A reagent set, a kit and a method for performing immunoassay using the same

Abstract

The present invention discloses a reagent set, a kit and a method for performing immunoassay using the same. Also disclosed is a method for preparing a latex enhanced immunoturbidimetry reagent, which comprises the following steps: after activating large-diameter and small-diameter latex microspheres respectively, adding an antibody against the object to be detected to the large-diameter latex microspheres, and adding the protein complex of the object to be detected to the small-diameter latex microspheres to perform a coupling reaction; after the reaction solution is respectively sealed, centrifuged, redissolved and resuspended, an R1 reagent containing antibody-coupled large-diameter latex microspheres and an R2 reagent containing small-diameter latex microspheres coupled to the object to be detected are obtained. The kit of the present invention comprises: the R1 reagent, the R2 reagent and a calibrator. The present invention gives full play to the advantages of latex microspheres of different particle sizes, and extremely overcomes the problems of low sensitivity and detection linear range that cannot be taken into account in the existing methods, and has the advantages of accurate measurement, simple and fast operation, can be used for outpatient and emergency detection, and the instrument has a wide range of applicability.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection, and in particular, relates to a reagent set, a test kit and a method for performing immunoassay using the same. Background Art

[0002] Latex enhanced turbidimetric immunoassay (LETIA) couples antibodies or antigens to the surface of nano-sized latex microspheres by covalent coupling or physical adsorption to form latex microsphere-antibody or latex microsphere-antigen complexes. The complex reacts with the antigen or antibody to be tested in the sample with high specificity, so that the antibody or antigen on the surface of the latex microsphere reacts with the test substance contained in the specimen to form microsphere aggregates, which enhances the signal during the reaction process, and can improve the sensitivity of the detection by several orders of magnitude, achieving accurate quantitative results.

[0003] Latex enhanced immunoturbidimetry can be performed in two modes: competitive and non-competitive. The competitive method can be used for both large and small molecule analytes, while the non-competitive method is generally only used to detect protein analytes containing multiple antigenic epitopes. Due to the limitation of molecular weight, it is generally difficult to establish a non-competitive immunoturbidimetric detection method for small molecule analytes, and only competitive immunoturbidimetry can be used for detection. The competitive latex enhanced turbidimetric method refers to the competition between the analyte contained in the sample and the fixed amount of analyte in the detection system for binding to a limited amount of anti-analyte antibodies, causing the absorbance value (A) of the immune response system at a specific wavelength to change. The concentration of the small molecule analyte in the sample is calculated by measuring the change, thereby achieving clinical purposes such as disease diagnosis, screening, and monitoring.

[0004] In latex immunoturbidimetric reagents, the sensitivity is directly reflected by the rate of change of the absorbance signal value and its test repeatability, which can be indirectly expressed by the minimum detection limit (LOD), that is, the lower the LOD, the higher the sensitivity. The linear range refers to the range that can be accurately measured according to the instructions. It is an important indicator for measuring the quality of the kit and one of the key indicators to ensure the use and identification of the kit.

[0005] Polystyrene (PS) nano latex microspheres with carboxyl functional groups on the surface are commonly used carriers for LETIA reagents. Currently, the latex-enhanced immunoturbidimetry reagents available on the market mainly use latex microspheres with a single particle size. Selecting latex microspheres with a small particle size, the microspheres have a large protein load and can be coupled with more immune binding reagents. Often, a wider range of analyte concentrations can be obtained, and the precision and linearity are relatively good. The linear range of detection can cover the high value area. However, the A value increment caused by the agglutination of small-particle microspheres is relatively small, and the detection sensitivity is correspondingly low. For analytes with extremely low content in the sample, especially for small molecule analytes with extremely low content in the sample and requiring competitive immunoassay detection, it is often difficult to achieve high-sensitivity detection using small-particle microspheres, the measured values ​​are inaccurate, and the coefficient of variation (CV) value is too large. Choosing microspheres with large particle sizes requires less antibody, which can effectively increase the absorbance signal value and exponentially increase the reagent sensitivity, which is beneficial for low-value detection to meet sensitivity requirements. However, it will cause the reagent blank to be too high, and the precision and linearity will be relatively poor. The linear range of microsphere detection is not enough, so high-value detection is inaccurate. In addition, large-particle latex usually reduces the reagent linear range and antigen excess capacity, and the production process of large-particle latex is more difficult to control. Summary of the invention

[0006] In order to overcome the problem of low sensitivity and inability to balance the detection linear range in the latex enhanced immunoassay in the prior art, the present invention provides a method for improving the linear range and sensitivity of the latex enhanced immunoturbidimetric method, which selects two carboxylated latex particles with different average particle sizes, and uses a chemical crosslinking method to simultaneously couple the antigen complex and the antibody with two fluorescent microspheres of different particle sizes, giving full play to the advantages of latex microspheres of different particle sizes, and effectively overcoming the problem of low sensitivity and inability to balance the detection linear range in the latex enhanced immunoassay. The present invention also provides a reagent and a kit with high sensitivity and a wide linear range, which include: reagent 1 (R1), reagent 2 (R2) and a calibrator, and has the advantages of accurate measurement, simple and fast operation, can be used for outpatient and emergency detection, and the instrument has a wide range of applicability.

[0007] In order to solve the above technical problems, one of the technical solutions used in the present invention is: a method for preparing a latex enhanced immunoturbidimetry reagent, the method comprising the following steps:

[0008] (1) mixing the large-size latex microspheres and the small-size latex microspheres with activation buffer respectively and then adding an activator for activation;

[0009] (2) adding an antibody against the analyte to the activated large-particle latex microspheres, preparing the analyte protein complex with the analyte sample and protein, and then adding the complex to the activated small-particle latex microspheres to perform coupling reactions respectively;

[0010] (3) adding a blocking solution to the reaction solution after the coupling reaction for blocking, discarding the supernatant after blocking, and re-dissolving and resuspending the solids with a buffer solution to obtain the R1 reagent containing the large-size latex microspheres coupled to the antibody, and the R2 reagent containing the small-size latex microspheres coupled to the analyte;

[0011] The latex microspheres are hydrophobic microspheres with negatively charged carboxyl groups on the surface; the large-diameter latex microspheres have a solid content of 5%-10% and a particle size of 275-310nm; the small-diameter latex microspheres have a solid content of 5%-10% and a particle size of 70-90nm.

[0012] In some embodiments, in step (1), the activation buffer is MES buffer; and / or, the activating agent is NHS and EDC; and / or, the total mass ratio of latex microspheres: NHS: EDC is 7:10:1; and / or, the activation temperature is 37° C., and the reaction time is 30-60 min; and / or, in step (3), after blocking, the supernatant is discarded by centrifugation;

[0013] In some preferred embodiments, the latex microspheres are carboxyl polystyrene latex microspheres; and / or, the large-particle latex microspheres have a solid content of 10% and a particle size of 275 nm; and / or, the small-particle latex microspheres have a solid content of 5% and a particle size of 70 nm; and / or, in step (1), the concentration of the MES buffer is 20 mmol / L and the pH value is 6.1; and / or, in step (3), the centrifugal speed is 16,000 rpm and the time is 40 min.

[0014] In some embodiments, the antibody described in step (2) is any one of a monoclonal antibody or a polyclonal antibody; the analyte protein complex is a complex of a drug and keyhole limpet hemocyanin; and / or the temperature of the coupling reaction is 25° C., and the reaction time is 10-90 min; preferably, the analyte is tacrolimus, rapamycin or cyclosporine.

[0015] In some embodiments, the concentration of the antibody after dilution with buffer is 0.5-5 mg / mL; preferably 1 mg / mL; and / or the concentration of the analyte protein complex after dilution with buffer is 20-100 μg / mL; preferably 50 μg / mL.

[0016] In some embodiments, in step (3), the blocking solution is bovine serum albumin, glycine or ethanolamine; and / or the blocking temperature is 37° C., the blocking time is 30-60 min, preferably under shaking conditions, such as 120 rpm.

[0017] In some embodiments, in step (3), the components of the buffer used to re-dissolve the antibody-coupled large-particle latex microspheres (also referred to as R1 buffer in the present invention) include:

[0018] -50-100mmol / L HEPES or Tris or PBS; preferably 100mmol / L HEPES;

[0019] -1% NaCl;

[0020] - Preservative: 0.1% NaN3 or Procline300 or Thimerosal; preferably 0.1% Procline300;

[0021] - Protective agent: 1-5% trehalose and 0.1-0.5% glycine; preferably 2% trehalose and 0.2% glycine;

[0022] The pH value is 7.0-8.0; preferably the pH value is 7.4; and / or,

[0023] The composition of the buffer used for re-dissolving the small-size latex microspheres coupled with the protein complex to be tested (also referred to as R2 buffer in the present invention) is:

[0024] -50-100mmol / L HEPES or Tris or PBS; preferably 50mmol / L HEPES;

[0025] -3% PEG, molecular weight 6,000-20,000; preferably 8,000;

[0026] - Preservative: 0.1% NaN3 or Procline300 or Thimerosal; preferably 0.1% Procline300;

[0027] - Protective agent: 1% glycerol and 2% sucrose;

[0028] The pH value is 6.5-8.5; the preferred pH value is 8.0.

[0029] The second technical solution used in the present invention is: a reagent set for latex enhanced immunoturbidimetry, which includes R1 reagent containing large-particle latex microspheres coupled with antibodies prepared by the method described in one of the technical solutions, and R2 reagent components for preparing small-particle latex microspheres coupled with protein complexes containing the object to be tested.

[0030] The third technical solution used in the present invention is: a kit for latex enhanced immunoturbidimetry, the kit comprising the reagent set as described in the second technical solution; preferably also comprising a calibrator; the calibrator is a standard solution or a lyophilized product containing a known concentration of a test substance, which is used to establish a calibration curve.

[0031] The fourth technical solution used in the present invention is: a method for detecting a sample using latex enhanced immunoturbidimetry, the method comprising the following steps: (1) preparing R1 reagent and / or R2 reagent according to the method described in one of the technical solutions; (2) mixing the R1 reagent prepared in (1), or the reagent set described in the second technical solution, or the R1 reagent in the kit described in the third technical solution with the R2 reagent prepared in (1), for example, in a ratio of 1:1, measuring the absorbance at a wavelength of 400-800nm, for example, 546nm, and calculating the concentration of the sample to be tested according to the calibration curve.

[0032] The fifth technical solution used in the present invention is: the use of the reagent set as described in the second technical solution or the kit as described in the third technical solution in the preparation of latex enhanced immune turbidimetric preparation.

[0033] The present invention has the following technical effects or advantages :

[0034] The technical solution of the present invention is to cross-link the antibody against the analyte on the surface of large-diameter polystyrene latex microspheres, react with the analyte protein complex coated on the small-diameter polystyrene latex microspheres, drive the polystyrene latex microspheres to aggregate and produce a certain turbidity, and the analyte in the whole blood will competitively bind to the monoclonal antibody on the surface of the polystyrene microspheres. Therefore, the turbidity of the reaction system is negatively correlated with the analyte in the sample within a certain range, and the concentration of the small molecule analyte in the sample can be quantified using a fully automatic biochemical analyzer at a wavelength of 400-800nm.

[0035] The monoclonal antibody cross-linked with large-size latex microspheres is conducive to the detection of low values ​​to meet the sensitivity requirements, and the protein complex of the analyte coated with small-size latex microspheres allows the linear range of detection to cover the high-value area. Therefore, the present invention can simultaneously meet the requirements of high sensitivity and wide detection linear range.

[0036] The present invention selects two carboxylated latex particles with different average particle sizes, and utilizes a chemical cross-linking method to simultaneously couple the two fluorescent microspheres with the protein complex and the antibody to be detected, thereby giving full play to the advantages of latex microspheres with different particle sizes, and effectively overcoming the problems of low sensitivity and inability to balance the detection linear range in latex enhanced immunoassay. DETAILED DESCRIPTION

[0037] The technical solution provided by the embodiment of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0038] The analyte is coupled to the protein to obtain a protein-analyte complex, and then the protein-analyte is coupled to the surface of the latex microspheres through chemical coupling between the reactive functional groups contained in the protein and the surface functional groups of the latex microspheres, thereby completing the indirect coupling of the analyte to the latex microspheres. A single carrier molecule contains multiple small molecules; due to the multivalency of the carrier-small molecule complex-microspheres and the antibody-latex microspheres, under appropriate conditions, the carrier-small molecule complex-microspheres and the antibody-latex microspheres can easily induce agglutination, and the small molecule analyte in the specimen inhibits this agglutination reaction by occupying the antibody binding sites on the surface of the latex microspheres. According to the degree of inhibition of the agglutination reaction, the concentration of the small molecule analyte in the specimen can be quantified at a wavelength of 400-800nm ​​using a fully automatic biochemical analyzer.

[0039] A method for improving the linear range and sensitivity of latex enhanced immunoturbidimetry of the present invention will be described in detail below in conjunction with the embodiments, reference examples and experimental data, but the present invention is not limited to the scope of the embodiments. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0040] Example 1: Tacrolimus assay kit (latex enhanced immunosuppression method)

[0041] Preparation method of tacrolimus-protein complex: weigh tacrolimus, add DMF to dissolve, and then dissolve in KLH. Use the eluent to pass through the Sephadex G50 column, collect the first peak as the target product according to the peak signal of the nucleic acid protein quantitative detector, and put it into the dialysis bag for dialysis. After the dialysis is completed, collect the product. For specific steps, refer to (Feng-Bo Wu, Yun-YunYang, Xue-Bin Wang, et al., A sample processing method for immunoassay of whole blood tacrolimus. Analytical Biochemistry. 2019, 576, 13-19).

[0042] The preparation method of the calibrator is as follows:

[0043] Fresh anticoagulated bovine whole blood was ultrasonically broken and then filtered, 0.1% gentamicin and 0.02% streptomycin sulfate were added as preservatives, and pure tacrolimus was added as needed. Tacrolimus calibrators of different concentrations were obtained.

[0044] The preparation method of tacrolimus antibody-coupled latex microspheres is as follows:

[0045] 1. Take a 1 mL centrifuge tube, add 0.1 mL (i.e. 10 mg) of 10% latex microspheres (JSR, #P0322, particle size 250-370 nm) with an average particle size of 310 nm, and dilute with 0.9 mL activation buffer (20 mmol / L MES, pH 6.1) to obtain a microsphere concentration of 1%;

[0046] 2. Add activator (100 mg / mL NHS solution: 10 mg / mL EDC solution = 142 μL: 142 μL) and shake well;

[0047] 3. Add 1 mg of tacrolimus, shake well immediately, and then place in a constant temperature shaker (25°C, 120 rpm) for 90 min;

[0048] 4. After ultrasonication for 1 min, add 5 mL of 10% glycine, mix well, and continue to place in a constant temperature shaker (37°C, 120 rpm) for 30 min;

[0049] 5. After blocking, centrifuge;

[0050] 6. After centrifugation, discard the supernatant, disperse the precipitate with 10 mL storage buffer (100 mmol HEPES, 1% NaCl, 0.1% Procline 300, 2% trehalose and 0.2% glycine, pH 7.4), suspend and sonicate for 5 min, and store at 2-8°C;

[0051] The preparation method of tacrolimus protein complex coupled latex microspheres is as follows:

[0052] 1. Take a 1mL centrifuge tube, add 0.2mL (i.e. 10mg) of 5% latex microspheres (JSR, #0011, particle size 70-110nm) with an average particle size of 90nm, and dilute with 0.8mL activation buffer (20mmol / L MES, pH6.1) to make the microsphere concentration at 1%;

[0053] 2. Add activator (100 mg / mL NHS solution: 10 mg / mL EDC solution = 714 μL: 714 μL) and shake well;

[0054] 3. Add 100 μg of tacrolimus protein complex, shake well immediately, and then place in a constant temperature shaker (25°C, 120 rpm) for 10 min;

[0055] 4. After ultrasonication for 1 min, add 5 mL of 10% ethanolamine, mix well, ultrasonicate for 5 min, and continue to place in a constant temperature shaker (37°C, 120 rpm) for 60 min;

[0056] 5. After blocking, centrifuge;

[0057] 6 After centrifugation, discard the supernatant, disperse the precipitate with 12 mL storage buffer (50 mmol / L HEPES, 3% PEG8000, 1% glycerol, 2% sucrose, 0.1% Procline300, pH 8.0), suspend and sonicate for 5 min, and store at 2-8°C;

[0058] The tacrolimus assay is as follows:

[0059] Before measuring the sample, extract tacrolimus from the whole blood using an extractant: transfer 300 μL of whole blood sample or calibrator to a 1.5 mL EP tube containing 300 μL of extractant, shake thoroughly on a vortex for 10 seconds, place in a 42°C water bath for 10 minutes, and centrifuge. After centrifugation, pour the supernatant from the centrifuge tube into a clean centrifuge tube and perform on-line testing.

[0060] Before testing, set the equipment parameters according to the data in the table below:

[0061] Table 1: Equipment setting parameters before measuring samples

[0062]

[0063] Set the detection parameters according to the data in the table below:

[0064] Table 2: Set the test parameters before measuring the sample

[0065]

[0066] Example 2

[0067] On the basis of Example 1, tacrolimus monoclonal antibody was replaced with tacrolimus polyclonal antibody, and other components and processes were the same as those in Example 1.

[0068] Example 3

[0069] Based on Example 1, the average particle size of the antibody-coated latex microspheres (JSR, #0221, particle size 220-330 nm) is 275 nm, and the other components and processes are the same as in Example 1.

[0070] Example 4

[0071] On the basis of Example 1, the average particle size of the tacrolimus protein complex-coated latex microspheres (JSR, #0001, particle size 50-90 nm) was 70 nm, and the other components and processes were the same as in Example 1.

[0072] Example 5

[0073] On the basis of Example 1, the antibody was directly mixed in R1 buffer without activation and cross-linking, and the other components and processes were the same as those in Example 1.

[0074] Example 6

[0075] Based on Example 1, the antigen complex was directly mixed in R2 buffer without activation and cross-linking. Other components and processes were the same as in Example 1.

[0076] Example 7

[0077] Based on Example 1, the volume ratio of reagents R1:R2 is 1:2 (Example 7-1) or 2:1 (Example 7-2). Other components and processes are the same as in Example 1.

[0078] Various performance tests of the detection kits in various embodiments:

[0079] 1. Linear relationship detection method: Use high concentration samples close to the upper limit of the linear range and low concentration samples close to the lower limit of the linear range to mix into 6 dilution concentrations (Xi), measure each concentration three times and take the average value (Yi), use the dilution concentration (Xi) as the independent variable and the test result mean (Yi) as the dependent variable to find the linear regression equation, and calculate the correlation coefficient (r) of the linear regression. Linear relationship detection, specific test data are shown in Table 3.

[0080] Table 3: Linear relationship test data

[0081]

[0082]

[0083] It can be seen from the detection data in Table 3 above that the linear coefficients of the three groups of samples in which the antibody was coated with large-size microspheres, the antigen complex was not activated and cross-linked in the small-size microspheres but was directly mixed in the R2 buffer (Example 6), and the R1:R2 volume ratio was 1:2 (Example 7-1) and the R1:R2 volume ratio was 2:1 (Example 7-2) were significantly smaller than those of the other groups, proving that the detection method provided by the present invention can significantly improve the linear relationship of the detection.

[0084] 2. Sensitivity test: Use calibrator A as the sample test reagent (kit), under the test wavelength of 546nm (main), 1cm light path, 37℃, record the absorbance (A2) about 5min (t) after R2 is added, the A2 test result is the reagent blank absorbance measurement value, the absorbance difference (ΔA); use the reagent (kit) to test the blank sample (calibrator A), and repeat the test 20 times. Calculate the average and standard deviation of the concentration of the 20 test results, and calculate the average + 2 standard deviations as the blank limit. The specific data of the sensitivity test are shown in Tables 4 and 5.

[0085] Table 4: Reagent absorbance difference

[0086] Example Absorbance difference Example 1 ≥1.5 Example 2 ≥1.5 Example 3 ≥1.5 Example 4 ≥1.5 Example 5 ≥1.0 Example 6 ≥1.5 Example 7-1 ≥1.0 Example 7-2 ≥1.0

[0087] Table 5: Blank limits for 20 tests of blank samples (calibrator A)

[0088]

[0089]

[0090] It can be seen from the detection data in Tables 4 and 5 above that the antigen complex was coated with small-particle microspheres, the antibody was not activated and cross-linked in the large-particle microspheres but was directly mixed in the R1 buffer (Example 5), and the absorbance and blank limit of the three groups of samples with a R1:R2 volume ratio of 1:2 (Example 7-1) and a R1:R2 volume ratio of 2:1 (Example 7-2) were significantly worse than those of the other groups, proving that the detection method provided by the present invention can be used to detect a variety of antibodies with high detection sensitivity.

[0091] 3. Precision: Take a sample of the test agent, measure the sample of a specific concentration 10 times continuously, and calculate the mean of the measurement results ( ) standard deviation (S) and coefficient of variation CV. The specific data of precision test are shown in Table 6.

[0092] Table 6: Precision test data

[0093]

[0094]

[0095] It can be seen from the detection data in Table 6 above that the precision of the three groups of samples where only large-size microspheres were used to coat the antibody, the antigen complex was not activated and cross-linked in the small-size microspheres but was directly mixed in the R2 buffer (Example 6), and the R1:R2 volume ratio was 1:2 (Example 7-1) and the R1:R2 volume ratio was 2:1 (Example 7-2) was significantly worse than that of the other groups, proving that the detection method provided by the present invention can be used to detect a variety of antibodies with high detection precision.

[0096] 4. Accuracy test: Use no less than 30 human samples with different concentrations within the test concentration range and compare them with the test system of imported products; each sample is tested according to the kit operation method and comparison method, and the correlation coefficient and relative deviation of the two groups of results are calculated using the linear regression equation. The specific data of the accuracy test are shown in Table 7.

[0097] Table 7: Accuracy test data

[0098]

[0099]

[0100] It can be seen from the detection data in Table 7 above that the precision of the four groups of samples using only single particle size microspheres (Examples 5 and 6), R1:R2 volume ratio of 1:2 (Example 7-1) and R1:R2 volume ratio of 2:1 (Example 7-2) is significantly worse than that of other groups, which proves that the detection method provided by the present invention can be used to detect multiple antibodies with high detection accuracy.

[0101] In summary, the present invention not only improves the sensitivity of detection, but also can significantly improve the linear range of detection.

Claims

1. A method for preparing a latex enhanced immunoturbidimetry reagent, characterized in that: The method comprises the following steps: (1) The large-size latex microspheres and the small-size latex microspheres were mixed with 20 mmol / L MES buffer at a pH of 6.1, and then activated by adding activators NHS and EDC, respectively; (2) adding an antibody against the analyte to the activated large-size latex microspheres, preparing the analyte and keyhole limpet hemocyanin into an analyte protein complex, and then adding the complex to the activated small-size latex microspheres, respectively performing coupling reactions to prepare antibody-coupled large-size latex microspheres and analyte protein complex-coupled small-size latex microspheres; (3) adding blocking solution to the reaction solution after the coupling reaction for blocking, discarding the supernatant after blocking, and re-dissolving and resuspending the solids with buffer to obtain R1 reagent containing large-particle latex microspheres coupled to the antibody, and R2 reagent containing small-particle latex microspheres coupled to the protein complex of the analyte; the analyte is tacrolimus; The latex microspheres are hydrophobic microspheres with negatively charged carboxyl groups on the surface; the large-size latex microspheres have a solid content of 5%-10% and a particle size of 275 nm; the small-size latex microspheres have a solid content of 5%-10% and a particle size of 90 nm; The buffer used to reconstitute the antibody-conjugated large particle size latex microspheres contains: -100 mmol / L HEPES; -1% NaCl; -0.1% Procline300; -2% trehalose and 0.2% glycine; - pH value is 7.4; The composition of the buffer used to reconstitute the small-size latex microspheres coupled to the analyte protein complex is: -50 mmol / L HEPES; -3% PEG 8000; -0.1% Procline300; -1% glycerol and 2% sucrose; - pH value is 8.0; The R1 reagent and the R2 reagent are mixed in a ratio of 1:

1.

2. The method according to claim 1, characterized in that In step (1), the total mass ratio of latex microspheres: NHS: EDC is 7:10:1; and / or, the activation temperature is 37° C., and the reaction time is 30-60 min; and / or, in step (3), after sealing, the supernatant is discarded by centrifugation.

3. The method according to claim 2, characterized in that The latex microspheres are carboxyl polystyrene latex microspheres; and / or, the solid content of the large-size latex microspheres is 10%; and / or, the solid content of the small-size latex microspheres is 5%; and / or, in step (3), the centrifugal speed is 16000 rpm and the time is 40 min.

4. The method according to claim 1, wherein: The antibody described in step (2) is any one of a monoclonal antibody or a polyclonal antibody; the temperature of the coupling reaction is 25° C., and the reaction time is 10-90 min.

5. The method according to claim 1, wherein In step (3), the blocking solution is bovine serum albumin, glycine or ethanolamine; and / or the blocking temperature is 37° C. and the blocking time is 30-60 min.

6. The method according to claim 5, characterized in that The closure is closed under shaking conditions.

7. A reagent set for latex enhanced immunoturbidimetry, characterized in that: The reagent set comprises the R1 reagent containing large-particle latex microspheres coupled with antibodies prepared by the method as described in any one of claims 1 to 6, and the components of the R2 reagent for preparing small-particle latex microspheres coupled with protein complexes containing test substances.

8. A kit for latex enhanced immunoturbidimetry, characterized in that: The kit comprises the reagent set as claimed in claim 7.

9. The kit according to claim 8, characterized in that The kit also includes a calibrator; the calibrator is a standard solution or a lyophilized product containing a known concentration of the substance to be tested, and is used to establish a calibration curve.

10. A method for detecting a sample using latex enhanced immunoturbidimetry, characterized in that: The method comprises the following steps: (1) preparing R1 reagent and R2 reagent according to the method described in any one of claims 1 to 6; (2) mixing the R1 reagent prepared in (1), the R1 reagent in the reagent set described in claim 7, or the R1 reagent in the kit described in claim 8 or 9 with the R2 reagent prepared in (1) in a ratio of 1:1, measuring the absorbance at a wavelength of 400-800 nm, and calculating the concentration of the sample to be tested according to the calibration curve.

11. The method according to claim 10, wherein: In the method described above, in step (2), the absorbance is measured at a wavelength of 546 nm, and the concentration of the sample to be tested is calculated based on the calibration curve.

12. Use of the reagent set as claimed in claim 7 or the kit as claimed in claim 8 or 9 in the preparation of latex enhanced immunoturbidimetric preparation.

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