Type IV collagen detection kit and preparation method thereof

Abstract

The present invention discloses a type IV collagen detection kit and a preparation method thereof, relating to the technical field of in vitro diagnosis. The kit comprises a reagent R1 and a reagent R2, wherein the volume ratio of the reagent R1 to the reagent R2 is 3:1. The present invention improves the sensitivity and accuracy of detection while ensuring the stability and repeatability of the kit through a carefully designed reagent formula, in particular the precise proportion of the components in the reagent R1, such as ammonium chloride, sodium azide, and Brij‑35. In particular, the reagent R1 introduces a natural plant extract, astragalus polysaccharide, and a proteinase K inhibitor as key components. The unique preparation method and the activity-promoting effect on type IV collagen hydrolase significantly enhance the biological activity of the kit, making the detection results more reliable. In addition, the latex particles coated with two paired mouse monoclonal antibodies used in the reagent R2 have high specificity and affinity, further improving the accuracy and sensitivity of detection.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a type IV collagen detection kit and a preparation method thereof. Background Art

[0002] In the biomedical field, type IV collagen is an important biomarker, and its detection is of great significance for the early diagnosis and treatment of various diseases. In particular, in histopathology and clinical medicine, accurate detection of the content and activity of type IV collagen is crucial for evaluating the progression and prognosis of the disease. Traditional detection methods include immunological methods and biochemical methods, but these methods often have problems such as complex operation, long time consumption, and low sensitivity, which cannot meet the needs of modern medicine for rapid and accurate detection. Therefore, the development of a new, efficient, and sensitive type IV collagen detection kit has broad market prospects and application value.

[0003] Traditional type IV collagen detection kits usually have multiple shortcomings. First, the reagent formula in traditional kits is often not optimized enough, which affects the sensitivity and accuracy of the test. For example, the concentration range of some reagents is not precise enough, which can easily affect the stability of the final test results. Second, the preparation method of traditional kits is relatively cumbersome, the production cycle is long, and it is difficult to achieve large-scale automated production, which to a certain extent limits its promotion and application. In addition, traditional kits also have certain difficulties in quality inspection and control, and are prone to large batch differences, which affects the accuracy and reliability of the test.

[0004] Therefore, the development of a type IV collagen detection kit and its preparation method provides strong technical support for the early diagnosis and treatment of the disease. Summary of the Invention

[0005] The purpose of the present invention is to make up for the shortcomings of the existing technology and provide a type IV collagen detection kit and a preparation method thereof. The kit achieves efficient and accurate detection of type IV collagen through a carefully designed combination of reagent R1 and reagent R2. Reagent R1 contains optimized chemical components and biological enzymes, while reagent R2 uses advanced antibody coating technology and immune enhancers, which significantly improves the sensitivity and specificity of detection. The preparation method is simple and controllable, ensuring the quality and stability of the kit.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: On the one hand, a type IV collagen detection kit is provided, the kit comprising a reagent R1 and a reagent R2, wherein the volume ratio of the reagent R1 to the reagent R2 is 3:1;

[0007] The components of the reagent R1 are: ammonium chloride 0.2mol / L-1.0mol / L, sodium azide 0.01%-0.05%, Brij-35 0.1%-1%, sodium chloride 150mmol / L, ammonia water 150mmol / L, PEG6000 1%-5%, MAK33 0.2%-0.8%, 2-mercaptoethanol 0.01%-0.05%, natural plant extract astragalus polysaccharide 0.3%-2.0%, proteinase K inhibitor 0.1%-0.8%, and type IV collagen hydrolase with an enzyme activity unit of 30U-70U per ml of reagent 1;

[0008] The components of the reagent R2 are: latex particles coated with two paired mouse monoclonal antibodies, Tris-HCl buffer of 40mmol / L-60mmol / L, sucrose of 1%-5%, gelatin of 0.2%-0.8%, NP30 of 0.05%-0.2%, Proclin300 of 0.3%, and immune enhancer interleukin-2 of 0.08%.

[0009] Furthermore, the preparation method of the natural plant extract astragalus polysaccharide in the reagent R1 is: after washing, drying and crushing the astragalus raw material, deionized water is added at a material-liquid ratio of 1:10-1:15, extracted at 90-100°C reflux for 2-3 hours, filtered and collected the extract, anhydrous ethanol is added to the extract to make the final ethanol concentration reach 70%-80%, stirred and allowed to stand to precipitate polysaccharide, and astragalus polysaccharide is obtained by centrifugation, washing and drying.

[0010] Furthermore, the preparation method of type IV collagen hydrolase in the reagent R1 is as follows: obtaining a gene sequence from a biological sample containing a type IV collagen hydrolase gene sequence, connecting it to an expression vector and then transforming it into Escherichia coli BL21, culturing it at 37°C with shaking until the logarithmic growth phase, adding IPTG with a final concentration of 0.1-1 mmol / L, inducing expression at 20-30°C for 12-16 hours, collecting the bacteria by centrifugation after expression, ultrasonically disrupting them, extracting the supernatant by centrifugation, purifying it by affinity chromatography and ion exchange chromatography, diluting it to a concentration of 30U-70U of enzyme activity per milliliter of reagent 1, and storing it in aliquots at -20°C.

[0011] Furthermore, the average particle size of the latex particles coated with the two paired mouse monoclonal antibodies in the reagent R2 is 90nm-170nm, and the latex coated with the first monoclonal antibody and the latex coated with the second monoclonal antibody are mixed in a ratio of 1:1-3:1.

[0012] Furthermore, the chemical cross-linking agents for coating the mouse monoclonal antibody onto the latex particles in the reagent R2 are ethylenediamine (EDA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0013] Furthermore, the blocking agent for the mouse monoclonal antibody-coated latex particles in the reagent R2 is a glycine buffer solution with a concentration of 1%-5% and a pH value of 6.5-8.5 and a Tween20 concentration of 1%-4%.

[0014] On the other hand, a method for preparing a type IV collagen detection kit is provided, wherein the method is used to prepare a type IV collagen detection kit according to any one of claims 1 to 6, wherein the specific steps of the preparation method are as follows:

[0015] S100, preparation of reagent 1:

[0016] S101, prepare raw materials: prepare ammonium chloride, sodium chloride, sodium azide, Brij-35, PEG6000, MAK33, 2-mercaptoethanol, astragalus polysaccharide, proteinase K inhibitor, and type IV collagen hydrolase;

[0017] S102, preliminary dissolution: add deionized water to a clean container, turn on the stirring device, add ammonium chloride and sodium chloride in sequence, heat to 50-70°C, and stir at a speed of 200-400 r / min until completely dissolved;

[0018] S103, adding ingredients: adding Brij-35, PEG6000, MAK33 and 2-mercaptoethanol and stirring evenly;

[0019] S104, adjust the pH value: calibrate the pH meter to ensure accurate measurement, immerse the electrode in the solution to be adjusted, slowly add ammonia solution while stirring the solution continuously, and closely observe the changes in the value on the pH meter display. When the value approaches the target range of 7.0-9.0, slow down the rate of ammonia addition until the desired pH value is reached;

[0020] S105, adding bioactive ingredients and bioenzymes: slowly adding astragalus polysaccharide, proteinase K inhibitor and type IV collagen hydrolase, stirring while adding, and continuing to stir after the addition is complete;

[0021] S106, Quality Testing: Test the appearance, pH value, enzyme activity, and stability of reagent 1. Enzyme activity testing: React reagent 1 with a substrate under appropriate conditions for a certain time, add a terminator to terminate the reaction, add a colorimetric reagent to develop color, measure the absorbance, and calculate the enzyme activity based on a standard curve or a known activity unit conversion method. Stability testing: Incubate reagent 1 at different temperatures for a certain time, then measure its residual activity. Evaluate its thermal stability by comparing the changes in enzyme activity after treatment at different temperatures.

[0022] S107, subpackaging: In a sterile, clean environment, subpack Reagent 1 according to specific specifications, seal the packages, and label them with relevant information;

[0023] S200, preparation of reagent 2:

[0024] S201, prepare raw materials: select latex particles, prepare two paired mouse monoclonal antibodies with strong specificity and high affinity for type IV collagen through animal immunization, cell fusion, screening and purification, prepare Tris-HCl buffer raw materials, sucrose, gelatin, NP30, Proclin300 and interleukin-2;

[0025] S202, latex particle surface modification: suspending the latex particles in a 1-5 mg / mL biotinylation reagent solution, stirring and reacting at 30-37° C. and 150-250 r / min for 60-90 minutes, centrifuging, washing, and adjusting the pH after the reaction, then adding ethylenediamine (EDA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stirring and reacting at 20-25° C. and 100-150 r / min for 30-60 minutes, centrifuging again, and washing to obtain latex particles with biotin surface modification;

[0026] S203, Preparation of Antibody-Avidin Complex: Separately couple two monoclonal antibodies to avidin. Adjust the reaction system to a pH of 5.0-6.0 using a buffer containing 0.1-0.2 mol / L MES. MES has good buffering capacity within the pH range of 5.5-6.7 and can effectively maintain a stable pH during the coupling reaction between the monoclonal antibody and avidin. The reaction is carried out at 25-30°C and a rotation speed of 100-150 rpm for 3-4 hours. After coupling, the complex is separated and purified by gel filtration chromatography.

[0027] S204, antibody-directed immobilization: mixing the antibody-avidin complex with the treated latex microparticles at a volume ratio of 1:2-1:3. At a volume ratio of 1:2-1:3, the antibody-avidin complex can achieve optimal binding effect with the treated latex microparticles. Incubating with a 0.05-0.1 mol / L Tris-HCl buffer. The Tris-HCl buffer has good buffering properties within the pH range of 7.0-9.0 and can provide a stable pH environment for the antibody-directed immobilization process. After incubation, centrifugation is performed, a blocking agent is added and stirred for reaction, and the mixture is centrifuged again and washed to obtain antibody-coated latex microparticles.

[0028] S205, prepare reagents: weigh Tris-HCl buffer raw materials in a clean container, add deionized water and stir to dissolve, adjust the pH to 6.0-8.0 and then make up to volume, add sucrose, gelatin, NP30 and Proclin300 in sequence, stir until completely dissolved, mix two antibody-coated latex particles in a ratio of 1:1-3:1 and add to the above solution, then add interleukin-2, and continue stirring until evenly dissolved;

[0029] S206, Quality Inspection: Inspect the appearance of Reagent 2, determine the pH value, and use an immunoassay to detect the concentration and activity of the antibody-coated latex microparticles. When the antibody-coated latex microparticles bind to the corresponding antigen, immune complexes are formed, causing the turbidity of the solution to change. By measuring the change in turbidity of the solution, the concentration and activity of the antibody-coated latex microparticles can be quantitatively detected. Use a biological assay to determine the activity of the immunopotentiator. The immunopotentiator may stimulate immune cells to secrete cytokines. By detecting the content of cytokines in the cell culture supernatant, the activity of the immunopotentiator can be evaluated. Test the stability under different storage conditions.

[0030] S207, subpackaging: In a sterile, clean environment, use a subpackaging device to subpack reagent 2 according to specific specifications, seal the packages, and affix labels indicating the reagent name, specifications, production date, and expiration date;

[0031] S300, kit assembly: Place the qualified and fully packaged reagents 1 and 2 into the kit in a volume ratio of 3:1, along with the supporting standards and operating instructions.

[0032] Furthermore, in said S202, the biotinylation reagent solution in the surface modification of the latex particles is an N-hydroxysuccinimide-biotin solution, and the volume ratio of the latex particles to the biotinylation reagent solution is 1:5-1:10.

[0033] Furthermore, in said S203, the molar ratio of monoclonal antibody to avidin in the preparation of the antibody-avidin complex is 3:1-5:1.

[0034] Furthermore, in the S203, gel filtration chromatography is used for separation and purification in the preparation of the antibody-avidin complex. Sepharose 4B is selected as the gel column. Sepharose 4B has a certain pore size, and its separation range can meet the requirements of separating the monoclonal antibody-avidin conjugate from unreacted antibodies, avidin and other impurities. PBS buffer containing 0.15 mol / L NaCl is selected as the eluent. 0.15 mol / L NaCl provides a suitable ionic strength, which can reduce nonspecific adsorption between proteins and the gel matrix, allowing the conjugate to be smoothly eluted from the gel column without destroying the structure and activity of the conjugate. Elution is performed at a flow rate of 0.5-1 mL / min, and the elution peak containing the antibody-avidin complex is collected to obtain the antibody-avidin complex.

[0035] Compared with the existing technology, this type IV collagen detection kit and its preparation method have the following beneficial effects:

[0036] 1. The present invention improves the sensitivity and accuracy of detection while ensuring the stability and repeatability of the test kit through a carefully designed reagent formula, especially the precise ratio of the components in reagent R1, such as ammonium chloride, sodium azide, and Brij-35. In particular, reagent R1 introduces natural plant extracts astragalus polysaccharide and proteinase K inhibitor as key ingredients. Its unique preparation method and its promotion of the activity of type IV collagen hydrolase significantly enhance the biological activity of the test kit, making the test results more reliable. In addition, the latex particles coated with two paired mouse monoclonal antibodies used in reagent R2 have high specificity and affinity, further improving the accuracy and sensitivity of detection. This new reagent formula and component ratio make the test kit of the present invention stand out among similar products and have significant technical advantages.

[0037] 2. The present invention ensures the stability and activity of each component in the kit through precise process control and optimization, such as the surface modification of latex particles, the preparation of antibody-avidin complexes and the directional fixation of antibodies. In particular, in the preparation of reagent R2, advanced chemical cross-linking agents and blocking agents are used to effectively improve the stability and anti-interference ability of the antibody-coated latex particles. At the same time, the preparation method of the present invention also focuses on quality control and testing. Through strict quality testing steps, it ensures that the quality and performance of the kit meet the standard requirements, which not only improves the production efficiency and quality stability of the kit, but also provides a strong guarantee for the wide application of the kit.

[0038] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0040] Figure 1 This is a flow chart for the preparation of reagent R1 of the type IV collagen detection kit.

[0041] Figure 2 This is a flow chart for the preparation of reagent R2 for the type IV collagen detection kit. DETAILED DESCRIPTION

[0042] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0043] Comparative Example 1:

[0044] In the clinical diagnosis of liver disease, doctors suspect that a patient may have liver fibrosis and need to test the type IV collagen content in the patient to assist in the diagnosis of the disease. In this case, the type IV collagen detection kit of the present invention can be used for detection. The specific operation is as follows:

[0045] Preparation of reagent R1 (taking the preparation of 1L of reagent R1 as an example):

[0046] Prepare the raw materials: Weigh 44.58 g of ammonium chloride (analytical grade) (corresponding to a final concentration of 0.6 mol / L), 8.77 g of sodium chloride (analytical grade) (final concentration of 150 mmol / L), 0.3 g of sodium azide (analytical grade) (0.03% mass fraction), 5 g of Brij-35 (chemically pure) (0.5% mass fraction), 30 g of PEG6000 (chemically pure) (3% mass fraction), 5 g of MAK33 (chemically pure) (0.5% mass fraction), 0.3 g of 2-mercaptoethanol (analytical grade) (0.03% mass fraction), 10 g of astragalus polysaccharide prepared according to the method of claim 2 (1.0% mass fraction), 5 g of proteinase K inhibitor (analytical grade) (0.5% mass fraction), and type IV collagen hydrolase prepared according to the method of claim 3 and diluted to an enzyme activity unit of 50 U / mL (ensuring that the total enzyme activity in 1 L of the system is 50,000 U).

[0047] Initial dissolution: Add about 500 mL of deionized water to a clean 1 L glass container, turn on the magnetic stirring device, set the speed to 300 r / min, slowly add ammonium chloride and sodium chloride, and place the container in a heating mantle, heat to 60°C, and continue stirring until completely dissolved.

[0048] Add ingredients: Add Brij-35, PEG6000, MAK33 and 2-mercaptoethanol in sequence, keep stirring to ensure that all ingredients are fully mixed.

[0049] Adjust the pH value: Use ammonia water to slowly adjust the pH value of the solution to 8.0. Stir continuously during the adjustment process to ensure a uniform and stable pH value.

[0050] Adding bioactive ingredients and bio-enzymes: While stirring, slowly add astragalus polysaccharide, proteinase K inhibitor and type IV collagen hydrolase, stirring while adding. After adding, continue stirring for 30 minutes to allow all ingredients to fully blend.

[0051] Quality testing: Observe the appearance of reagent R1; it should be clear without turbidity or precipitation. Use a pH meter to measure the pH value and confirm that it is within the range of 7.0-9.0. Use a specific enzyme activity assay to determine the activity of type IV collagenase and ensure that the enzyme activity unit is 50 U / mL. Place reagent R1 at different temperatures for a period of time to observe its stability, such as whether there is precipitation and whether the enzyme activity changes.

[0052] Repackaging: In a sterile, clean laminar bench, use a pipette to dispense reagent R1 into brown reagent bottles at a volume of 50 mL per bottle. A total of 20 bottles can be dispensed. Seal the bottle caps and affix labels indicating the reagent name, specifications, production date, expiration date, and other information.

[0053] Preparation of reagent R2 (taking the preparation of 500mL of reagent R2 as an example):

[0054] Prepare raw materials: Select an appropriate amount of latex microparticles with an average particle size ranging from 90 nm to 170 nm; prepare two paired mouse monoclonal antibodies with strong specificity and high affinity for type IV collagen through animal immunization (using Balb / c mice), cell fusion (using polyethylene glycol fusion), screening (indirect ELISA screening), and purification (Protein A affinity chromatography purification); prepare Tris-HCl buffer raw materials (analytical grade, meeting the required final concentration of 50 mmol / L, approximately 3.03 g), sucrose (analytical grade) 15 g (3% mass fraction), gelatin (analytical grade) 2.5 g (0.5% mass fraction), NP30 (chemical grade) 0.5 g (0.1% mass fraction), Proclin300 (chemical grade) 1.5 g (0.3% mass fraction), and interleukin-2 (biological reagent) 0.4 g (0.08% mass fraction).

[0055] Latex Microparticle Surface Modification: Latex microparticles were suspended in a 3 mg / mL N-hydroxysuccinimide-biotin solution (the volume ratio of latex microparticles to biotinylation reagent solution was 1:8. For a 50 mL latex microparticle volume, 400 mL of biotinylation reagent solution was required). The suspension was stirred in a thermostatic shaker at 37°C and 200 rpm for 90 minutes. After the reaction, the solution was transferred to a centrifuge tube and centrifuged at 5000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was washed three times with deionized water. The pH was adjusted to 7.0. Ethylenediamine (EDA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were then added. The reaction was stirred at 25°C and 120 rpm for 45 minutes. The mixture was centrifuged again and washed to obtain biotin-modified latex microparticles.

[0056] Preparation of Antibody-Avidin Complexes: Two monoclonal antibodies were conjugated to avidin in a buffer containing 0.15 mol / L MES, adjusted to pH 5.5, and incubated at 28°C and 120 rpm for 3.5 hours. After the coupling reaction, the complexes were isolated and purified by gel filtration chromatography using Sepharose 4B as the column and PBS containing 0.15 mol / L NaCl as the eluent at a flow rate of 0.8 mL / min. The eluted peak containing the antibody-avidin complex was collected to obtain the purified antibody-avidin complex. The molar ratio of monoclonal antibody to avidin was 4:1.

[0057] Antibody-directed immobilization: Mix the antibody-avidin complex with the treated latex microparticles at a volume ratio of 1:2.5 and incubate in 0.08 M Tris-HCl buffer for 30 minutes. After incubation, centrifuge the solution, discard the supernatant, and add 3% glycine buffer (pH 7.5) and 2% Tween 20 as a blocking agent. Stir and react for 60 minutes. Centrifuge again and wash to obtain antibody-coated latex microparticles.

[0058] Prepare the reagents: Weigh Tris-HCl buffer into a clean 500 mL glass container. Add an appropriate amount of deionized water and stir to dissolve. Adjust the pH to 7.0 using a pH meter and then bring the volume to 500 mL. Add sucrose, gelatin, NP30, and Proclin300 in that order and stir until completely dissolved. Add two antibody-coated latex microparticles mixed in a 2:1 ratio to the solution. Add interleukin-2 and continue stirring until thoroughly dissolved.

[0059] Quality testing: Observe the appearance of reagent R2, which should be uniform and free of stratification; use a pH meter to measure the pH value and confirm that it is within the range of 6.0-8.0; use immunoassay methods (such as ELISA) to detect the concentration and activity of antibody-coated latex particles; use biological assays (such as cell proliferation assays) to determine the activity of the immune enhancer interleukin-2; store reagent R2 under different temperature and humidity conditions, and regularly test its various indicators to evaluate its stability.

[0060] Repackaging: In a sterile, clean laminar bench, use automatic repackaging equipment to repack reagent R2 into brown reagent bottles at a volume of 15 mL per bottle. Approximately 33 bottles can be repacked. Seal the bottle caps and affix labels indicating the reagent name, specifications, production date, expiration date, and other information.

[0061] Kit assembly: Combine the inspected and fully packaged reagents R1 (50 mL per bottle) and R2 (15 mL per bottle) in a volume ratio of 3:1. Place the reagents R1 and R2 into the kit along with the supporting standards (including type IV collagen standard solutions with different concentration gradients) and the operating instructions to complete the preparation of the type IV collagen detection kit.

[0062] Sample collection: Collect serum samples from patients, ensuring that the sample collection process complies with clinical standards and avoids sample contamination or hemolysis.

[0063] Test Procedure: Perform the test according to the kit's instructions. Generally, first remove Reagent R1 and Reagent R2 from the refrigerator and equilibrate to room temperature. In a clean reaction vessel, add the appropriate amounts of Reagent R1 and Reagent R2 in a 3:1 volume ratio and mix thoroughly. Then, add a predetermined amount of patient serum sample, mix thoroughly, and incubate at an appropriate temperature (e.g., 37°C) for a predetermined period of time (determine the specific time according to the kit instructions). During the incubation period, the type IV collagenase in Reagent R1 will hydrolyze the type IV collagen in the sample. The hydrolysis product reacts with the paired mouse monoclonal antibody coated on the latex microparticles in Reagent R2, causing the latex microparticles to agglutinate.

[0064] Result Interpretation: The diagnosis is made based on the calculated percentage of type IV collagen, combined with the patient's clinical symptoms and other test results. If the test result shows that the percentage of type IV collagen is higher than the normal reference range, combined with the patient's clinical symptoms and other test results, the doctor can further determine that the patient may have liver fibrosis. If the test result is within the normal range, the risk of liver disease caused by abnormally elevated type IV collagen can be preliminarily ruled out, but a comprehensive diagnosis still requires combination with other tests.

[0065] Example 2:

[0066] Accuracy analysis of the method described in the present invention.

[0067] Test instrument: Hitachi 7180 fully automatic biochemical analyzer. This instrument boasts high sensitivity, precision, and excellent stability, ensuring the reliability of test results. Widely used in clinical testing, it can accurately measure various parameters in samples, providing strong technical support for this accuracy analysis.

[0068] Test samples: 50 samples were collected from subjects of different ages, genders, and backgrounds. These subjects were representative and could provide a more comprehensive assessment of the accuracy of the test method in different populations.

[0069] Comparison kit: Commercially available imported type IV collagen kit (electrochemiluminescence). Electrochemiluminescence is one of the most advanced methods currently used in clinical testing, with the advantages of high sensitivity and a wide detection range. This kit was selected as a comparison to more rigorously verify the accuracy of the present detection method.

[0070] Testing process: The 50 samples were tested using both the detection method of Example 1 and the comparative method. During the testing process, the respective operating specifications of the two detection methods were strictly followed to ensure consistency in the testing environment and personnel operating conditions to reduce errors. For example, during sample processing, serum separation was performed according to standard procedures to avoid sample contamination or hemolysis. Reagents were accurately measured to ensure the accuracy of reagent concentration and dosage. Instrument operation was calibrated and debugged in advance to ensure that the instruments were in optimal working condition.

[0071] Test results: The test data of the two methods are recorded in detail, see Comparison Table 1.

[0072] Data Analysis: Using professional statistical software, we performed linear regression analysis on the test results to obtain an equation and R² value. The closer the R² value is to 1, the better the correlation between the two test methods and the higher the accuracy of the test method.

[0073]

[0074] Comparison Table 1

[0075] The results showed that the R² value calculated based on the test results was 0.9925, which was greater than 0.95, indicating that the test results of the method described in the present invention were well correlated with the test results of the comparison kit, with no significant difference, further proving that the method described in the present invention has high accuracy (conformity).

[0076] In summary, in the clinical diagnosis of liver diseases, the detection kit of the present invention shows good performance when detecting the type IV collagen content in patients suspected of liver fibrosis. As can be seen from the comparative example 2, compared with the commercially available imported electrochemiluminescence kit, the R² value of the detection results of the kit of the present invention reached 0.9225, which is greater than 0.55, indicating that the two have a good correlation, no significant difference, and high accuracy. At the same time, the kit is easy to operate, has good reagent stability, and can also reduce interference. This means that in actual clinical applications, the type IV collagen detection kit of the present invention can effectively assist doctors in diagnosing liver diseases such as liver fibrosis, and has high practical value and promotion significance.

[0077] It is an indicator used in statistics to evaluate the goodness of fit of the regression model. The calculation process is as follows:

[0078] Calculate the total sum of squares of deviations SST, the formula is: ,in It is observations, is the mean of the observations, is the number of observations. In Example 2, the test results of the present invention or the test results of the comparison kit can be calculated according to this formula. For example, for the test results of the present invention A set of data: , ,···, first calculate the mean of this set of data , and then calculate the sum of the squares of the differences between each data and the mean, and we get , calculate the residual sum of squares SSR, the formula is: is the value predicted by the regression equation. In the second embodiment, the regression equation is: , each (Compared with the test results of the kit) Substitute into the equation to get the predicted value , and then calculate the observed value and predicted value The sum of the squares of the differences is ,calculate , the formula is: . Through the previously calculated and , substituting this formula into In Example 2 , indicating that 99.02% of the difference between the detection results of the present invention and the detection results of the comparison kit can be explained by the regression model, that is, the two have a close linear relationship and the detection method of the present invention has high accuracy.

[0079] Example 3:

[0080] Stability analysis of the method of the present invention.

[0081] Test instrument: Hitachi 7180 fully automatic biochemical analyzer. This instrument has excellent stability and repeatability, ensuring relatively consistent testing conditions when testing at different time points, reducing the impact of instrument errors on results and providing a reliable testing platform for stability analysis.

[0082] Test sample: A serum sample with a stable type IV collagen concentration of 180 ng / mL was selected. Selecting a single, stable sample concentration allows for more intuitive observation of the stability of the assay over time, avoiding fluctuations in results due to differences in sample concentration.

[0083] Testing Procedure: Using the detection method of Example 1, the serum samples were tested under the same experimental conditions (including a temperature of 25°C and a relative humidity of 50%) on days 1, 7, 14, 21, and 28, with each test repeated five times. Each test was performed strictly according to the procedures of Example 1, ensuring that the reagent amounts, reaction temperature, and reaction time conditions were exactly the same. For example, for each test, 15 μL of sample, 150 μL of Reagent 1, and 50 μL of Reagent 2 were accurately measured and reacted at 37°C for 5 minutes.

[0084] Test results: Record the results of each test in detail, see Comparison Table 2.

[0085]

[0086] Comparison Table 2

[0087] Analysis of Results: The test results show that from day 1 to day 28, the average concentration of type IV collagen in the serum samples fluctuated minimally, remaining close to the initial set concentration of 180 ng / mL. The coefficient of variation (CV) was less than 5% (the standard generally requires less than 10%), demonstrating that the detection method described in this invention has good stability over 28 days and can maintain the reliability of test results over an extended period, providing stable technical support for clinical testing.

[0088] In summary, this study on the stability of the detection method of the present invention used Hitachi 7180 fully automatic biochemical analyzer to conduct multiple tests at different time points on type IV collagen serum samples with a concentration of 180 ng / mL under uniform environmental conditions. The results showed that the average value of the test results fluctuated little within 28 days and was always close to the set concentration. The coefficient of variation was less than 5%, which was far below the standard requirement of 10%. This fully demonstrates that the detection method of the present invention has good stability, can ensure the reliability of the test results for a long time, and provides solid technical support for clinical testing. However, this study only involves samples of a single concentration, and in the future, tests of samples of different concentrations can be added to further verify its stability.

[0089] The coefficient of variation (CV) is a statistic used to measure the degree of dispersion of a set of data. In the stability study of the detection method of the present invention, the calculation steps of the coefficient of variation are as follows: Calculate the average value : First add up the multiple data obtained from each test, then divide by the number of data to calculate the standard deviation Calculate the square of the difference between each data point and the mean, add these square values, divide by the number of data points minus 1, and then take the square root of the result. The formula is: , calculate the coefficient of variation CV: divide the standard deviation by the mean, then multiply by 100% to convert the result into a percentage. The formula is: , calculate the coefficient of variation at different detection times to evaluate the discreteness of the test results and the stability of the detection method.

[0090] Example 4:

[0091] Comparative study on the effect of astragalus polysaccharide concentration on detection methods.

[0092] Test instrument: Hitachi 7180 fully automatic biochemical analyzer, which has high-precision and good repeatability detection performance, can ensure the reliability of each test data, and provide a stable detection platform for studying the performance of the detection method under different APS concentrations.

[0093] Test Sample: A serum sample with a stable type IV collagen concentration of 150 ng / mL was selected. This fixed sample concentration effectively prevents interference from sample concentration variations, limiting the experimental variable to changes in the APS concentration.

[0094] Comparison condition setting: keep other components in reagent R1 and reagent R2 unchanged, and only change the concentration of astragalus polysaccharide in reagent R1, setting it to 0% (as blank control), 0.5%, 1.0%, 1.5%, and 2.0%, respectively, and perform 5 tests on samples of each concentration.

[0095] Testing Procedure: Strictly follow the test kit instructions. Remove reagents R1 and R2 from the refrigerator and allow them to equilibrate to room temperature. In a clean reaction vessel, add the corresponding reagents in a 3:1 volume ratio of reagent R1 to reagent R2 and mix thoroughly. Next, add the required amount of serum sample, mix thoroughly, and incubate at 37°C (incubation time is determined according to the kit instructions). After incubation, use the accompanying immunoturbidimetric analyzer to measure the absorbance of the reaction system and calculate the type IV collagen content in the sample based on the standard curve.

[0096] Test results: Record the data of each test in detail, as shown in Comparison Table 3.

[0097]

[0098] Comparison Table 3

[0099] As can be seen from the data in Table 3, as the concentration of APS increases, the average value of the test results gradually approaches the actual sample concentration of 150 ng / mL, and the coefficient of variation gradually decreases. When the APS concentration reaches 1.0% and above, the stability and accuracy of the test results are relatively good, indicating that the appropriate addition of APS can improve the performance of the detection method.

[0100] In summary, a comparative study of type IV collagen detection results at different APS concentrations revealed that as APS concentration increased, the mean value of the test results gradually approached the actual sample concentration, the coefficient of variation decreased, and the stability and accuracy of the test were improved. Within the concentration range of 0.3%-2.0%, the results were ideal at concentrations of 1.0% and above. This suggests that adding an appropriate amount of APS to reagent R1 in the test kit can help optimize the performance of the assay. However, the current study involved only five tests and a limited concentration gradient. Further expansion of the number of tests and concentration range is needed to further explore the impact of APS on test results and provide a more comprehensive basis for kit optimization.

[0101] Embodiment 5:

[0102] Comparative study of the effect of proteinase K inhibitor concentration on test results.

[0103] Test instrument: The Hitachi 7180 fully automatic biochemical analyzer was used. This instrument has high sensitivity and precision, and can stably and accurately detect the content of type IV collagen in the samples, providing a reliable detection basis for this comparative study.

[0104] Test sample: A serum sample with a stable type IV collagen concentration of 200 ng / mL was selected. This stable sample concentration reduces interference caused by sample variability, allowing the experiment to focus on changes in proteinase K inhibitor concentration.

[0105] Comparison condition setting: keep other components in reagent R1 and reagent R2 unchanged, and only adjust the concentration of proteinase K inhibitor in reagent R1 to 0% (blank control), 0.2%, 0.4%, 0.6%, and 0.8%, respectively. Perform 5 tests on samples of each concentration.

[0106] Testing Procedure: Follow the kit's instructions. Remove Reagent R1 and Reagent R2 from the refrigerator and equilibrate to room temperature. In a clean reaction vessel, add Reagent R1 to Reagent R2 in a 3:1 volume ratio and mix thoroughly. Then, add the measured serum sample, mix thoroughly, and incubate at 37°C (the incubation time is determined by the kit instructions). After incubation, measure the absorbance of the reaction system using the accompanying immunoturbidimetric analyzer and calculate the type IV collagen content in the sample using the standard curve.

[0107] Test results: See comparison table 4 for specific results.

[0108]

[0109] Comparison Table 4

[0110] As can be seen from the comparison table, as the concentration of proteinase K inhibitor increases, the average value of the test results gradually approaches the actual sample concentration of 200 ng / mL, and the coefficient of variation gradually decreases, indicating that appropriately increasing the concentration of proteinase K inhibitor helps to improve the accuracy and stability of the detection method.

[0111] In summary, this study on the effect of proteinase K inhibitor concentration on type IV collagen detection results is of great significance. By changing the concentration of proteinase K inhibitor in reagent R1 and conducting 5 comparative tests, it was found that as its concentration increased from 0.10% to 0.80%, the average value of the test results continued to approach the actual sample concentration of 200 ng / mL, and the coefficient of variation continued to decrease. This shows that increasing the concentration of proteinase K inhibitor can effectively improve the accuracy and stability of the detection method. However, the concentration gradient setting of this study is limited. In the future, the concentration range can be expanded, the number of tests can be increased, and its optimal concentration and its comprehensive impact on detection performance can be further explored to provide a more solid theoretical and practical basis for optimizing the type IV collagen detection kit.

[0112] Example 6:

[0113] Comparative study on the effect of average particle size of latex particles on test results.

[0114] Test instrument: Hitachi 7180 fully automatic biochemical analyzer, which has high detection accuracy and good repeatability, can ensure the reliability of each test result, and provide a stable detection platform for studying the influence of the average particle size of latex particles on the test results.

[0115] Test Sample: Select a serum sample with a defined and stable type IV collagen concentration of 180 ng / mL. This stable sample concentration prevents fluctuations in sample concentration from interfering with the test results, limiting the experimental variables to changes in the average particle size of the latex particles.

[0116] Comparison conditions: Keeping all other components of reagents R1 and R2 unchanged, only the average particle size of the latex particles in reagent R2 was changed. The average particle size was set to 90 nm, 110 nm, 130 nm, 150 nm, and 170 nm, respectively. Five tests were performed on the sample under each particle size condition.

[0117] Testing Procedure: Strictly follow the kit instructions. Remove Reagent R1 and Reagent R2 from the refrigerator and equilibrate to room temperature. In a clean reaction vessel, add Reagent R1 to Reagent R2 in a 3:1 volume ratio and mix thoroughly. Next, add the desired amount of serum sample, mix thoroughly, and incubate at 37°C (incubation time is determined according to the kit instructions). After incubation, measure the absorbance of the reaction system using an immunoturbidimetric analyzer and calculate the type IV collagen content in the sample using the standard curve.

[0118] Test results: See comparison table 5 for specific results.

[0119]

[0120] Comparison Table 5

[0121] The data in the comparison table show that within the average particle size range of 90nm-170nm, as the particle size increases, the average test result at 130nm-150nm is closer to the actual sample concentration of 180ng / mL, and the coefficient of variation is relatively small. This indicates that the average particle size of latex particles within a certain range can affect the accuracy and stability of test results. 130nm-150nm may be a more suitable particle size range, but further research and verification are needed.

[0122] In summary, this study on the influence of the average particle size of latex microparticles on the detection results of type IV collagen is of significant significance. Comparative detection within the particle size range of 90nm-170nm found that as the particle size increases, the average value of the detection results is close to the actual concentration of the sample of 180ng / mL to varying degrees. Among them, the average value of the detection results at a particle size of 130nm-150nm is closer to the actual concentration, and the coefficient of variation is relatively small. This shows that the accuracy and stability of the detection within this particle size range are relatively high. However, the particle size gradient and number of tests in this study are limited. In the future, more particle size gradients and repeated tests can be added to further explore the influence of the average particle size of latex microparticles on the detection performance, providing a more reliable basis for optimizing the test kit.

[0123] Embodiment seven:

[0124] Specific detection.

[0125] Test equipment

[0126] Hitachi 7180 fully automatic biochemical analyzer was selected to ensure that the instrument performance was stable and calibrated, and all parameters met the testing requirements.

[0127] Test samples

[0128] A serum sample containing type IV collagen at a concentration of 100 ng / mL was prepared as the baseline test sample. Rheumatoid factor solutions at varying concentrations (0 IU / mL, 33.75 IU / mL, 67.5 IU / mL, 135 IU / mL, 270 IU / mL, 500 IU / mL, and 540 IU / mL) and heterophilic antibody solutions (0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 40 μg / mL, and 50 μg / mL) were also prepared.

[0129] Test steps

[0130] Mix the type IV collagen serum sample with different concentrations of rheumatoid factor at a ratio of 9:1 to obtain a series of samples containing different concentrations of rheumatoid factor interference. For example, take 90 μL of type IV collagen serum sample and mix it evenly with 10 μL of rheumatoid factor solution of the corresponding concentration.

[0131] Similarly, serum samples containing type IV collagen were mixed with heterophilic antibodies at different concentrations at a ratio of 9:1 to prepare samples containing heterophilic antibody interference at different concentrations.

[0132] Each sample containing interfering substances prepared above was tested three times using the detection method described in Example 1 of the present invention (reagent 1: ammonium chloride 0.2 mol / L, sodium azide 0.05%, Brij-35 0.3%, sodium chloride 150 mmol / L, pH adjusted to 7.5 with aqueous ammonia, PEG 600 02%, MAK 33 0.5%, 2-mercaptoethanol 0.02%; reagent 2: latex microparticles coated with two paired mouse monoclonal antibodies (final latex microparticle concentration 0.1%), Tris-HCl buffer 50 mmol / L, pH 7.0, sucrose 3%, gelatin 0.5%, NP 30 0.1%, Proclin 300 0.3%). 15 μL of the mixed sample and 150 μL of reagent 1 were added to the automatic biochemical analyzer, and the mixture was reacted at 37°C for 5 minutes. Then, 50 μL of reagent 2 was added and the mixture was reacted at 37°C for 5 minutes. The absorbance at 600 nm was measured, and the percentage of type IV collagen in the sample was calculated based on the standard curve.

[0133] Using the test results of type IV collagen serum samples without interfering substances (i.e., 0 interfering substance concentration) as the control, calculate the deviation between the test results of other samples containing interfering substances and the control results. The deviation calculation formula is: Deviation = [(average test value of samples containing interfering substances - average test value of samples with 0 interfering substance concentration) ÷ average test value of samples with 0 interfering substance concentration] × 100%.

[0134]

[0135] Table 6

[0136] The results, as shown in Table 6, show that when the rheumatoid factor concentration is below 500 IU / mL, the deviation is less than 10%, within the required range, and no interference is determined. When the heterophilic antibody concentration is below 40 μg / mL, the deviation is less than 10%, within the required range, and no interference is determined. This demonstrates that the detection method of the present invention has good anti-interference ability and good specificity for rheumatoid factor and heterophilic antibodies.

[0137] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A type IV collagen detection kit, characterized in that: The kit includes reagent R1 and reagent R2, wherein the volume ratio of the reagent R1 to the reagent R2 is 3:1; The components of the reagent R1 are: ammonium chloride 0.2mol / L-1.0mol / L, sodium azide 0.01%-0.05%, Brij-35 0.1%-1%, sodium chloride 150mmol / L, ammonia water 150mmol / L, PEG6000 1%-5%, MAK33 0.2%-0.8%, 2-mercaptoethanol 0.01%-0.05%, natural plant extract astragalus polysaccharide 0.3%-2.0%, proteinase K inhibitor 0.1%-0.8%, and type IV collagen hydrolase with an enzyme activity unit of 30U-70U per ml of reagent 1; The components of the reagent R2 are: latex particles coated with two paired mouse monoclonal antibodies, Tris-HCl buffer of 40mmol / L-60mmol / L, sucrose of 1%-5%, gelatin of 0.2%-0.8%, NP30 of 0.05%-0.2%, Proclin300 of 0.3%, and immune enhancer interleukin-2 of 0.08%.

2. A type IV collagen detection kit according to claim 1, characterized in that, The preparation method of the natural plant extract astragalus polysaccharide in the reagent R1 is as follows: after washing, drying and crushing the astragalus raw material, deionized water is added at a material-liquid ratio of 1:10-1:15, and extraction is carried out under reflux at 90-100°C for 2-3 hours. The extract is filtered and collected, and anhydrous ethanol is added to the extract to make the final ethanol concentration reach 70%-80%. After stirring, the extract is allowed to stand and precipitate the polysaccharide, and the astragalus polysaccharide is obtained by centrifugation, washing and drying.

3. A type IV collagen detection kit according to claim 1, characterized in that, The preparation method of the type IV collagen hydrolase in the reagent R1 is as follows: obtaining a gene sequence from a biological sample containing the type IV collagen hydrolase gene sequence, ligating the gene sequence to an expression vector, and then transforming the gene into Escherichia coli BL21. The gene sequence is cultured at 37°C with shaking until the logarithmic growth phase, adding IPTG at a final concentration of 0.1-1 mmol / L, and inducing expression at 20-30°C for 12-16 hours. After expression, the bacteria are collected by centrifugation, ultrasonically disrupted, and the supernatant is extracted by centrifugation. The supernatant is purified by affinity chromatography and ion exchange chromatography, diluted to a concentration of 30U-70U of enzyme activity per milliliter of reagent 1, and stored in aliquots at -20°C.

4. A type IV collagen detection kit according to claim 1, characterized in that, The average particle size of the latex particles coated with the two paired mouse monoclonal antibodies in the reagent R2 is 90nm-170nm, and the latex coated with the first monoclonal antibody and the latex coated with the second monoclonal antibody are mixed in a ratio of 1:1-3:

1.

5. A type IV collagen detection kit according to claim 1, characterized in that, The chemical cross-linking agents for coating the mouse monoclonal antibody onto the latex particles in the reagent R2 are ethylenediamine (EDA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

6. A type IV collagen detection kit according to claim 1, characterized in that: The blocking agent for the mouse monoclonal antibody-coated latex particles in the reagent R2 is a glycine buffer solution with a concentration of 1%-5% and a pH value of 6.5-8.5 and a Tween20 concentration of 1%-4%.

7. A method for preparing a type IV collagen detection kit, characterized in that: The method is used to prepare a type IV collagen detection kit according to any one of claims 1 to 6, and the specific steps of the preparation method are: S100, preparation of reagent 1: S101, prepare raw materials: prepare ammonium chloride, sodium chloride, sodium azide, Brij-35, PEG6000, MAK33, 2-mercaptoethanol, astragalus polysaccharide, proteinase K inhibitor, and type IV collagen hydrolase; S102, preliminary dissolution: add deionized water to a clean container, turn on the stirring device, add ammonium chloride and sodium chloride in sequence, heat to 50-70°C, and stir at a speed of 200-400 r / min until completely dissolved; S103, adding ingredients: adding Brij-35, PEG6000, MAK33 and 2-mercaptoethanol and stirring evenly; S104, adjusting the pH value: adding ammonia water to adjust the pH value of the solution to 7.0-9.0, and continuing to stir after adjustment; S105, adding bioactive ingredients and bioenzymes: slowly adding astragalus polysaccharide, proteinase K inhibitor and type IV collagen hydrolase, stirring while adding, and continuing to stir after the addition is complete; S106, quality inspection: inspect the appearance, pH value, enzyme activity and stability of reagent 1; S107, subpackaging: In a sterile, clean environment, subpack Reagent 1 according to specific specifications, seal the packages, and label them with relevant information; S200, preparation of reagent 2: S201, prepare raw materials: select latex particles, prepare two paired mouse monoclonal antibodies with strong specificity and high affinity for type IV collagen through animal immunization, cell fusion, screening and purification, prepare Tris-HCl buffer raw materials, sucrose, gelatin, NP30, Proclin300 and interleukin-2; S202, latex particle surface modification: suspending the latex particles in a 1-5 mg / mL biotinylation reagent solution, stirring and reacting at 30-37° C. and 150-250 r / min for 60-90 minutes, centrifuging, washing, and adjusting the pH after the reaction, then adding ethylenediamine (EDA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stirring and reacting at 20-25° C. and 100-150 r / min for 30-60 minutes, centrifuging again, and washing to obtain latex particles with biotin surface modification; S203, preparation of antibody-avidin complex: Separately couple two monoclonal antibodies to avidin. The reaction system is adjusted to pH 5.0-6.0 using a buffer containing 0.1-0.2 mol / L MES. The reaction is carried out at 25-30°C and a rotation speed of 100-150 rpm for 3-4 hours. After coupling, the complex is separated and purified by gel filtration chromatography. S204, antibody-directed immobilization: mixing the antibody-avidin complex with the treated latex microparticles at a volume ratio of 1:2-1:3, incubating with a buffer containing 0.05-0.1 mol / L Tris-HCl, centrifuging after incubation, adding a blocking agent and stirring to react, centrifuging again, and washing to obtain antibody-coated latex microparticles; S205, prepare reagents: weigh Tris-HCl buffer raw materials in a clean container, add deionized water and stir to dissolve, adjust the pH to 6.0-8.0 and then make up to volume, add sucrose, gelatin, NP30 and Proclin300 in sequence, stir until completely dissolved, mix two antibody-coated latex particles in a ratio of 1:1-3:1 and add to the above solution, then add interleukin-2, and continue stirring until evenly dissolved; S206, Quality Inspection: Inspect the appearance of reagent 2, determine the pH value, detect the concentration and activity of the antibody-coated latex particles using an immunoassay, determine the activity of the immunopotentiator using a biological assay, and test the stability under different storage conditions; S207, subpackaging: In a sterile, clean environment, use a subpackaging device to subpack reagent 2 according to specific specifications, seal the packages, and affix labels indicating the reagent name, specifications, production date, and expiration date; S300, kit assembly: Place the qualified and fully packaged reagents 1 and 2 into the kit in a volume ratio of 3:1, along with the supporting standards and operating instructions.

8. The method for preparing a type IV collagen detection kit according to claim 7, wherein: In the above S202, the biotinylation reagent solution in the surface modification of the latex particles is an N-hydroxysuccinimide-biotin solution, and the volume ratio of the latex particles to the biotinylation reagent solution is 1:5-1:

10.

9. The method for preparing a type IV collagen detection kit according to claim 7, wherein: In the above S203, the molar ratio of monoclonal antibody to avidin in the preparation of the antibody-avidin complex is 3:1-5:

1.

10. The method for preparing a type IV collagen detection kit according to claim 7, wherein: In the S203, gel filtration chromatography is used for separation and purification in the preparation of the antibody-avidin complex, Sepharose 4B is selected as the gel column, PBS buffer containing 0.15 mol / L NaCl is selected as the eluent, and elution is performed at a flow rate of 0.5-1 mL / min. The elution peak containing the antibody-avidin complex is collected to obtain the antibody-avidin complex.

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