IV type collagen detection kit and preparation method thereof

Abstract

The invention discloses an IV-type collagen detection kit and a preparation method thereof, and relates to the technical field of in vitro diagnos.The kit comprises a reagent R1 and a reagent R2, and the volume ratio of the reagent R1 to the reagent R2 is 3: 1; through an elaborately designed reagent formula, especially the accurate proportion of all components in the reagent R1, such as ammonium chloride, sodium azide and Brij-35, the detection sensitivity and accuracy are improved, the stability and repeatability of the kit are also ensured, and particularly, the natural plant extract astragalus polysaccharide and the protease K inhibitor are introduced into the reagent R1 as key components, so that the detection sensitivity and accuracy are improved, and the detection sensitivity and repeatability are improved. According to the present invention, the unique preparation method and the activity promotion effect on the IV-type collagen hydrolase significantly enhance the biological activity of the kit, such that the detection result is reliable, and the two latex particles coated with the paired mouse monoclonal antibodies adopted in the reagent R2 have high specificity and affinity, such that the kit can be used for the detection of the IV-type collagen hydrolase. And the detection accuracy and sensitivity are further improved.

Description

Technical Field

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

[0002] In the field of biomedicine, type IV collagen, as an important biomarker, its detection is of great significance for the early diagnosis and treatment of various diseases. Especially in histopathology and clinical medicine, accurately detecting the content and activity of type IV collagen is crucial for evaluating the progression and prognosis of diseases. 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, making it difficult to meet the requirements of modern medicine for rapid and accurate detection. Therefore, developing a new type, efficient, and sensitive type IV collagen detection kit has broad market prospects and application values.

[0003] Traditional type IV collagen detection kits usually have deficiencies in multiple aspects. First of all, the reagent formulations in traditional kits are often not optimized enough, resulting in the sensitivity and accuracy of detection being affected. For example, the concentration ranges of some reagents are not precise enough, which easily affects the stability of the final detection results. Secondly, the preparation methods of traditional kits are relatively cumbersome, with a long production cycle and it is difficult to achieve large-scale automated production, which to a certain extent limits their promotion and application. In addition, there are also certain difficulties in quality inspection and control of traditional kits, and it is easy to have large differences between batches, thus affecting the accuracy and reliability of detection.

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

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

[0006] To solve the above technical problems, the present invention provides the following technical solutions: On the one hand, a type IV collagen detection kit, which includes reagent R1 and reagent R2, and the volume ratio of the reagent R1 to the reagent R2 is 3:1; The components of the reagent R1 are as follows: ammonium chloride is 0.2 mol / L - 1.0 mol / L, sodium azide is 0.01% - 0.05%, Brij-35 is 0.1% - 1%, sodium chloride is 150 mmol / L, ammonia water is 150 mmol / L, PEG6000 is 1% - 5%, MAK33 is 0.2% - 0.8%, 2-mercaptoethanol is 0.01% - 0.05%, natural plant extract astragalus polysaccharide is 0.3% - 2.0%, protease K inhibitor is 0.1% - 0.8%, and type IV collagenase with an enzyme activity unit of 30 U - 70 U per milliliter of reagent 1; The components of the reagent R2 are as follows: latex particles coated with 2 pairs of paired mouse monoclonal antibodies, Tris-Hcl buffer is 40 mmol / L - 60 mmol / L, sucrose is 1% - 5%, gelatin is 0.2% - 0.8%, NP30 is 0.05% - 0.2%, Proclin300 is 0.3%, and immune enhancer interleukin-2 is 0.08%.

[0007] Furthermore, the preparation method of the natural plant extract astragalus polysaccharide in the reagent R1 is as follows: after washing, drying, and pulverizing the astragalus raw material, add deionized water according to a material-liquid ratio of 1:10 - 1:15, extract at 90 - 100 °C under reflux for 2 - 3 hours, filter and collect the extract, add absolute ethanol to the extract to make the final ethanol concentration reach 70% - 80%, stir and then let it stand to precipitate polysaccharides, and obtain astragalus polysaccharide through centrifugation, washing, and drying.

[0008] Furthermore, the preparation method of the type IV collagenase in the reagent R1 is as follows: obtain the gene sequence from a biological sample containing the type IV collagenase gene sequence, ligate it to an expression vector and then transform it into Escherichia coli BL21, culture it with shaking at 37 °C until the logarithmic growth phase, add IPTG with a final concentration of 0.1 - 1 mmol / L, induce expression at 20 - 30 °C for 12 - 16 hours, after expression, centrifuge to collect the bacterial cells, ultrasonically disrupt them, centrifuge to extract the supernatant, purify it by affinity chromatography and ion exchange chromatography methods, dilute it to make the enzyme activity unit per milliliter of reagent 1 between 30 U and 70 U, and dispense and store it at -20 °C.

[0009] Furthermore, the average particle size of the latex particles coated with 2 pairs of paired mouse monoclonal antibodies in the reagent R2 is 90 nm - 170 nm, 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.

[0010] Furthermore, the chemical cross-linking agents for the mouse monoclonal antibody-coated latex particles in the reagent R2 are ethylenediamine EDA and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC.

[0011] 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 Tween 20 with a concentration of 1%-4%.

[0012] On the other hand, a method for preparing a type IV collagen detection kit, which is used to prepare a type IV collagen detection kit according to any one of claims 1-6. The specific steps of the preparation method are as follows: S100, Preparation of reagent 1: S101, Preparation of raw materials: Prepare ammonium chloride, sodium chloride, sodium azide, Brij-35, PEG6000, MAK33, 2-mercaptoethanol, astragalus polysaccharide, protease K inhibitor, and type IV collagen hydrolase; S102, Preliminary dissolution: Add deionized water to a clean container, turn on the stirring device, and sequentially add ammonium chloride and sodium chloride. Heat to 50-70°C and stir at a speed of 200-400 r / min until completely dissolved; S103, Addition of components: Add Brij-35, PEG6000, MAK33, and 2-mercaptoethanol and stir evenly; S104, Adjustment of pH value: Calibrate the pH meter to ensure accurate measurement. Immerse the electrode in the solution to be adjusted, slowly add ammonia water while constantly stirring the solution, and closely observe the change in the value on the pH meter display screen. When the value approaches the target range of 7.0-9.0, slow down the dropping speed of ammonia water until the required pH value is reached; S105, Addition of bioactive components and bioenzymes: Slowly add astragalus polysaccharide, protease K inhibitor, and type IV collagen hydrolase while stirring. After adding, continue to stir; S106, Quality inspection: Detect the appearance, pH value, bioenzyme activity, and stability of reagent 1. Enzyme activity detection: React reagent 1 with the substrate under appropriate conditions for a certain period of time, add a terminator to terminate the reaction, add a chromogenic agent for color development, measure the absorbance, and calculate the enzyme activity according to the standard curve or the known activity unit conversion method; Stability detection: Incubate reagent 1 at different temperatures for a certain period of time, and then measure its remaining activity. Evaluate its thermal stability by comparing the changes in enzyme activity after different temperature treatments; S107, Sub-packaging: Under sterile and clean conditions, sub-package reagent 1 according to specific specifications, seal the package and label it with relevant information; S200, Preparation of reagent 2: S201, Prepare raw materials: Select latex microparticles, and prepare 2 pairs of murine monoclonal antibodies with strong specificity and high affinity for type IV collagen through animal immunization, cell fusion, screening, and purification. Prepare raw materials for Tris-Hcl buffer solution, sucrose, gelatin, NP30, Proclin300, and interleukin-2; S202, Surface modification of latex microparticles: Suspend the latex microparticles in a biotinylation reagent solution at 1 - 5 mg / mL, stir and react at 30 - 37 °C and a rotation speed of 150 - 250 r / min for 60 - 90 minutes. After the reaction, centrifuge, wash, and adjust the pH. Then add ethylenediamine EDA and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, stir and react at 20 - 25 °C and a rotation speed of 100 - 150 r / min for 30 - 60 minutes. Centrifuge and wash again to obtain latex microparticles with biotin on the surface; S203, Preparation of antibody-avidin complex: Couple the 2 monoclonal antibodies with avidin respectively. The reaction system uses a buffer solution containing 0.1 - 0.2 mol / L MES, and adjust the pH to 5.0 - 6.0. MES has good buffering capacity in the pH range of 5.5 - 6.7, and can effectively maintain the pH stability of the reaction system during the coupling reaction of monoclonal antibody and avidin. React at 25 - 30 °C and a rotation speed of 100 - 150 r / min for 3 - 4 hours. After coupling, separate and purify by gel filtration chromatography; S204, Oriented immobilization of antibody: Mix the antibody-avidin complex and the treated latex microparticles at a volume ratio of 1:2 - 1:3. At this volume ratio, the antibody-avidin complex can achieve the best binding effect with the treated latex microparticles. Incubate with a buffer solution containing 0.05 - 0.1 mol / L Tris-HCl. Tris-HCl buffer has good buffering performance in the pH range of 7.0 - 9.0, and can provide a stable pH environment for the oriented immobilization of the antibody. After incubation, centrifuge, add a blocking agent and stir to react. Centrifuge and wash again to obtain antibody-coated latex microparticles; S205, Prepare the reagent: Weigh the raw materials for Tris-Hcl buffer solution in a clean container, add deionized water and stir to dissolve. Adjust the pH value to 6.0 - 8.0 and then make up the volume. Add sucrose, gelatin, NP30, and Proclin300 in sequence, stir until completely dissolved. Mix two kinds of antibody-coated latex microparticles at a ratio of 1:1 - 3:1 and add them to the above solution, then add interleukin-2, and continuously stir evenly; S206, Quality inspection: Check the appearance of Reagent 2, measure the pH value, and use immunoassay methods to detect the concentration and activity of antibody-coated latex particles. When the antibody-coated latex particles bind to the corresponding antigen, immune complexes will be formed, resulting in changes in the turbidity of the solution. By measuring the change in turbidity of the solution, the concentration and activity of antibody-coated latex particles can be quantitatively detected. Use biological detection methods 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. Detect the stability under different storage conditions; S207, Sub-packaging: In a sterile and clean environment, use sub-packaging equipment to sub-package Reagent 2 according to specific specifications, seal and label it, indicating information such as the name of the reagent, specifications, production date, and expiration date; S300, Kit assembly: Put the qualified and completely packaged Reagent 1 and Reagent 2 into the kit according to the volume ratio of Reagent 1 to Reagent 2 of 3:1, together with the supporting standard product and operation manual.

[0013] Furthermore, in the S202, surface modification of latex particles, the biotinylation reagent solution is N-hydroxysuccinimide biotin solution, and the volume ratio of latex particles to biotinylation reagent solution is 1:5 - 1:10.

[0014] Furthermore, in the S203, preparation of antibody-avidin complex, the molar ratio of monoclonal antibody to avidin is 3:1 - 5:1.

[0015] Furthermore, in the S203, preparation of antibody-avidin complex, gel filtration chromatography is used for separation and purification. Sepharose 4B is selected as the gel column. Sepharose 4B has a certain pore size, and its separation range can meet the separation requirements of monoclonal antibody-avidin conjugates 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 the non-specific adsorption between proteins and the gel matrix, enabling the conjugate to be eluted from the gel column smoothly without destroying the structure and activity of the conjugate. Elute at a flow rate of 0.5 - 1 mL / min, collect the elution peak containing the antibody-avidin complex, and obtain the antibody-avidin complex.

[0016] Compared with the prior art, the type IV collagen detection kit and its preparation method have the following beneficial effects: I. Through a carefully designed reagent formula, especially the precise ratio of each component in reagent R1, such as ammonium chloride, sodium azide, and Brij-35, the present invention not only improves the sensitivity and accuracy of detection, but also ensures the stability and repeatability of the kit. In particular, natural plant extract astragalus polysaccharide and protease K inhibitor are introduced as key components in reagent R1. Their unique preparation method and the promoting effect on the activity of 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 pairs of paired murine 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 kit of the present invention stand out among similar products and have significant technical advantages.

[0017] II. Through fine process control and optimization, such as the surface modification of latex particles, the preparation of antibody-avidin complexes, and the antibody orientation fixation step, the present invention ensures the stability and activity of each component in the kit. In particular, when preparing reagent R2, advanced chemical cross-linking agents and blocking agents are used, effectively improving the stability and anti-interference ability of antibody-coated latex particles. At the same time, the preparation method of the present invention also pays attention to quality control and detection. Through strict quality detection steps, it ensures that the quality and performance of the kit meet the standard requirements, not only improving the production efficiency and quality stability of the kit, but also providing a strong guarantee for the wide application of the kit.

[0018] Other advantages, objectives, and features of the present invention will be described to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 Flow chart for the preparation of reagent R1 for the type IV collagen detection kit.

[0021] Figure 2 Flow chart for the preparation of reagent R2 for the type IV collagen detection kit. DETAILED DESCRIPTION OF THE INVENTION

[0022] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation manners, structures, features, and their effects of the present invention as follows.

[0023] Comparative Example 1: In the clinical liver disease diagnosis scenario, when a doctor suspects that a patient may have liver fibrosis and needs to detect the content of type IV collagen in the patient's body to assist in diagnosing the condition, the type IV collagen detection kit of the present invention can be used for detection. The specific operation is as follows: Preparation of reagent R1 (taking the preparation of 1L of reagent R1 as an example): Prepare raw materials: Weigh 44.58 g of ammonium chloride (analytical pure) (corresponding to a final concentration of 0.6 mol / L), 8.77 g of sodium chloride (analytical pure) (final concentration of 150 mmol / L), 0.3 g of sodium azide (analytical pure) (mass fraction of 0.03%), 5 g of Brij-35 (chemical pure) (mass fraction of 0.5%), 30 g of PEG6000 (chemical pure) (mass fraction of 3%), 5 g of MAK33 (chemical pure) (mass fraction of 0.5%), 0.3 g of 2-mercaptoethanol (analytical pure) (mass fraction of 0.03%), 10 g of astragalus polysaccharide prepared according to the method of claim 2 (mass fraction of 1.0%), 5 g of protease K inhibitor (analytical pure) (mass fraction of 0.5%), and type IV collagenase 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 the 1L system is 50,000 U).

[0024] Preliminary dissolution: Add approximately 500 mL of deionized water to a clean 1L glass container, turn on the magnetic stirring device, set the rotation speed to 300 r / min, slowly add ammonium chloride and sodium chloride, and at the same time place the container in a heating jacket and heat to 60 °C, and continue stirring until completely dissolved.

[0025] Add components: Add Brij-35, PEG6000, MAK33, and 2-mercaptoethanol in sequence, keep stirring, and make each component fully mixed and uniform.

[0026] Adjust the pH value: Slowly adjust the pH value of the solution to 8.0 using ammonia water, and keep stirring during the adjustment process to ensure that the pH value is uniform and stable.

[0027] Add bioactive components and bioenzymes: Slowly add astragalus polysaccharide, protease K inhibitor, and type IV collagenase under stirring, stir while adding, and continue stirring for 30 minutes after adding to make each component fully blend.

[0028] Quality inspection: Observe the appearance of reagent R1, which 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 detection method to measure the activity of type IV collagenase, ensuring that the enzyme activity unit is 50 U / mL; place reagent R1 under different temperature conditions for a period of time and observe its stability, such as whether precipitation occurs and whether the enzyme activity changes, etc.

[0029] Sub-packaging: Inside a sterile and clean laminar flow hood, use a pipette to sub-package reagent R1 into brown reagent bottles according to the specification of 50 mL per bottle. 20 bottles can be sub-packaged. After sealing the bottle mouths, affix labels indicating the reagent name, specification, production date, expiration date and other information.

[0030] Preparation of reagent R2 (taking the preparation of 500 mL of reagent R2 as an example): Prepare raw materials: Select an appropriate amount of latex particles with an average particle size in the range of 90 nm - 170 nm; prepare 2 pairs of 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 method), screening (screening by indirect ELISA method) and purification (purification by Protein A affinity chromatography); prepare Tris-Hcl buffer raw materials (analytical pure, meeting the final concentration requirement of 50 mmol / L, about 3.03 g), sucrose (analytical pure) 15 g (3% mass fraction), gelatin (analytical pure) 2.5 g (0.5% mass fraction), NP30 (chemically pure) 0.5 g (0.1% mass fraction), Proclin300 (chemically pure) 1.5 g (0.3% mass fraction) and interleukin-2 (biological reagent) 0.4 g (0.08% mass fraction).

[0031] Surface modification of latex particles: Suspend the latex particles in an N-hydroxysuccinimide biotin solution with a concentration of 3 mg / mL (the volume ratio of latex particles to biotinylation reagent solution is 1:8. Assuming the volume of latex particles is 50 mL, then 400 mL of biotinylation reagent solution is required), place it in a constant temperature shaker, and stir and react at 37 °C and a rotation speed of 200 r / min for 90 minutes. After the reaction is completed, transfer the solution to a centrifuge tube, centrifuge at a rotation speed of 5000 r / min for 15 minutes, discard the supernatant, wash the precipitate 3 times with deionized water, and adjust the pH value to 7.0. Then add ethylenediamine EDA and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, stir and react at 25 °C and a rotation speed of 120 r / min for 45 minutes, centrifuge and wash again to obtain latex particles with biotin modified on the surface.

[0032] Preparation of antibody-avidin complex: Two monoclonal antibodies were conjugated with avidin separately. The reaction system used a buffer containing 0.15 mol / L MES, adjusted the pH to 5.5, and reacted at 28 °C and a rotation speed of 120 r / min for 3.5 hours. After the conjugation reaction, gel filtration chromatography was used for separation and purification. Sepharose 4B was selected as the gel column, and PBS buffer containing 0.15 mol / L NaCl was used as the eluent. The flow rate was controlled at 0.8 mL / min, and the elution peak containing the antibody-avidin complex was collected to obtain the purified antibody-avidin complex. Among them, the molar ratio of monoclonal antibody to avidin was 4:1.

[0033] Antibody directed immobilization: The antibody-avidin complex and the treated latex particles were mixed at a volume ratio of 1:2.5 and incubated with a buffer containing 0.08 mol / L Tris-HCl for 30 minutes. After incubation, the solution was centrifuged, the supernatant was discarded, and glycine buffer with a concentration of 3% and a pH value of 7.5 and Tween 20 with a concentration of 2% were added as blocking agents, and the reaction was stirred for 60 minutes. Then it was centrifuged and washed again to obtain antibody-coated latex particles.

[0034] Reagent preparation: Weigh the raw materials of Tris-Hcl buffer in a clean 500 mL glass container, add an appropriate amount of deionized water and stir to dissolve. Use a pH meter to adjust the pH value to 7.0 and then make up the volume to 500 mL. Sucrose, gelatin, NP30 and Proclin 300 were added in sequence and stirred until completely dissolved. Two kinds of antibody-coated latex particles were mixed in a ratio of 2:1 and added to the above solution, and then interleukin-2 was added and continuously stirred evenly.

[0035] Quality inspection: Observe the appearance of reagent R2, which should be uniform and without stratification; use a pH meter to measure the pH value to 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 detection methods (such as cell proliferation experiments) to determine the activity of the immune enhancer interleukin-2; store reagent R2 under different temperature and humidity conditions and regularly detect its various indicators to evaluate the stability.

[0036] Sub-packaging: In a sterile and clean laminar flow hood, use an automatic sub-packaging device to sub-package reagent R2 into brown reagent bottles according to the specification of 15 mL per bottle. Approximately 33 bottles can be sub-packaged. After sealing the bottle mouth, label it with information such as reagent name, specification, production date, and expiration date.

[0037] Kit Assembly: Combine the qualified and intactly packaged Reagent R1 (50 mL per bottle) and Reagent R2 (15 mL per bottle) in a ratio of 3:1 by volume of Reagent R1 to Reagent R2, and then put them together with the supporting standard product (including type IV collagen standard solutions with different concentration gradients) and the operation manual into the kit to complete the preparation of the type IV collagen detection kit.

[0038] Sample Collection: Collect the serum samples of patients, ensuring that the sample collection process complies with clinical specifications to avoid sample contamination or hemolysis.

[0039] Detection Operation: Conduct the detection according to the operation manual of the kit. Generally, first take out Reagent R1 and Reagent R2 from the refrigerator and equilibrate them to room temperature. In a clean reaction container, add appropriate amounts of Reagent R1 and Reagent R2 in a ratio of 3:1 by volume of Reagent R1 to Reagent R2, and mix them evenly. Then add a certain amount of the patient's serum sample, mix well, and incubate at an appropriate temperature (such as 37°C) for a certain period of time (determined according to the kit instruction manual). During the incubation process, the type IV collagen hydrolase in Reagent R1 will hydrolyze the type IV collagen in the sample, and the hydrolysis product will have an immune reaction with the paired mouse monoclonal antibody coated on the latex particles in Reagent R2, causing the latex particles to agglutinate.

[0040] Result Judgment: Make a diagnosis based on the calculated percentage content of type IV collagen in combination with the patient's clinical symptoms and other examination results. If the test result shows that the percentage content of type IV collagen is higher than the normal reference range, combined with the patient's clinical symptoms and other examination results, doctors can further judge that the patient may have liver fibrosis liver disease; if the test result is within the normal range, the risk of liver disease caused by abnormal elevation of type IV collagen can be initially excluded, but a comprehensive diagnosis still needs to be combined with other examinations.

[0041] Example 2: Accuracy Analysis of the Method of the Present Invention.

[0042] Test Instrument: Hitachi 7180 Automatic Biochemical Analyzer. This instrument has high sensitivity, precision, and good stability, can ensure the reliability of test results, is widely used in the field of clinical testing, can accurately measure various indicators in samples, and provides strong technical support for this accuracy analysis.

[0043] Test Samples: Select 50 samples from physical examination subjects. These physical examination subjects come from different ages, genders, and life backgrounds, are representative, and can more comprehensively evaluate the accuracy of the detection method of the present invention in different populations.

[0044] Comparison method kit: A commercially available imported type IV collagen kit (electrochemiluminescence method). The electrochemiluminescence method is one of the commonly used advanced methods in current clinical detection, with the advantages of high sensitivity and wide detection range. Selecting this kit for comparison can more strictly verify the accuracy of the detection method of the present invention.

[0045] Detection process: Use the detection method of Example 1 and the comparison method to measure these 50 samples respectively. During the detection process, strictly follow the respective operation specifications of the two detection methods to ensure that the detection environment and personnel operation conditions are consistent to reduce errors. For example, during sample processing, serum separation is carried out according to the standard process to avoid sample contamination or hemolysis; in the use of reagents, the reagents are accurately measured to ensure the accuracy of reagent concentration and dosage; in terms of instrument operation, the instrument is calibrated and debugged in advance to ensure that the instrument is in the best working condition.

[0046] Detection results: The detection data of the two methods are recorded in detail, as shown in Comparison Table 1.

[0047] Data analysis: Through professional data statistical software, linear regression analysis is performed on the detection results to obtain the equation and R² value. The closer the R² value is to 1, the better the correlation between the two detection methods, and the higher the accuracy of the detection method of the present invention.

[0048] Comparison Table 1 The results show that the R² value calculated based on the detection results is 0.9925, which is greater than 0.95, indicating that the detection results of the method of the present invention and the detection results of the comparison kit have a good correlation and no obvious difference, further proving that the method of the present invention has a high accuracy (conformance).

[0049] In summary, in the clinical diagnosis of liver diseases, when detecting the content of type IV collagen in patients suspected of liver fibrosis, the detection kit of the present invention shows good performance. As can be seen from Comparative Example 2, compared with the commercially available imported electrochemiluminescence method kit, the R² value of the detection results of the kit of the present invention reaches 0.9225, which is greater than 0.55, indicating that there is a good correlation and no obvious difference between the two, and the accuracy is relatively high. At the same time, the kit has simple operation, good reagent stability, and can also reduce interference. This means that in clinical practical applications, the type IV collagen detection kit of the present invention can effectively assist doctors in diagnosing liver fibrosis liver diseases, and has high practical value and popularization significance.

[0050] It is an index used in statistics to evaluate the goodness of fit of a regression model. The calculation process of Calculate the total sum of squared deviations SST, and the formula is: , where is the th observation value, is the mean value of the observation values, is the number of observation values. In Example 2, for the detection results of the present invention or the detection results of the comparative kit, they can all be calculated according to this formula. For example, for the detection results of the present invention a set of data: , , ···, first calculate the mean value of this set of data, and then calculate the sum of squares of the differences between each data and the mean value, that is, obtain . Calculate the sum of squared residuals SSR, and the formula is: is the value predicted according to the regression equation. In Example 2, the regression equation is: . Substitute each (detection results of the comparative kit) into the equation to obtain the predicted value , and then calculate the sum of squares of the differences between the observation value and the predicted value , which is . Calculate , and the formula is: . Through the and calculated previously and substituting them into this formula, the value can be obtained. In Example 2 , indicating that 99.02% of the differences between the detection results of the present invention and the detection results of the comparative kit can be explained by the regression model, that is, the linear relationship between the two is close, and the accuracy of the detection method of the present invention is high.

[0051] Example 3: Stability analysis of the method of the present invention.

[0052] Test instrument: Hitachi 7180 automatic biochemical analyzer. This instrument has good stability and repeatability, and can ensure that the detection conditions are relatively consistent when detecting at different time points, reduce the influence of instrument error on the results, and provide a reliable detection platform for stability analysis.

[0053] Test sample: Select a sample of type IV collagen serum with a stable concentration of 180 ng / mL. Selecting a sample with a single stable concentration can more intuitively observe the detection stability of the detection method of the present invention at different times and avoid result fluctuations caused by sample concentration differences.

[0054] Detection process: The detection method of Example 1 was adopted. Under the same experimental environmental conditions (including a temperature of 25°C and a relative humidity of 50%), the serum sample was detected on the 1st day, 7th day, 14th day, 21st day, and 28th day respectively, and each detection was repeated 5 times. During each detection, the operation steps of Example 1 were strictly followed to ensure that the usage amount of the reagent, reaction temperature, and reaction time conditions were exactly the same. For example, during each detection, 15 μL of the sample, 150 μL of Reagent 1, and 50 μL of Reagent 2 were accurately measured and reacted at 37°C for 5 minutes respectively.

[0055] Detection results: The results of each detection were recorded in detail, as shown in Comparative Table 2.

[0056] Comparative Table 2 Result analysis: It can be seen from the detection results that during the detection process from the 1st day to the 28th day, the average value of the type IV collagen concentration in the serum sample fluctuated slightly and was always close to the initial set concentration of 180 ng / mL. The coefficient of variation (CV) was less than 5% (the standard requirement is generally less than 10%), indicating that the detection method described in the present invention has good stability within 28 days and can maintain the reliability of the detection results for a long time, providing stable technical support for clinical detection.

[0057] To sum up, in this study on the stability of the detection method of the present invention, with the help of Hitachi 7180 automatic biochemical analyzer, under unified environmental conditions, the type IV collagen serum sample with a concentration of 180 ng / mL was detected multiple times at different time points. The results showed that within 28 days, the average value of the detection results fluctuated slightly, always close to the set concentration, and the coefficient of variation was less than 5%, far lower than the standard requirement of 10%. This fully indicates that the detection method of the present invention has good stability, can ensure the reliability of the detection results for a long time, and provides solid technical support for clinical detection. However, this study only involved samples of a single concentration, and different concentration samples can be added for testing in the future to further verify its stability.

[0058] The coefficient of variation CV is a statistic used to measure the degree of dispersion of a set of data. In the study on the stability 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 detection, and then divide by the number of data to calculate the standard deviation Calculate the square of the difference between each data and the average value, add up these squared values and divide by the number of data 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 average value and then multiply by 100% to convert the result into a percentage form. The formula is: , the coefficient of variation at different detection times is calculated to evaluate the dispersion degree of the detection results and the stability of the detection method.

[0059] Example 4: Comparative study on the influence of astragalus polysaccharide concentration on the detection method.

[0060] Test instrument: Hitachi 7180 fully automatic biochemical analyzer, which has high-precision and good repeatability detection performance, can ensure the reliability of each detection data, and provides a stable detection platform for studying the performance of the detection method under different astragalus polysaccharide concentrations.

[0061] Detection sample: Select a type IV collagen serum sample with a stable concentration, and the concentration is determined to be 150 ng / mL. Fixing the sample concentration can effectively avoid the interference of the sample's own concentration difference on the detection results, making the experimental variables only focus on the change of astragalus polysaccharide concentration.

[0062] Setting of comparison conditions: Keep other components in reagent R1 and reagent R2 unchanged, only change the concentration of astragalus polysaccharide in reagent R1, and set them to 0% (as a blank control), 0.5%, 1.0%, 1.5%, 2.0% respectively, and perform 5 detections on the samples of each concentration.

[0063] Detection process: Strictly follow the operation instructions of the kit to perform the detection steps. After taking out reagent R1 and reagent R2 from the refrigerator, let them equilibrate to room temperature. In a clean reaction container, add the corresponding reagents according to the volume ratio of reagent R1 to reagent R2 of 3:1, and mix well. Then add a certain amount of serum sample, mix well, and incubate at 37 °C (the incubation time is determined according to the kit instructions). After incubation, use the supporting immunoturbidimetric analyzer to detect the absorbance of the reaction system, and calculate the content of type IV collagen in the sample according to the standard curve.

[0064] Detection results: Record the data of each detection in detail, such as in comparison table 3.

[0065] Comparison table 3 It can be seen from the data in comparison table 3 that as the concentration of astragalus polysaccharide increases, the average value of the detection results gradually approaches the actual sample concentration of 150 ng / mL, and the coefficient of variation gradually decreases. When the concentration of astragalus polysaccharide reaches 1.0% and above, the stability and accuracy of the detection results are relatively good, indicating that appropriately adding astragalus polysaccharide can improve the performance of the detection method.

[0066] In summary, through the comparative study of the detection results of type IV collagen at different concentrations of astragalus polysaccharide, it can be seen that as the concentration of astragalus polysaccharide increases, the average value of the detection results gradually approaches the actual concentration of the sample, the coefficient of variation decreases, and the stability and accuracy of the detection are improved. In the concentration range of 0.3% - 2.0%, the effect is relatively ideal when the concentration is 1.0% or above. This indicates that adding an appropriate amount of astragalus polysaccharide to reagent R1 of the detection kit helps to optimize the performance of the detection method. However, the current study only involves 5 detections and limited concentration gradients. Subsequently, the number of detections and the concentration range can be further increased to deeply explore the influence of astragalus polysaccharide on the detection results and provide a more sufficient basis for the optimization of the kit.

[0067] Example 5: Comparative study on the influence of the concentration of proteinase K inhibitor on the detection results.

[0068] Test instrument: Hitachi 7180 automatic biochemical analyzer is used. This instrument has high sensitivity and precision, and can stably and accurately detect the content of type IV collagen in the sample, providing a reliable detection basis for this comparative study.

[0069] Test sample: A serum sample of type IV collagen with a stable concentration of 200 ng / mL is selected. The stable sample concentration can reduce the interference caused by the differences in the samples themselves, focusing the experiment on the changes in the concentration of the proteinase K inhibitor.

[0070] Setting of comparative conditions: Keep other components in reagent R1 and reagent R2 unchanged, and only adjust the concentration of the proteinase K inhibitor in reagent R1, which are set to 0% (blank control), 0.2%, 0.4%, 0.6%, and 0.8% respectively. Each concentration of the sample is detected 5 times.

[0071] Detection process: Follow the operation manual of the kit. Take reagent R1 and reagent R2 out of the refrigerator and equilibrate to room temperature. In a clean reaction vessel, add the reagents according to the volume ratio of reagent R1 to reagent R2 of 3:1 and mix evenly. Then add a quantitative serum sample, mix well, and incubate at 37°C (the incubation time depends on the kit manual). After incubation, use the supporting immunoturbidimetric analyzer to detect the absorbance of the reaction system, and calculate the content of type IV collagen in the sample according to the standard curve.

[0072] Detection results: See comparative table 4 for specific results.

[0073] Comparative table 4 As can be seen from this comparison table, as the concentration of the proteinase K inhibitor increases, the average value of the detection results gradually approaches the actual concentration of the sample, 200 ng / mL, and the coefficient of variation gradually decreases, indicating that appropriately increasing the concentration of the proteinase K inhibitor helps to improve the accuracy and stability of the detection method.

[0074] In summary, this study on the effect of the proteinase K inhibitor concentration on the detection results of type IV collagen is of great significance. By changing the concentration of the proteinase K inhibitor in reagent R1 and conducting 5 comparative detections, it was found that as the concentration increased from 0.10% to 0.80%, the average value of the detection results continuously approached the actual concentration of the sample, 200 ng / mL, and the coefficient of variation continued to decrease. This indicates that increasing the concentration of the proteinase K inhibitor can effectively improve the accuracy and stability of the detection method. However, the concentration gradient set in this study is limited. In the future, the concentration range can be expanded and the number of detections can be increased to further explore its optimal concentration and the overall impact on the detection performance, providing a more solid theoretical and practical basis for optimizing the type IV collagen detection kit.

[0075] Example 6: Comparative study on the effect of the average particle size of latex particles on the detection results.

[0076] Test instrument: Hitachi 7180 automatic biochemical analyzer, which has high detection accuracy and good repeatability, can ensure the reliability of each detection result, and provides a stable detection platform for studying the effect of the average particle size of latex particles on the detection results.

[0077] Test sample: Select a type IV collagen serum sample with a determined and stable concentration, set at 180 ng / mL. The stable sample concentration can avoid interference with the detection results caused by the fluctuation of the sample's own concentration, making the experimental variable focus only on the change in the average particle size of the latex particles.

[0078] Setting of comparison conditions: Keep other components in reagent R1 and reagent R2 unchanged, and only change the average particle size of the latex particles in reagent R2. The average particle sizes are set at 90 nm, 110 nm, 130 nm, 150 nm, and 170 nm respectively, and the samples under each particle size condition are detected 5 times.

[0079] Detection process: Strictly follow the operation manual of the kit. Take reagent R1 and reagent R2 out of the refrigerator and equilibrate to room temperature. In a clean reaction vessel, add the reagents according to the volume ratio of reagent R1 to reagent R2 of 3:1, and mix well. Then add a quantitative serum sample, mix thoroughly, and incubate at 37 °C (the incubation time is determined according to the kit manual). After incubation, use the supporting immunoturbidimetric analyzer to detect the absorbance of the reaction system, and calculate the content of type IV collagen in the sample according to the standard curve.

[0080] Test results: See comparison table 5 for specific results.

[0081] Comparison Table 5 From the data in the comparison table, it can be seen that within the average particle size range of 90 nm - 170 nm of latex particles, as the particle size increases, the average value of the test results is closer to the actual concentration of 180 ng / mL of the sample when the particle size is between 130 nm - 150 nm, and the coefficient of variation is relatively small. This indicates that the average particle size of latex particles within a certain range will affect the accuracy and stability of the test results. 130 nm - 150 nm may be a more suitable particle size range, but further research and verification are still needed.

[0082] In summary, the research on the influence of the average particle size of latex particles on the detection results of type IV collagen in this study is of great significance. Through comparative detection within the particle size range of 90 nm - 170 nm, it is found that as the particle size increases, the degree to which the average value of the test results approaches the actual concentration of 180 ng / mL of the sample is different. Among them, the average value of the test results at a particle size of 130 nm - 150 nm is closer to the actual concentration, and the coefficient of variation is also 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 the number of detections in this study are limited. In the future, more particle size gradients can be added and repeated detections can be carried out to deeply explore the influence of the average particle size of latex particles on the detection performance, providing a more reliable basis for optimizing the test kit.

[0083] Example Seven: Specific detection.

[0084] Test instrument Select the Hitachi 7180 fully automatic biochemical analyzer to ensure stable instrument performance and calibration, and all parameters meet the detection requirements.

[0085] Test sample Prepare a type IV collagen serum sample with a concentration of 100 ng / mL as the basic test sample. At the same time, prepare rheumatoid factor solutions with different concentration gradients (0 IU / mL, 33.75 IU / mL, 67.5 IU / mL, 135 IU / mL, 270 IU / mL, 500 IU / mL, 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, 50 μg / mL).

[0086] Test procedure Mix the type IV collagen serum samples with rheumatoid factors at different concentrations in a ratio of 9:1 to obtain a series of samples with different concentrations of rheumatoid factor interference. For example, take 90 μL of the type IV collagen serum sample and mix it evenly with 10 μL of the rheumatoid factor solution at the corresponding concentration.

[0087] Similarly, mix the type IV collagen serum samples with heterophilic antibodies at different concentrations in a ratio of 9:1 to prepare samples with different concentrations of heterophilic antibody interference.

[0088] Use 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, adjust the pH value to 7.5 with ammonia water, PEG6000 2%, MAK33 0.5%, 2-mercaptoethanol 0.02%; Reagent 2: latex particles coated with 2 pairs of paired murine monoclonal antibodies (final concentration of latex particles 0.1%), Tris-Hcl buffer 50 mmol / L, pH 7.0, sucrose 3%, gelatin 0.5%, NP30 0.1%, Proclin300 0.3%) to detect each of the above-prepared samples containing interfering substances 3 times. Add 15 μL of the mixed sample, 150 μL of Reagent 1 by the automatic biochemical analyzer, react at 37°C for 5 minutes, then add 50 μL of Reagent 2 and react at 37°C for 5 minutes, measure the absorbance value at 600 nm, and calculate the percentage content of type IV collagen in the sample according to the standard curve.

[0089] Taking the detection result of the type IV collagen serum sample without interfering substances (i.e., 0 concentration of interfering substances) as a control, calculate the deviation between the detection results of other samples containing interfering substances and the control result. The deviation calculation formula is: Deviation = [(Average detection value of the sample containing interfering substances - Average detection value of the sample with 0 concentration of interfering substances) ÷ Average detection value of the sample with 0 concentration of interfering substances] × 100%.

[0090] Table 6 The results show that, as shown in Table 6, when the concentration of rheumatoid factor is below 500 IU / mL, the deviation is less than 10%, within the required range, and it is determined that there is no interference; when the heterophilic antibody is below 40 μg / mL, the deviation is less than 10%, within the required range, and it is determined that there is no interference. This indicates that the detection method described in the present invention has good anti-interference ability and good specificity for rheumatoid factor and heterophilic antibody.

[0091] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above in the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content without departing from the technical solution of the present invention. However, as long as it does not depart from the technical solution content of the present invention, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall 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, and the volume ratio of reagent R1 to reagent R2 is 3:1; The components of reagent R1 are as follows: ammonium chloride is 0.2 mol / L - 1.0 mol / L, sodium azide is 0.01% - 0.05%, Brij-35 is 0.1% - 1%, sodium chloride is 150 mmol / L, ammonia water is 150 mmol / L, PEG6000 is 1% - 5%, MAK33 is 0.2% - 0.8%, 2-mercaptoethanol is 0.01% - 0.05%, natural plant extract astragalus polysaccharide is 0.3% - 2.0%, protease K inhibitor is 0.1% - 0.8%, and type IV collagenase with an enzyme activity unit of 30 U - 70 U per milliliter of reagent 1; The components of reagent R2 are as follows: latex particles coated with 2 pairs of paired mouse monoclonal antibodies, Tris-Hcl buffer is 40 mmol / L - 60 mmol / L, sucrose is 1% - 5%, gelatin is 0.2% - 0.8%, NP30 is 0.05% - 0.2%, Proclin300 is 0.3%, and immune enhancer interleukin-2 is 0.08%.

2. The type IV collagen detection kit according to claim 1, wherein The preparation method of astragalus polysaccharide in reagent R1 is as follows: after cleaning, drying, and pulverizing astragalus raw materials, add deionized water at a solid-liquid ratio of 1:10 - 1:15, extract at 90 - 100 °C under reflux for 2 - 3 hours, filter and collect the extract, add absolute ethanol to the extract to make the final concentration of ethanol reach 70% - 80%, stir and then let it stand to precipitate polysaccharides, and obtain astragalus polysaccharide through centrifugation, washing, and drying.

3. The type IV collagen detection kit according to claim 1, characterized in that, The preparation method of type IV collagenase in reagent R1 is as follows: obtain the gene sequence from a biological sample containing the type IV collagenase gene sequence, ligate it to an expression vector and then transform it into Escherichia coli BL21, shake and culture at 37 °C until the logarithmic growth phase, add IPTG with a final concentration of 0.1 - 1 mmol / L, induce expression at 20 - 30 °C for 12 - 16 hours, after expression, centrifuge to collect the thalli, ultrasonically disrupt, centrifuge to extract the supernatant, purify it by affinity chromatography and ion exchange chromatography methods, dilute it to an enzyme activity unit of 30 U - 70 U per milliliter of reagent 1, and aliquot and store at -20 °C.

4. The type IV collagen detection kit according to claim 1, characterized in that The average particle size of the latex particles coated with 2 pairs of paired mouse monoclonal antibodies in reagent R2 is 90 nm - 170 nm, 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. The type IV collagen detection kit according to claim 1, wherein, The chemical cross-linking agents for the latex particles coated with mouse monoclonal antibodies in reagent R2 are ethylenediamine EDA and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC.

6. The type IV collagen detection kit according to claim 1, characterized in that, The blocking agents for the latex particles coated with mouse monoclonal antibodies in reagent R2 are glycine buffer with a concentration of 1% - 5% and a pH value of 6.5 - 8.5 and Tween 20 with a concentration of 1% - 4%.

7. A method for preparing a type IV collagen detection kit, characterized in that, This method is used to prepare a type IV collagen detection kit according to any one of claims 1 - 6, and the specific steps of this preparation method are as follows: S100, Preparation of Reagent 1: S101, Prepare raw materials: Prepare ammonium chloride, sodium chloride, sodium azide, Brij-35, PEG6000, MAK33, 2-mercaptoethanol, astragalus polysaccharide, protease K inhibitor, and type IV collagenase; S102, Preliminary dissolution: Add deionized water to a clean container, turn on the stirring device, sequentially add ammonium chloride and sodium chloride, heat to 50 - 70 °C, and stir at a speed of 200 - 400 r / min until completely dissolved; S103, Add components: Add Brij-35, PEG6000, MAK33, and 2-mercaptoethanol, and stir evenly; S104, Adjust pH value: Add ammonia water to adjust the pH value of the solution to 7.0 - 9.0, and continue stirring after adjustment; S105, Add bioactive components and bioenzymes: Slowly add astragalus polysaccharide, protease K inhibitor, and type IV collagenase while stirring, and continue stirring after addition; S106, Quality inspection: Detect the appearance, pH value, bioenzyme activity, and stability of Reagent 1; S107, Sub-packaging: Under sterile and clean environment, sub-package Reagent 1 according to specific specifications, seal the package and label it with relevant information; S200, Preparation of Reagent 2: S201, Prepare raw materials: Select latex particles, prepare 2 pairs of mouse monoclonal antibodies with strong specificity and high affinity for type IV collagen through animal immunization, cell fusion, screening, and purification, and prepare raw materials for Tris-Hcl buffer, sucrose, gelatin, NP30, Proclin300, and interleukin-2; S202, Surface modification of latex particles: Suspend the latex particles in a biotinylation reagent solution at 1 - 5 mg / mL, stir and react at 30 - 37 °C and a speed of 150 - 250 r / min for 60 - 90 minutes. After the reaction, centrifuge, wash, and adjust the pH. Then add ethylenediamine EDA and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, stir and react at 20 - 25 °C and a speed of 100 - 150 r / min for 30 - 60 minutes, centrifuge and wash again to obtain latex particles with biotin-modified surfaces; S203, Preparation of antibody-avidin complex: Couple the 2 monoclonal antibodies with avidin respectively. The reaction system uses a buffer containing 0.1 - 0.2 mol / L MES, adjust the pH to 5.0 - 6.0, react at 25 - 30 °C and a speed of 100 - 150 r / min for 3 - 4 hours, and separate and purify by gel filtration chromatography after coupling; S204, Antibody directed immobilization: Mix the antibody-avidin complex and the treated latex particles at a volume ratio of 1:2 - 1:3, incubate with a Tris-HCl buffer containing 0.05 - 0.1 mol / L, centrifuge after incubation, add a blocking agent and stir to react, centrifuge and wash again to obtain antibody-coated latex particles; S205, Reagent Preparation: Weigh the Tris-Hcl buffer raw materials in a clean container, add deionized water and stir to dissolve. Adjust the pH value to 6.0 - 8.0 and then make up the volume. Sequentially add sucrose, gelatin, NP30, and Proclin300, and stir until completely dissolved. Mix two kinds of antibody-coated latex particles in a ratio of 1:1 - 3:1 and add them to the above solution, then add interleukin-2, and continuously stir evenly; S206, Quality Inspection: Check the appearance of Reagent 2, measure the pH value, detect the concentration and activity of antibody-coated latex particles by immunoassay methods, measure the activity of immunopotentiator by biological detection methods, and detect the stability under different storage conditions; S207, Sub-packaging: Under sterile and clean environment, use sub-packaging equipment to sub-package Reagent 2 according to specific specifications, seal and label it, indicating the information of reagent name, specification, production date, and expiration date; S300, Kit Assembly: Put the qualified and completely packaged Reagent 1 and Reagent 2 into the kit according to the volume ratio of Reagent 1 to Reagent 2 of 3:1, together with the supporting standard product and operation manual.

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

10.

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

1.

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

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