Detection kit for chitosanase 3-like protein 1 and preparation method thereof

Abstract

The kit for detecting the chitosanase 3-like protein 1 (CHI3L1) is developed by adopting a magnetic bead chemiluminescence method and is matched with a full-automatic chemiluminescence analyzer for use, and human intervention is basically not needed. Compared with the existing CHI3L1 detection kit of Hangzhou Purpoval Biotechnology Co., the kit provided by the invention does not need to add a diluent to dilute a sample by 20 times and then detect the sample on a machine, so that the step of diluting the sample is simplified, and the detection error caused by diluting the sample is avoided. According to the invention, the detection time is shortened, the detection efficiency is improved, the lowest detection limit is 0.028 ng / mL, the linear R2 is equal to 0.9999, the anti-interference performance is good, the stability is improved to 27 months, the correlation R2 with the marketed kit is equal to 0.9986, and the kit is superior to a detection kit on the market.

Description

Technical Field

[0001] The present application belongs to the field of clinical in vitro diagnosis and medical immunology, and relates to an immunoassay reagent, and specifically to a magnetic bead chemiluminescence detection kit for detecting the content of human chitinase 3-like protein 1 (CHI3L1) using magnetic bead chemiluminescence technology. Background Art

[0002] Chitinase-3-like protein 1 (CHI3L1), also known as human cartilage glycoprotein 39 (YK-40), was discovered in the human osteosarcoma cell line MG63 as early as 1992. It is a secretory glycoprotein located on chromosome 1 with a molecular weight of approximately 40 kDa.

[0003] CHI3L1 can be derived from a variety of cells, such as macrophages, neutrophils, vascular smooth muscle cells, cancer cells, etc., and has the effects of affecting cell proliferation, migration, invasion, apoptosis and epithelial-mesenchymal transition; promoting extracellular matrix degradation and tissue remodeling; promoting angiogenesis, and participating in embryonic development to a certain extent. Studies have confirmed that CHI3L1 expression increases in various inflammatory diseases and fibrotic diseases. In chronic liver diseases, alcoholic liver disease, non-alcoholic liver disease, chronic liver fibrosis, and cirrhosis have all been found to have higher CHI3L1 levels than normal people. The 2019 edition of the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B" pointed out that CHI3L1 can predict moderate and severe liver fibrosis in patients with normal or mildly elevated ALT, indicating that peripheral blood CHI3L1 levels are closely related to liver fibrosis. In recent years, studies have confirmed that CHI3L1 may affect cell survival, proliferation, tissue remodeling and angiogenesis, indicating that it can promote the invasion and metastasis of tumor cells. Further studies have shown that CHI3L1 levels in patients with hepatitis C-related liver cancer are significantly higher than those in patients with cirrhosis and hepatitis. In summary, the level of CHI3L1 in plasma is of great significance for the detection of liver cirrhosis, liver fibrosis, liver cancer, etc.

[0004] The existing methods for detecting CHI3L1 mainly include enzyme-linked immunosorbent assay, immunochromatography, chemiluminescence, etc., but these methods cannot meet clinical needs due to their complex process, narrow linear range, too long detection time of 60 minutes, and high price.

[0005] Therefore, there is a need for a chitinase 3-like protein 1 (CHI3L1) detection kit and a method for use with low pollution, wide linear range, high sensitivity, simple operation, high degree of automation, good specificity, low cost and fast detection speed. Summary of the invention

[0006] The present invention aims to develop a magnetic bead chemiluminescence detection kit for determining the content of human chitinase 3-like protein 1 (CHI3L1) which has the advantages of simple operation, wide linear range, fast detection speed, high sensitivity, no pollution, good specificity and low cost.

[0007] Based on the above purpose, the present invention provides a magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1, characterized in that it includes the following components: Biotinylated conjugate labeled with chitinase 3-like protein 1 monoclonal antibody; an enzyme-labeled conjugate labeled with a monoclonal antibody against chitinase 3-like protein 1; Magnetic bead buffer containing streptavidin magnetic beads; Luminescent substrate solution.

[0008] The magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as described above is characterized in that the biotinylated conjugate labeled with chitinase 3-like protein 1 monoclonal antibody and the enzyme-labeled conjugate labeled with chitinase 3-like protein 1 monoclonal antibody are prepared into an antibody solution using NT buffer, and the NT buffer includes 0.005% defoaming agent 204, 0.236% dipotassium hydrogen phosphate trihydrate, 0.54% anhydrous potassium dihydrogen phosphate, 0.1% preservative ProClin300, 0.1% 1% potassium ferrocyanide, 3% bovine serum albumin, 0.04% ANS, and a pH value of 6.5±0.1.

[0009] The magnetic bead chemiluminescent detection kit for detecting the content of chitinase 3-like protein 1 as described above is characterized in that the concentration of the biotinylated conjugate labeled with the chitinase 3-like protein 1 monoclonal antibody is 2 ng / mL, and the concentration of the enzyme-labeled conjugate labeled with the chitinase 3-like protein 1 monoclonal antibody is 1.5 ng / mL.

[0010] The magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as mentioned above is characterized in that it also includes a standard dilution solution.

[0011] Another object of the present invention is to provide a method for preparing the magnetic bead chemiluminescent detection kit for detecting the content of chitinase 3-like protein 1 as described above, characterized in that it comprises the following steps: (1) Conjugating CHI3L1 antibody to biotin; (2) CHI3L1 antibody is coupled to an enzyme-labeled conjugate; (3) Prepare CHI3L1 biotinylated conjugate antibody solution and CHI3L1 enzyme-labeled conjugate antibody solution: dilute with NT buffer to make the final concentrations of CHI3L1 biotinylated conjugate antibody solution and CHI3L1 enzyme-labeled conjugate antibody solution 2 ng / mL and 1.5 ng / mL, respectively; (4) Prepare magnetic bead buffer containing streptavidin magnetic beads and standard dilution solution.

[0012] The preparation method of the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as mentioned above is characterized in that the concentration of the magnetic bead buffer is 2 mg / mL, and the magnetic bead buffer comprises the following components: 2.56 g of sodium dihydrogen phosphate, 0.43 g of disodium hydrogen phosphate, 9 g of sodium chloride, 10 g of bovine serum albumin, 20 g of mannitol, 0.02 g of 4-aminoantipyrine, 0.1 mL of Triton X-100, 1 mL of Proclin 300, and water is added to make the volume to 1L.

[0013] The preparation method of the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as mentioned above is characterized in that the standard dilution solution includes CH3L1 solutions with concentrations of 0 ng / mL, 3 ng / mL, 6 ng / mL, 18 ng / mL, 54 ng / mL, and 120 ng / mL.

[0014] Another object of the present invention is to provide the use of the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as described above in detecting the content of chitinase 3-like protein 1.

[0015] The present invention uses magnetic bead chemiluminescence to develop a chitinase 3-like protein 1 (CHI3L1) detection kit, which is used in conjunction with a fully automatic chemiluminescence analyzer and basically does not require human intervention. Compared with the existing CHI3L1 detection kit of Hangzhou Puwang Biotechnology Co., Ltd., the present invention no longer needs to add diluent to dilute the sample 20 times before testing on the machine. It simplifies the steps of diluting the sample and avoids the detection error caused by diluting the sample. The present invention shortens the detection time and improves the detection efficiency. The minimum detection limit is 0.028 ng / mL, and the linear R 2 =0.9999, good anti-interference performance, stability increased to 27 months, and correlation with the kits already on the market is R 2 =0.9986, which is better than the detection kits on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the detection process of the chitinase 3-like protein 1 detection reagent of the present invention.

[0017] Figure 2 Schematic diagram of magnetic bead concentration for the CHI3L1 detection kit developed by the present invention; Figure 3 Schematic diagram of the linearity results of the CHI3L1 detection kit developed for the present invention; Figure 4 This is the result of correlation between the kit developed by the present invention and the Puwang kit sample test purchased from the hospital. DETAILED DESCRIPTION

[0018] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0019] The following are the main materials and equipment used in the implementation: CHI3L1 antigen, calibrators, and luminescent substrate were provided by Jiangsu Baiming Biotechnology Co., Ltd. (Wuxi, China); Antibodies (Cat. No.: CH1001, CH1002) were provided by Zhenjiang Arenac Biotechnology Co., Ltd. (Zhenjiang, China); Streptavidin magnetic beads reagent was purchased from Thermo Fisher Scientific (China), catalog number 88817; The fully automatic chemiluminescence analyzer (model: BM 1800) was purchased from Jiangsu Baiming Biotechnology Co., Ltd. (Wuxi, China); The pH meter was purchased from Mettler Toledo (Switzerland) Example

[0020] CHI3L1 antibody (CH1001) coupled with biotin: Place the mixer in a 37 °C constant temperature incubator and adjust the concentration of CHI3L1 antibody (CH1001) to 1 mg mL with PBS buffer. -1 , take 250 μL of 1 mg·mL -1 Dissolve the Sulfo-NHS-LC-Biotin solid in PBS buffer and adjust the concentration to 1 mg mL -1 . Take 5 μL of the dissolved biotin solution and add it to the antibody at the bottom of the centrifuge tube. Gently vortex and shake, then seal with sealing film and incubate on a mixer in a constant temperature incubator at 37°C for 1 hour. After incubation, dialyze in 500 mL PBS buffer using a 14000 Da dialysis bag for 4 hours, then replace the PBS buffer and continue dialysis overnight (16 to 18 hours). After dialysis, use UV OD 280 The antibody concentration was measured at nm / 1.4 and then stored by adding 0.1% BSA and 0.1% Proclin 300.

[0021] CHI3L1 antibody (CH1002) coupled with horseradish peroxidase: After dialyzing CHI3L1 antibody (CH1002) in carbonate buffer at pH 9.6 for 4 h, the carbonate buffer was replaced and the dialysis was continued overnight. The total dialysis time did not exceed 24 h. After the dialysis was completed, the absorbance was measured at 280 nm using a UV spectrophotometer and the OD 280nm / 1.35 to calculate the concentration of the antibody and ensure that the concentration of the antibody is 1 mg·mL -1 Weigh 10 mg of solid sodium periodate and add it to 0.5 mL of PBS buffer with a pH of 7.0 to dissolve it in a dark environment. Weigh 5 mg of horseradish peroxide and add it to 1.25 mL of water to make a 4 mg mL -1 300 μL of sodium periodate solution was then added to the HRP solution and vortexed for 20 minutes. After the reaction was completed, 2 μL of ethylene glycol was added and vortexed for 5 minutes to terminate the oxidation reaction. The reaction mixture was dialyzed with a 14000 Da dialysis membrane for 5 h, during which the solution was changed twice. 0.5 mg of antibody was added to 1, 2, and 4 times the volume of HRP solution, and the reaction lasted for 1 h. Then 0.1 mL of 4 mg mL -1 The sodium borohydride solution was placed at 4°C in the dark for 3 h. After the reaction was completed, it was dialyzed in PBS buffer using a 14000 Da dialysis membrane and the solution was changed twice. Finally, the UV absorbance at 280 nm was measured using a UV spectrophotometer. 280nm / 1.35 to calculate the concentration of the conjugate and store it at 4°C until use.

[0022] Prepare NT buffer: 0.005% defoamer 204, 0.236% potassium phosphate trihydrate, 0.54% potassium dihydrogen phosphate anhydrous, 0.1% preservative ProClin300, 0.1% 1% potassium ferrocyanide, 3% bovine serum albumin, 0.04% ANS. After stirring, test the pH value to 6.5±0.1, filter, and store in a refrigerator at 4°C; Preparation of CHI3L1 biotinylated conjugate antibody solution: Take out the biotinylated conjugate labeled with CHI3L1 monoclonal antibody and add it to the prepared NT buffer to prepare a final concentration of 2 ng / mL, shake at room temperature for 15-20 min to obtain a mixed solution; Preparation of CHI3L1 enzyme-labeled conjugate antibody solution: Take out the CHI3L1 monoclonal antibody enzyme-labeled conjugate and add it to the prepared NT buffer to prepare a final concentration of 1.5 ng / mL. Shake at room temperature for 15-20 min to obtain a mixed solution.

[0023] The magnetic bead buffer includes the following components (based on 1L): 2.56 g sodium dihydrogen phosphate, 0.43 g disodium hydrogen phosphate, 9 g sodium chloride, 10 g bovine serum albumin, 20 g mannitol, 0.02 g 4-aminoantipyrine, 0.1 mL Triton X-100, 1 mL Proclin 300, pH 7.15-7.25.

[0024] The present invention provides a CHI3L1 complex calibrant diluent, the preparation ratio of which is as follows: 1.482% sodium dihydrogen phosphate dihydrate, 0.124% potassium dihydrogen phosphate trihydrate, 2% casein, 0.2% Tween-20, 0.1% ProClin 300. After stirring, the pH value was tested to be 5.5±0.1, filtered, and stored at 4°C.

[0025] Example 2 Screening of reagent concentrations of this kit After screening the paired antibodies, it is necessary to explore their optimal working concentration. The CHI3L1 complex was diluted into calibration solutions S1-S6 of different concentrations, with concentrations of 0 ng / mL, 3 ng / mL, 6 ng / mL, 18 ng / mL, 54 ng / mL, and 120 ng / mL, respectively, and stored at 20°C for use. According to the chessboard method, different concentrations of calibrators (S1-S6) were used as samples for the experiment. As shown in Table 1, when the biotinylated antibody concentration of CHI3L1 was 2 ng / mL and the HRP antibody concentration was 1.5 ng / mL, the ratio difference was small and close, so the signal value was most suitable. Under the premise of ensuring the experimental effect, we chose a lower antibody concentration to reduce costs. Therefore, this study determined this antibody concentration combination as the standard working concentration for subsequent experiments, and the results are shown in Table 1.

[0026] Table 1: Comparison of experimental results of different concentrations of CHI3L1 calibrators

[0027] According to the selected antibody concentration, the magnetic bead concentration was screened. The magnetic bead concentrations were 2 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. After reducing the magnetic bead concentration, the curve luminescence value was lower, so there was no need to lower the magnetic bead concentration. The CHI3L1 test results are shown in Table 2. Figure 2 shown.

[0028] Table 2: Comparison of luminescence values ​​of CHI3L1 at different magnetic bead concentrations CHI3L1 Calibrator Calibrator concentration (ng / mL) Magnetic beads concentration 2mg / mL Magnetic beads concentration 1mg / mL Magnetic bead concentration 0.75 mg / mL Magnetic bead concentration 0.5 mg / mL Magnetic bead concentration 0.25 mg / mL S1 0 9802 8109 7958 7034 6356 S2 3 15645 10231 9564 8754 7012 S3 6 39234 30293 25638 19283 15273 S4 18 90231 78394 63452 51237 40293 S5 54 149467 112390 93842 83232 63849 S6 120 234123 192389 152394 98230 78390 Example 3 The operating procedure of this kit is as follows Take 30 μL of the sample to be tested, add 80 μL of CHI3L1 biotinylated antibody and enzyme-labeled antibody, incubate at 37 °C for 30 min, then add 30 μL of streptavidin-coated magnetic beads solution, mix thoroughly and shake for 5 min, wash 3 times to wash away the unbound antibody, add 100 μL of luminescent substrate, record the luminescence value, and calculate the concentration of CHI3L1 in the sample according to the calibration curve.

[0029] Example 4 The performance of this kit is evaluated as follows 1. Standard curve: The CHI3L1 complex described in Example 1 was diluted with a standard diluent to prepare calibration solutions S1-S6 of different concentrations, with concentrations of 0 ng / mL, 3 ng / mL, 6 ng / mL, 18 ng / mL, 54 ng / mL, and 120 ng / mL, respectively, and stored at -20°C for use. The calibrators were then tested using the kit provided in Example 1 of the present invention, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The standard curve was obtained by fitting with concentration as the abscissa and luminescence intensity as the ordinate.

[0030] 2. Linear The basic serum sample (concentration ≤ blank limit) or the calibration diluent (try to use the real high-value serum sample) with added antigen is used as the high-concentration sample, and the basic serum sample or the calibration diluent is used as the low-concentration sample. The high-value sample close to the upper limit of the linear range is diluted to several concentrations according to a certain ratio (for example: linear sample preparation table), and the low-value concentration sample must be close to the lower limit of the linear range. The sample of each concentration is tested 2-3 times and the average value is calculated. The concentration value of the analyte (as the horizontal axis) and the average value of the test result (as the vertical axis) are fitted with a straight line using the least squares method. And calculate the correlation coefficient r within the linear range, and the acceptance standard is correlation coefficient r ≥ 0.99.

[0031] Figure 2 This is the linear analysis of the CHI3L1 magnetic bead chemiluminescence assay kit in the present invention. The results show that the linear relationship between the theoretical concentration and the actual concentration of the sample is good, and the linear regression equation after fitting is y=1.014x-0.2578, R 2 =0.9999.

[0032] 3. Minimum detection limit Use the zero-concentration calibrator dilution as the sample for detection, repeat the measurement 20 times, and obtain the light signal values ​​of the 20 measurement results. Calculate the mean (M) and standard deviation (SD) to obtain M+2SD. Perform two-point regression fitting on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibrator to obtain a linear equation. Substitute the light signal value of M+2SD into the equation to find the corresponding concentration value, which is the minimum detection limit.

[0033] The results are shown in Table 3. The concentration values ​​of CHI3L1 were 0.02 ng / mL, 0.028 ng / mL, and 0.017 ng / mL, respectively, and the minimum detection limit of CHI3L1 was 0.028 ng / mL.

[0034] Table 3: Validation results of detection limit of CHI3L1 Test 1 Test 2 Test 3 M 1409.20 1431.50 1357.55 SD 55.87 76.12 76.29 M+2SD 1520.99 1587.74 1510.14 LOD(ng / mL) 0.02 0.028 0.017 4. Accuracy Prepare samples with two concentration levels, high and low, using the CHI3L1 complex. Repeat the measurement three times and calculate the relative deviation between the average value (denoted as M) and the labeled value. Calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples with two concentrations, high and low, that have been determined by the reference method. Repeat the measurement three times for each concentration sample, take the average of the test results, and calculate its relative deviation from the labeled value.

[0035] B=(MT) / T×100%---------(1) Where: B—relative deviation; M—average value of measured concentration; T—indicative value; The above experimental results are shown in Table 4. The relative deviation of the CHI3L1 test kit is within 5%, which has good accuracy and is better than similar test kits in the domestic market.

[0036] Table 4: Comparison of CHI3L1 accuracy measurement results Concentration (ng / mL) Test 1 Test 2 Test 3 Average value (ng / mL) Relative deviation (%) 3.00 2.98 3.11 2.91 3.01 1% 100.00 102.10 99.1 101.6 100.35 -2% 5. Precision The test reagent (same batch or multiple batches) is used to test the same batch of samples multiple times to see if the results are consistent. This indicator is used as precision. Usually, the CV value within the batch is within 8%, and the CV value between batches is within 10%. According to the NCCLSEP5-A2 standard, this technology was used to measure two plasma samples of different concentrations. CHI3L1 sample 1: a low-concentration sample with a concentration of 3.8 ng / mL, CHI3L1 sample 2: a high-concentration sample with a concentration of 101 ng / mL.

[0037] (1) Intra-batch precision: The two samples were measured 20 times using three batches of reagents respectively, and the average M and standard deviation SD of the 20 measured concentrations were calculated, and the CV was calculated according to Formula 2.

[0038] CV=M / SD×100%---------(2) Where CV is the coefficient of variation and M is the mean; SD is standard deviation.

[0039] (2) Inter-batch precision: Two samples were measured using three batches of reagents, 20 times each. The CV value was calculated according to Formula 2.

[0040] The precision of this kit is within 5%, which meets clinical requirements. See Table 5 Table 5: Comparison of CHI3L1 precision measurement results Low value quality control High value quality control 1 3.12 100.1 2 3.29 101.3 3 2.98 102.4 4 2.88 101.3 5 3.02 100.1 6 3.11 101.3 7 3.24 101.4 8 3.12 100.3 9 3.29 102.3 10 2.99 101.7 Fitting concentration M 3.104 101.12 Fitted concentration SD 0.131848 2.2121483 Fitting concentration CV 4.2% 1.8% Fitting concentration deviation 3% 3% 5. Thermal stability test The reagent must meet the quality standards of the product within the validity period, and the various performance indexes of the product must be stable. The stability test includes: thermal stability when stored at 2-8 ℃ for 15 months and 37 ℃ for 10 days, and the stability of each index is determined by a three-day simulation experiment. At the same time, the stability of the standard sample and the quality control material was evaluated, and the stability of the sample stored at 2-8 ℃ for 14-15 months was determined, the stability of the sample after 20 days of opening the bottle was determined, and the stability of the simulated transport sample for 3 days was evaluated. In addition, the stability of clinical samples placed under different conditions also needs to be considered. To verify the stability of the reagent, a standard experimental process needs to be carried out in a storage environment of 37 ℃, so that the experimental determination can be completed quickly. The detailed operation process requires three different batches of plasma reagents to be stored in an incubator, and the stability of the samples is determined at different time points. The sample can be tested on the 1st, 3rd, 6th and 10th days as 4 different measurement time points, and then the final test results are compared and analyzed with the reagents stored at 4 ℃, and each performance index is evaluated.

[0041] The prepared CHI3L1 calibrator was placed at 4°C and 37°C for 1 day, 3 days, 6 days, 10 days, 20 days, 25 days, and 30 days, and the luminescence value was tested. The relative error (B%) was within ±20%. The accelerated stability of the CHI3L1 kit was fully analyzed and met the test requirements. The results are shown in Table 6 Table 6: CHI3L1 accelerated stability test results

[0042] The CHI3L1 kit was taken out at the 3rd, 6th, 9th, 12th and 15th month, and the blank quality control and quality control products S2, S4 and S6 were tested, and the CV values ​​were within 10%, which met the specified standards. Therefore, the CHI3L1 kit can be effectively stored at room temperature for 12 months.

[0043] 6. Interference test In the actual determination of CHI3L1 concentration, due to the diverse sources of samples, there are various interfering factors in the samples. When using in vitro diagnostic reagents, interfering substances are one of the important factors causing errors in the measurement results. The so-called "interference test" is to use experimental methods to find out substances that may affect the measurement results of in vitro diagnostic reagents. Interfering substances refer to endogenous and exogenous substances that can interfere with the pH value of the system, reagent stability, etc. These interfering substances of CHI3L1 in human blood samples can be type III procollagen (PC Ⅲ) and alpha-fetoprotein (AFP), etc.

[0044] Fresh plasma was collected from healthy volunteers who had never taken any medication, and the CHI3L1 content was detected using a reference reagent. The specific method was as follows: 5 samples with a concentration below 50 ng / mL were selected and mixed to form a low-concentration sample; 5 samples with a concentration of 50-80 ng / mL were selected and the 5 samples were evenly mixed to form a medium-concentration sample; and 5 samples with a concentration of 80-100 ng / mL were mixed to obtain a high-concentration sample, and then the concentration of each sample was measured using a reference reagent. Each sample was divided into two groups. First, 50 µg / mL of type III procollagen (PC Ⅲ) and 600 µg / mL of alpha-fetoprotein (AFP) were added to the experimental group. No substance was added to the control group, and the measurement was repeated three times. The obtained data is processed by regression analysis: the average error of the tested sample and the control sample is used to characterize the interference effect. It is calculated in the form of formula 3: d obs =Mt-Mc---------(3) Where Mt is the mean value of the experimental samples and Mc is the mean value of the control samples.

[0045] Calculate the threshold value dc: The threshold value calculation formula for a two-sided test is Formula 4, and the threshold value calculation formula for a one-sided test is Formula 5.

[0046] ---------(4) ---------(5) where d null is the value of the null hypothesis, usually 0, Z (1−α / 2) is the percentage of normal distribution relative to the 100(1-α)% confidence level of the two-sided test, Z (1−α) is the percentage of normal distribution relative to the 100(1-α)% confidence level of the one-sided test, and S is the standard deviation of the intra-batch repeatability of the evaluated method; n is the number of sample repetitions.

[0047] In this experiment, the z value was selected as 1.282, n was 3, and the bilateral test boundary value calculation formula was used.

[0048] d obs ≤dc proves that the deviation caused by the measured interference meets the requirements, that is, there is no interference. obs >dc proves that the interfering substance has a significant interference effect on the measured method and is an interfering substance.

[0049] The CHI3L1 kit was used to test interferences including high concentrations of type III procollagen and alpha-fetoprotein, with each test repeated three times. When the added concentration of type III procollagen was less than 50 μg / mL and triglycerides were less than 600 μg / mL, the test results showed no interference. Example 5 Clinical Performance Evaluation In order to better verify the detection effect of the kit developed in this study, clinical performance evaluation is required. 150 samples of patient plasma (provided by the Affiliated Hospital of Jiangnan University) were collected and tested using the chitinase 3-like protein 1 detection kit (magnetic particle chemiluminescence method; model CHI-100Z) and instruments provided by the hospital and purchased from Hangzhou Puwang Biotechnology Co., Ltd., and the data were exported. The concentration in plasma was determined using the self-developed magnetic bead immunochemiluminescence method and compared with the measured values ​​of the Affiliated Hospital of Jiangnan University. The values ​​measured by the homemade magnetic bead immunochemiluminescence method were used as the horizontal axis, and the hospital's measured values ​​were used as the vertical axis. This method was used to fit the corresponding method. The obtained linear regression results are shown in Figure 4 The linear correlation coefficients of CHI3L1 were R 2 =0.9986, greater than 0.95. Its detection effect is well linearly correlated with the results of existing detection reagents in the laboratory of the Affiliated Hospital of Jiangnan University, and is better than other current test kits, indicating that the developed test kit can meet the needs of clinical testing.

Claims

1. A magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1, characterized in that: The components include: Biotinylated conjugate labeled with chitinase 3-like protein 1 monoclonal antibody; an enzyme-labeled conjugate labeled with a monoclonal antibody against chitinase 3-like protein 1; Magnetic bead buffer containing streptavidin magnetic beads; Luminescent substrate solution.

2. The magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 according to claim 1, characterized in that: The biotinylated conjugate labeled with chitinase 3-like protein 1 monoclonal antibody and the enzyme-labeled conjugate labeled with chitinase 3-like protein 1 monoclonal antibody are prepared into an antibody solution using NT buffer, wherein the NT buffer includes 0.005% defoamer 204, 0.236% dipotassium hydrogen phosphate trihydrate, 0.54% anhydrous potassium dihydrogen phosphate, 0.1% preservative ProClin300, 0.1% 1% potassium ferrocyanide, 3% bovine serum albumin, 0.04% ANS, and a pH value of 6.5±0.

1.

3. The magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 according to claim 1 or 2, characterized in that: The concentration of the biotinylated conjugate labeled with the monoclonal antibody against chitinase 3-like protein 1 is 2 ng / mL, and the concentration of the enzyme-labeled conjugate labeled with the monoclonal antibody against chitinase 3-like protein 1 is 1.5 ng / mL.

4. The magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 according to claim 1 or 2, characterized in that: Also included are standard diluents.

5. The preparation method of the magnetic bead chemiluminescent detection kit for detecting the content of chitinase 3-like protein 1 according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Conjugating CHI3L1 antibody to biotin; (2) CHI3L1 antibody is coupled to an enzyme-labeled conjugate; (3) Prepare CHI3L1 biotinylated conjugate antibody solution and CHI3L1 enzyme-labeled conjugate antibody solution: dilute with NT buffer to make the final concentrations of CHI3L1 biotinylated conjugate antibody solution and CHI3L1 enzyme-labeled conjugate antibody solution 2 ng / mL and 1.5 ng / mL, respectively; (4) Prepare magnetic bead buffer containing streptavidin magnetic beads and standard dilution solution.

6. The method for preparing the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 according to claim 5, characterized in that: The concentration of the magnetic bead buffer is 2 mg / mL, and the magnetic bead buffer includes the following components: 2.56 g of sodium dihydrogen phosphate, 0.43 g of disodium hydrogen phosphate, 9 g of sodium chloride, 10 g of bovine serum albumin, 20 g of mannitol, 0.02 g of 4-aminoantipyrine, 0.1 mL of Triton X-100, and 1 mL of Proclin 300, and water is added to make the volume to 1L.

7. The method for preparing the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 according to claim 5, characterized in that: The standard dilution solutions include CH3L1 solutions with concentrations of 0 ng / mL, 3 ng / mL, 6 ng / mL, 18 ng / mL, 54 ng / mL, and 120 ng / mL.

8. Use of the magnetic bead chemiluminescence detection kit for detecting the content of chitinase 3-like protein 1 as described in any one of claims 1 to 4 in detecting the content of chitinase 3-like protein 1.

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