor necrosis factor alpha antibody 1; the pH of the R1 reagent is 7.2-7.6; and / or, the R2 reagent comprises MES buffer containing 0.05%-0.15% (m / v) bovine serum albumin, 0.05%-0.15% (v / v) Proclin 300 and 0.1-0.9 μg / ml alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2; the pH of the R2 reagent is 5.8-6.2; and / or, the R3 reagent comprises HEPES buffer containing 0.5-1.5% (m / v) sodium caseinate, 0.05-0.15% (v / v) Proclin 300 and 0.1-0.6 mg / ml streptavidin-coated magnetic particle suspension; the pH of the R3 reagent is 7.2-7.6; Preferably, the R1 reagent comprises Tris buffer containing 0.1% (m / v) bovine serum albumin, 0.1% (v / v) Proclin 300 and 0.5-0.8 μg / ml biotin-labeled anti-tumor necrosis factor alpha antibody 1; the pH of the R1 reagent is 7.4; and / or, the R2 reagent comprises MES buffer containing 0.1% (m / v) bovine serum albumin, 0.1% (v / v) Proclin 300 and 0.2-0.8 μg / ml alkaline phosphatase-labeled anti-tumor necrosis factor alpha antibody 2; the pH of the R2 reagent is 6.0; and / or, the R3 reagent comprises HEPES buffer containing 1% (m / v) sodium caseinate, 0.1% (v / v) Proclin 300 and 0.2-0.5 mg / ml streptavidin-coated magnetic particle suspension; the pH of the R3 reagent is 7.

4.

6. The kit of claim 5, wherein The kit further comprises an R4 reagent, which comprises a chemiluminescent substrate; Preferably, the chemiluminescent substrate comprises 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane disodium salt, luminol and 3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; More preferably, the chemiluminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane disodium salt.

7. A method of detecting tumor necrosis factor alpha, characterized by, The method comprises the step of incubating a sample to be tested using the kit according to any one of claims 1-6; Preferably, the sample to be tested is a serum sample.

8. The method of claim 7, wherein, The method comprises the following steps: S1: adding the R1 reagent and the R2 reagent to the sample to be tested, and incubating at 35-40°C for 8-12 min; S2: adding the R3 reagent, and incubating at 35-40°C for 1-10 min; S3: after removing unbound substances, adding R4 reagent, incubating at 35-40°C for 1-10 min; Preferably, the method comprises the following steps: S1: adding the R1 reagent and the R2 reagent to the sample to be tested, incubating at 37°C for 10 min; S2: adding R3 reagent, incubating at 37°C for 5 min; S3: after removing unbound substances, adding R4 reagent, incubating at 37°C for 5 min.

9. The method of claim 8, wherein, The method comprises the following steps: the volume ratio of the sample to be tested, the R1 reagent and the R2 reagent is 1:1:1; and / or, the volume ratio of the sample to be tested and the R3 reagent is 1:1; the sample to be tested is loaded in an amount of 30-70 μL; Preferably, the sample to be tested is loaded in an amount of 50 μL.

10. Use of the kit of any one of claims 1-6 in detecting tumor necrosis factor alpha.

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