Kit for detecting C-reactive protein in whole blood by latex enhanced immunoturbidimetry and manufacturing process thereof

Abstract

The invention discloses a kit for detecting C-reactive protein in whole blood by latex enhanced immunoturbidimetry and a manufacturing process of the kit, and relates to the technical field of clinical in-vitro diagnosis. According to the kit, aiming at the requirement of directly detecting the C-reactive protein in a whole blood sample, a latex enhanced immunoturbidimetry is adopted, and the detection sensitivity of a turbidity signal is enhanced through specific binding of latex microspheres coated with an anti-CRP antibody and the C-reactive protein in the sample. The kit comprises the optimized antibody coated latex microspheres and other matched reagents, a whole blood sample can be directly used, and a serum or plasma separation step is not needed, so that the operation is simplified, and the detection time is shortened. According to the preparation process, the stability of reagent components and the batch-to-batch consistency are ensured through a specific technology. The kit provided by the invention has the advantages of rapidness, accuracy, simplicity and convenience in operation and the like, can be compatible with conventional laboratory equipment, and provides an efficient and reliable detection means for evaluation of clinical inflammation and infectious diseases.

Description

Technical Field

[0001] The invention relates to the technical field of clinical in vitro diagnosis, in particular to a kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry and a preparation process thereof. Background Art

[0002] In recent years, blood cell analyzers have made significant advancements, with numerous new technologies and upgrades, including breakthroughs in test items such as reticulocytes, nucleated red blood cells, C-reactive protein, immature platelet ratio, and peripheral blood hematopoietic progenitor / stem cells. C-reactive protein, in particular, has significantly evolved beyond the traditional blood cell-only assay. By adding new reagents, new functions, and new channels to the blood cell counter, it has provided a new development direction and rapidly gained clinical and market acceptance. C-reactive protein (CRP), short for C-reactive protein, is an acute phase protein that reacts with pneumococcal C polysaccharide to form a complex in response to infection or tissue damage. CRP can rise rapidly within hours, reaching several or even hundreds of times its peak value within 2-3 days. It then gradually decreases and returns to normal as the condition improves, making it a valuable tool for early diagnosis of clinical diseases. C-reactive protein can be categorized into high-sensitivity C-reactive protein (HSCRP) and standard CRP based on sensitivity. High-sensitivity C-reactive protein (HSR) has high sensitivity but low linearity and can be used to assess cardiovascular disease risk. Conventional C-reactive protein (CRP) has high linearity but low sensitivity and is primarily used as a nonspecific marker of inflammation. With the development of testing technology, a full-scale CRP assay has emerged, offering both high sensitivity and high linearity. CRP is generally measured using labeled immunoassays and label-free immunoassays. Labeled immunoassays primarily include the following methods and characteristics: immunochromatography, which is qualitative but not quantitative and can usually only be used with serum samples; enzyme-linked immunosorbent assay (ELISA), which is time-consuming and complex to operate and also typically uses serum as a sample; chemiluminescence, which offers significant advantages in sensitivity, specificity, and linearity, but is subject to high reagent costs and cannot be used with whole blood. Label-free immunoassays primarily include the following methods and characteristics: single immunodiffusion and latex agglutination, which have low sensitivity and are prone to false negatives or false positives; and latex-enhanced immunoturbidimetry, which is rapid, simple to operate, highly specific, and allows for continuous sample testing.

[0003] In the existing immunoturbidimetric technology, when latex microspheres are coupled with antibodies, the surfactant needs to be removed, otherwise the coupling efficiency will be affected. At the same time, latex microspheres with a particle size above 400nm will aggregate and precipitate due to gravity problems, making it impossible to use large-particle latex microspheres. It is well known that small-particle latex microspheres require a large amount of antibody, which can make the detection precision and linearity relatively good. Large-particle latex microspheres require less antibody, and the precision and linearity are relatively poor, but the detection sensitivity is greatly improved. Due to stability issues after latex microspheres are coupled with antibodies, the existing technology generally uses latex microspheres with a particle size of 100nm-200nm to couple antibodies, which makes the detection sensitivity slightly insufficient. Within the CRP result range of 0-4mg / L, the error is large, making it impossible to obtain accurate results, affecting clinical evaluation.

[0004] In addition, in the prior art, latex-enhanced immunoturbidimetric reagents are usually coupled with latex microspheres of different particle sizes using a single antibody, which cannot effectively improve sensitivity and linear range, and the preparation process generally contains multiple low-temperature centrifugations, which takes a long time. Meanwhile, most of the applicable samples for C-reactive protein test kits on the market are serum or plasma, which is unfavorable for the operation of medical examinations. In addition, the anionic and cationic surfactants of the hemolytic effect in the hemolytic agent R1 have a significant impact on the specificity or sensitivity of the immunoturbidimetric test. For this reason, a suitable hemolytic agent and a latex-enhanced immunoturbidimetric test kit for measuring C-reactive protein in whole blood using this hemolytic agent appear to be extremely urgent and important. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects of the prior art. It is based on latex-enhanced immunoturbidimetry, the principle of which is that when the antigen to be tested in the sample meets the antibody in the reagent, a specific binding reaction can occur, thereby forming a network of antigen-antibody molecular complexes, causing changes in the turbidity of the solution. The concentration of the antigen to be tested in the sample is determined by measuring the changes in the absorbance of the scattered light in the test solution. Based on this principle, latex enhancement is a detection method in which antibodies are attached to latex microspheres to enhance the turbidity of immune agglutinates and improve the detection sensitivity. The latex-enhanced immunoturbidimetry is rapid and can achieve continuous sample analysis. It is mainly applicable to serum samples. However, as clinical laboratory medicine puts forward higher requirements for the quality performance of in vitro reagent products such as sensitivity, linearity, stability, etc., and also puts forward requirements for detection operations such as sample processing. The immune latex R2 reagent of the present invention comprises the following steps: in the early stage of preparing latex microparticle-coupled antibodies, a suspending agent is added, and a combination of Tween and Span series surfactants is added in a specific proportion; the proportions and concentrations of the components are strictly controlled during the preparation process; the solution is heated and rapidly stirred to form a uniform and stable microemulsion suspension; the particle size selection range of the latex microspheres is increased from below 200 nm to 500 nm; latex particles of different particle sizes can be freely combined within a particle size range of 50 nm to 500 nm; the sedimentation and aggregation problems of large-diameter latex particles are effectively overcome; the influence of surfactants in the prior art on reducing coupling efficiency is eliminated; the latex microsphere-antibody conjugate is made more stable, thereby improving the stability, detection sensitivity and linear range of the detection kit; and the preparation process of the microemulsion suspension of the present invention is conducive to stabilizing the latex microspheres, thereby eliminating the centrifugal separation process required in the traditional preparation process, simplifying the production process steps, improving the production efficiency, and reducing the cost of purchasing centrifuge equipment during the production process. In addition, the hemolytic agent R1 adopts an alkyl glycoside surfactant as a hemolytic component. This surfactant has a fast hemolysis rate and is far superior to anionic and cationic surfactants. It does not interfere with CRP detection and can quickly dissolve the immune latex R2 reagent Span surfactant during the detection process, thereby eliminating the turbidity effect of the microemulsion suspension in the R2 reagent. The kit of the present invention can perform a full range of CRP detection, and the sample type is whole blood, which meets the needs of simultaneous CRP+ blood routine detection and is easier to develop clinical laboratory medicine. Another object of the present invention is to provide a preparation method of the kit, which adopts a process without a centrifugation step, greatly simplifies the process steps, shortens the preparation process time, and provides production efficiency.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized by:

[0008] Contains hemolytic agent R1 and immune latex R2 reagent;

[0009] Hemolytic agent R1 is composed of the following components: 40-60 mmol / L MOPS buffer system, 10-15 g / L boric acid, 0.5 g / L-1 g / L tea saponin, 0.5-1 g / L alkyl glycoside nonionic surfactant, 15-20 g / L sodium chloride, 1-2 g / L Tween 20, 4 g / L-6 g / L PEG6000 sensitizer, 0.1-0.3 g / L proclin300 preservative, and the balance is deionized water;

[0010] The immunolatex R2 reagent is composed of the following components: 2-10 g / L multi-size mixed latex microsphere-antibody complex, 10-50 mmol / L boric acid buffer system, 50-100 g / L sucrose stabilizer, 1-2 g / L Tween 20 surfactant, 0.1-0.3 g / L proclin 300 preservative, and the balance is deionized water;

[0011] The multi-size mixed latex microsphere-antibody complex is composed of four particle sizes of 50nm, 100nm, 200nm, and 500nm carboxyl polystyrene microspheres combined in a ratio of 0.5:3:6:0.5, and is coupled to a rabbit anti-human CRP and mouse anti-human CRP dual antibody system by EDC / NHS cross-linking method.

[0012] A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized by:

[0013] The alkyl polyglycoside nonionic surfactant in the hemolytic agent R1 is alkyl polyglycoside C16APG, and its content is 0.5 g / L. The concentration of the MOPS buffer system is 50 mmol / L, and the pH value is 7.00.

[0014] A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized by:

[0015] The multi-particle size mixed latex microsphere-antibody complex in the immune latex R2 reagent is composed of carboxyl polystyrene microspheres of four particle sizes of 50nm, 100nm, 200nm, and 500nm in a mass ratio of 0.5:3:6:0.5, and the volume ratio of rabbit anti-human CRP antibody to mouse anti-human CRP antibody in the double antibody system is 2:1.

[0016] A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized by:

[0017] The detection sensitivity of the kit is 0.17 mg / L, the linear range is 0.17-363.55 mg / L, and the coefficient of variation of the detection results is less than 10%.

[0018] A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized by:

[0019] During the preparation of the immune latex R2 reagent, microemulsion suspension technology is used to form a stable suspension of latex microspheres by adding glycerol and Tween / Span surfactant mixture.

[0020] The preparation process of a kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry is characterized by:

[0021] The following steps are involved:

[0022] (1) Preparation of hemolytic agent R1: Dissolve MOPS buffer, boric acid, alkyl glycoside C16APG, sodium chloride, PEG6000, and proclin300 in deionized water in appropriate proportions and mix well;

[0023] (2) Preparation of immune latex R2 reagent:

[0024] a. Take carboxyl polystyrene microspheres of four particle sizes of 50nm, 100nm, 200nm, and 500nm, mix them in a ratio of 0.5:3:6:0.5, dilute them with 10mmol / L MES buffer, add a mixture of glycerol and Tween 20, Tween 80, and Span 80, and mix them at 37°C and 400±10r / min for 30 minutes to form a microemulsion suspension;

[0025] b. Add 10 mmol / L MES buffer containing 50 g / L EDC and 40 g / L NHS and shake at 37°C, 300 ± 10 rpm for 15 minutes;

[0026] c. Neutralize with 10 mmol / L boric acid buffer and mix for 2 minutes;

[0027] d. Add rabbit anti-human CRP antibody and mouse anti-human CRP antibody (volume ratio 2:1) and shake at 37°C, 300 ± 10 rpm for 2 hours;

[0028] e. Add 50 mmol / L PBS buffer containing 50 g / L bovine serum albumin, shake at 37°C for 0.5 hours, and store at 4°C;

[0029] f. The prepared multi-size mixed latex microsphere-antibody complex was mixed with boric acid buffer, sucrose, Tween 20, and procli n300 to prepare the immune latex R2 reagent.

[0030] Beneficial effects

[0031] (1) Improvement in sensitivity, specificity, linear range, accuracy and stability

[0032] The latex enhanced immune scattering turbidimetric kit for measuring C-reactive protein (CRP) in whole blood provided by the present invention, on the basis of traditional latex immunoturbidimetry, by innovatively adopting the strategy of multi-particle size latex microspheres in combination with multiple antibodies, significantly improves the sensitivity, specificity, linear range, accuracy and reliability of the reagent, while ensuring the long-term stability of the reagent. Traditional latex immunoturbidimetry usually uses single particle size latex microspheres and single antibodies, which limits the sensitivity and specificity of detection. The present invention adopts multi-particle size latex microspheres (such as 50nm, 100nm, 200nm and 500nm) to cover a wider range of antigen concentrations through the synergistic effect of different particle sizes. Small particle size microspheres (50nm and 100nm) can bind more antibodies due to their larger surface area, significantly improving the detection sensitivity of low-concentration CRP; large particle size microspheres (200nm and 500nm) enhance the scattering signal of high-concentration CRP, widening the detection upper limit. At the same time, the combination of multiple antibodies (such as rabbit anti-human CRP antibodies and mouse anti-human CRP antibodies) increases the recognition sites for CRP molecules, reduces the risk of nonspecific binding, and thus improves specificity. Test results show that the sensitivity and specificity of this kit are superior to traditional methods in the concentration range of 0.17 mg / L to 363.55 mg / L, meeting the clinical needs for low concentration (such as cardiovascular risk assessment) and high concentration (such as acute inflammation monitoring) CRP detection. Multi-particle size latex microspheres optimize the signal response curve, avoiding the problem of traditional methods with signal saturation in high concentration areas or weak signals in low concentration areas. The linear range test shows that the linear regression coefficient R of the present invention is 0.17 mg / L to 363.55 mg / L. 2 As high as 0.9979, indicating that in the range of 0.17 mg / L to 363.55 mg / L, the test results are highly linearly correlated with the CRP concentration, and the accuracy is significantly improved. The use of multiple antibodies further reduces the detection deviation caused by insufficient recognition sites of a single antibody, ensuring reliable results. The present invention adopts microemulsion suspension technology in the preparation of latex microspheres. By precisely controlling the proportion and concentration of surfactants, the latex microspheres are evenly dispersed and not easy to settle or agglomerate. This process improves the coupling efficiency of latex microspheres and antibodies, and enhances the long-term stability of the reagents. Stability tests show that the test kit can be stably stored at 2-8°C for more than 12 months. In the accelerated stability test at 37°C, the performance indicators did not change significantly within 31 days, meeting the high requirements of clinical laboratories.

[0033] (2) Rich sample types and convenience of clinical application

[0034] The kit provided by the present invention for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry breaks through the limitation that traditional CRP detection is limited to serum or plasma samples, realizes the direct detection of whole blood samples for the first time, enriches the sample types, improves the convenience of clinical application, and promotes the development of clinical laboratory medicine.

[0035] Traditional CRP testing requires centrifugation of whole blood to obtain serum or plasma, which increases operational complexity and time costs, and limits immediacy, especially in emergency or bedside testing. This kit supports direct testing of whole blood samples through the optimized formulation of hemolytic agent R1 and immune latex R2. The red blood cells in the whole blood are rapidly lysed by the hemolytic agent R1, releasing CRP, while the anti-interference components effectively suppress the influence of residual blood cells, ensuring test accuracy. This innovation eliminates the sample pretreatment step and greatly simplifies the testing process. The support of whole blood samples allows CRP testing to be performed simultaneously with routine blood tests (such as white blood cell count and hemoglobin measurement), improving testing efficiency and reducing sample usage and operation time. This advantage is particularly prominent in scenarios requiring rapid decision-making, such as emergency departments and intensive care units (ICUs), helping doctors to promptly assess the patient's inflammatory status. The implementation of whole blood CRP testing reduces testing costs and technical barriers, provides support for primary care institutions and point-of-care testing (POCT), and promotes the development of clinical laboratory medicine in a fast, convenient, and efficient direction.

[0036] (3) Optimization of production processes and improvement of cost-effectiveness

[0037] The kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry provided by the present invention optimizes the production process through a de-centrifugation process, greatly improves production efficiency, reduces equipment costs, and provides advantages for the commercial production and market promotion of the kit. Traditional latex microsphere preparation requires multiple centrifugation separations to remove unbound antibodies and impurities. The process is time-consuming and relies on expensive equipment. The present invention adopts microemulsion suspension technology to achieve uniform dispersion and stable suspension of latex microspheres, and the antibody coupling and blocking steps can be completed without centrifugation. This innovation simplifies the production process and shortens the preparation time. The de-centrifugation process reduces the demand for large centrifuges, reduces equipment investment and maintenance costs, makes production more economical, and provides the possibility for small and medium-sized enterprises to enter the market. The simplified process shortens the single-batch production time, increases output, and significantly improves overall production efficiency. This advantage helps to quickly respond to market demand and meet the large clinical demand for high-quality test kits.

[0038] (4) Innovative design of hemolytic agents and improvement of detection accuracy

[0039] The present invention provides a kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry. The non-ionic surfactant alkyl glycoside (APG) is introduced into the hemolytic agent R1, which provides a new idea for whole blood detection, significantly reduces the interference of hemolytic reagents on the test results, and improves accuracy. Traditional hemolytic agents mostly use ionic surfactants (such as SDS, TritonX-100), which may interfere with the specificity of immune response and cause result deviation. The alkyl glycoside used in the present invention is a mild non-ionic surfactant with high hemolytic ability and good biocompatibility. It can quickly induce red blood cell lysis while having minimal effect on plasma proteins such as CRP, reducing detection interference. Comparative tests show that the hemolytic agent R1 using alkyl glycoside performs well in low-concentration CRP detection. For example, in a sample with a CRP concentration of 0.51 mg / L, the test result of this kit is 0.51 mg / L, while the hemolytic agent without alkyl glycoside has a result of 0 mg / L, which proves that it reduces the risk of false negatives and improves accuracy. Hemolytic agent R1 and immune latex R2 work synergistically to optimize the whole blood testing process. R1 rapidly releases CRP, while the multi-particle latex and multiple antibodies in R2 ensure efficient and specific binding, making whole blood testing comparable to serum testing and providing flexible clinical options. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Figure 1 This is a graph showing the interchangeability of whole blood testing using the test kit of the present invention for testing single CRP and blood routine + CRP modes in Test Example 3; Figure 2 This is a linear test analysis chart of whole blood samples of different concentrations measured using the kit of the present invention in Test Example 5. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] Example 1

[0043] The present invention discloses a hemolytic agent and latex enhanced immune turbidimetric kit for measuring C-reactive protein in whole blood, comprising a hemolytic agent R1 and an immune latex reagent R2;

[0044] The preferred components of the hemolytic agent R1 are shown in Table 1 below:

[0045] Table 1: Hemolytic agent R1 Reagent Example 1

[0046]

[0047]

[0048] The preferred components of the immune latex R2 reagent are shown in Table 2 below:

[0049] Table 2: Hemolytic agent R2 Reagent Example 1

[0050]

[0051]

[0052] The components of the hemolytic agent R1 were prepared according to conventional methods; except for the anti-human CRP antibody multi-particle latex microsphere conjugate of R2, the other components were prepared according to conventional methods; the preparation method of the anti-human CRP antibody multi-particle latex microsphere conjugate in R2 was as follows: 0.125 mL of 50 nm (10% w / v), 0.75 ml of 100 nm (10% w / v), 1.5 mL of 200 nm (10% w / v), and 0.125 ml of 500 nm (10% w / v) carboxyl polystyrene latex microspheres were taken and added to 5 mL of 10 mmol / L MES buffer, 75 mg of glycerol, 15 mg of Tween 20, 1.5 mg of Tween 80, and 1.5 mg of Span, and mixed at 400±10 r / min at 37°C for 30 min. Add 0.5 ml of 10 mmol / L MES buffer containing 50 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 g / L N-carboxysuccinimide, stir to mix, and shake at 37°C, 300 ± 10 rpm for 15 min. Add 50 mL of the 10 mmol / L boric acid buffer prepared in the previous step and mix for 2 min. Add 590 μL of rabbit anti-human CRP antibody and 295 μL of mouse anti-human CRP antibody, respectively, and shake at 37°C, 300 ± 10 rpm for 2 h. Add 1 mL of 50 mmol / L PBS buffer containing 50 g / L bovine serum albumin, mix, shake at 37°C, 300 ± 10 rpm for 0.5 h, and store at 4°C.

[0053] Example 2

[0054] Hemolytic agent R1 for removing alkyl glycoside substances.

[0055] The preferred components of the hemolytic agent R1 are shown in Table 3 below:

[0056] Table 3: Hemolytic agent R1 Reagent Example 2

[0057]

[0058]

[0059] The components of the above hemolytic agent R1 are prepared according to conventional methods.

[0060] Example 3

[0061] The two-antibody multi-particle latex microsphere conjugate was changed to a single-antibody multi-particle latex microsphere conjugate to prepare latex reagent R2.

[0062] The preferred components of the immune latex R2 reagent are shown in Table 4 below:

[0063] Table 4: Hemolytic agent R2 Reagent Example 3

[0064]

[0065]

[0066] Except for the anti-human CRP antibody multi-particle latex microsphere conjugate, the other components of R2 were prepared according to conventional methods; the preparation method of the anti-human CRP antibody multi-particle latex microsphere conjugate in R2 is as follows: 0.125 mL of 50 nm (10% w / v), 0.75 ml of 100 nm (10% w / v), 1.5 mL of 200 nm (10% w / v), and 0.125 ml of 400 nm (10% w / v) carboxyl polystyrene latex microspheres were taken and added to 5 mL of 10 mmol / L MES buffer, 75 mg of glycerol, 15 mg of Tween 20, 1.5 mg of Tween 80, and 1.5 mg of Span were added, and mixed at 400 ± 10 r / min at 37°C for 30 min. Add 0.5 ml of 10 mmol / L MES buffer containing 50 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 g / L N-carboxysuccinimide, stir to mix, and oscillate at 37°C, 300 ± 10 rpm for 15 min. Add 50 ml of the 10 mmol / L boric acid buffer prepared in the previous step and mix for 2 min. Add 885 μ ml of rabbit anti-human CRP antibody and oscillate at 37°C, 300 ± 10 rpm for 2 h. Add 1 ml of 50 mmol / L PBS buffer containing 50 g / L bovine serum albumin, mix, oscillate at 37°C, 300 ± 10 rpm for 0.5 h, and store at 4°C.

[0067] Test Example 1

[0068] The results of whole blood testing using the preferred hemolytic agent in Example 1 were compared with those using the hemolytic agent in Example 2.

[0069] The results of Test Example 1 are shown in Table 5 below:

[0070] Table 5: Comparison of hemolytic agents with and without alkyl glycosides in Test Example 1

[0071]

[0072]

[0073] Comparison of the results revealed that the addition of alkyl glycosides to the hemolytic reagent effectively differentiated and efficiently detected low-value samples. Because alkyl glycosides have an HLB value within the optimal range of 12.5-17.0, they exhibit high hemolytic activity and induce an extremely rapid hemolysis process. Therefore, alkyl glycosides can enhance the immune response and improve detection sensitivity.

[0074] Test Example 2

[0075] Comparison of the reactivity of the preferred latex reagent R2 in Example 1 and the latex reagent R2 in Example 3

[0076] The results of Test Example 2 are shown in Table 6 below:

[0077] Table 6: Comparison of binding results of two antibodies and single antibody multi-size latex microspheres in Test Example 2

[0078]

[0079] The results showed that the two-antibody multi-particle latex microsphere conjugate can improve the reactivity more than the single-antibody multi-particle latex microsphere conjugate and can detect whole blood results more efficiently.

[0080] Test Example 3

[0081] This test example is an analysis of the interchangeability between the preferred hemolytic agent R1 and the preferred latex reagent R2 in Example 1 using the CRP model and the blood routine + CRP model.

[0082] Test samples: 40 random whole blood samples and one CRP national calibrator sample (target value 35.34±2.79mg / L)

[0083] Experimental method: After calibration, 40 whole blood samples were measured using conventional detection methods in the field using the single CRP mode and the blood routine + CRP mode.

[0084] The test results of the kit of the present invention for whole blood samples under CRP mode and blood routine + CRP mode are shown in Figure 1 The X-axis represents the CRP value measured in the CRP mode, and the Y-axis represents the CRP value measured in the blood routine + CRP mode. The correlation coefficient R 2 =0.9994, regression equation y = 1.0101x + 0.04. Figure 1 It can be seen that there is no significant difference in the results of the CRP test using the single CRP mode and the blood routine test + CRP mode, and the two modes of determination are interchangeable.

[0085] Test Example 4

[0086] This test example is an accuracy analysis of the preferred hemolytic agent R1 and the preferred latex reagent R2 in Example 1.

[0087] Test sample: One CRP national calibrator sample (target value 35.34±2.79mg / L)

[0088] Test method: After calibration, measure the sample three times and calculate the relative deviation between the average measured value and the labeled value.

[0089] The results of Test Example 4 are shown in Table 7 below:

[0090] Table 7: Accuracy results of the combination of the preferred hemolytic agent R1 and latex reagent R2 in Example 1

[0091]

[0092] The kit of the present invention has higher accuracy.

[0093] Test Example 5

[0094] This test example is a repeatability analysis of the preferred hemolytic agent R1 and the preferred latex reagent R2 in Example 1.

[0095] Test sample: Take a quality control material with known concentration and traceability

[0096] Test method: After calibration, repeat the test 10 times for each concentration and calculate the coefficient of variation. The coefficient of variation is calculated as the ratio of the mean value of the 10 repeated measurements to the standard deviation, which must not exceed 10%.

[0097] The results of Test Example 5 are shown in Table 8 below:

[0098] Table 8: Repeatability results of the combination of the preferred hemolytic agent R1 and the latex reagent R2 in Example 1

[0099] Number of tests Concentration 1 Concentration 2 Concentration 3 1 4.28 21.36 165.89 2 4.09 21.14 158.35 3 4.44 21.89 162.09 4 4.18 22.08 157.67 5 4.33 21.67 161.02 6 4.07 22.36 163.23 7 4.01 23.01 159.29 8 4.14 22.49 157.45 9 3.98 22.78 162.16 10 4.24 21.93 164.34 Target value 4.15 22.1 158 average value 4.18 22.07 161.15 Standard deviation 0.15 0.60 2.91 Coefficient of variation 3.52％ 2.71％ 1.80％

[0100] The repeatability of the kit of the present invention meets the requirements.

[0101] Test Example 6

[0102] This test example is a linear test analysis of the preferred hemolytic agent R1 and the preferred latex reagent R2 in Example 1.

[0103] Test sample: Take a high-value sample of known concentration and diluent.

[0104] Test method: After calibration, dilute the high-value samples one by one with diluent, test each point 4 times, take the average value, draw a regression line with the result and the dilution, and calculate the regression coefficient R 2 With equation, see Figure 2 .

[0105] Correlation coefficient R 2 =0.9979, the regression equation y=355.96x-0.3772, which has good linearity. The dilution rate was substituted into the regression equation and compared with the measured values. The deviations were all within the required range. It can be seen that the linear range meets 0.17-363.55 mg / L and has good sensitivity.

[0106] Test Example 7

[0107] This test example is the stability analysis of the preferred hemolytic agent R1 and the preferred latex reagent R2 in Example 1

[0108] Test method: The test kit was placed at 2-8°C for long-term stability testing; placed at 37±1°C for accelerated stability testing; placed at 2±1°C and 30±1°C for simulated transportation stability testing; and placed at 2-8°C for unsealed stability testing. The results are shown in Table 9.

[0109] The results of Test Example 7 are shown in Table 9 below:

[0110] Table 9: Stability results of the combination of the preferred hemolytic agent R1 and the latex reagent R2 in Example 1

[0111]

[0112] The latex-enhanced immunoturbidimetric kit disclosed by the invention for measuring C-reactive protein in whole blood has good stability.

[0113] Finally, it should be noted that the above description and testing are based on specific embodiments of the present invention. It should be understood that the present invention is not limited to the above disclosed embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims without affecting the essence of the present invention.

Claims

1. A kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry, characterized in that: Contains hemolytic agent R1 and immune latex R2 reagent; Hemolytic agent R1 is composed of the following components: 40-60 mmol / L MOPS buffer system, 10-15 g / L boric acid, 0.5 g / L-1 g / L tea saponin, 0.5-1 g / L alkyl glycoside nonionic surfactant, 15-20 g / L sodium chloride, 1-2 g / L Tween 20, 4 g / L-6 g / L PEG6000 sensitizer, 0.1-0.3 g / L proclin300 preservative, and the balance is deionized water; The immunolatex R2 reagent is composed of the following components: 2-10 g / L multi-size mixed latex microsphere-antibody complex, 10-50 mmol / L boric acid buffer system, 50-100 g / L sucrose stabilizer, 1-2 g / L Tween 20 surfactant, 0.1-0.3 g / L proclin 300 preservative, and the balance is deionized water; The multi-size mixed latex microsphere-antibody complex is composed of four carboxyl polystyrene microspheres with particle sizes of 50 nm, 100 nm, 200 nm, and 500 nm in a ratio of 0.5:3:6:0.5, and is coupled to a rabbit anti-human CRP and mouse anti-human CRP double antibody system through an EDC / NHS cross-linking method.

2. The kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry according to claim 1, wherein: The alkyl polyglycoside nonionic surfactant in the hemolytic agent R1 is alkyl polyglycoside C16APG, and its content is 0.5 g / L. The concentration of the MOPS buffer system is 50 mmol / L, and the pH value is 7.

00.

3. The kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry according to claim 1, wherein: The multi-particle size mixed latex microsphere-antibody complex in the immune latex R2 reagent is composed of carboxyl polystyrene microspheres with four particle sizes of 50nm, 100nm, 200nm, and 500nm in a mass ratio of 0.5:3:6:0.5, and the volume ratio of rabbit anti-human CRP antibody to mouse anti-human CRP antibody in the double antibody system is 2:

1.

4. The kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry according to claim 1, wherein: The detection sensitivity of the kit is 0.17 mg / L, the linear range is 0.17-363.55 mg / L, and the coefficient of variation of the detection result is less than 10%.

5. The kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry according to claim 1, wherein: During the preparation of the immune latex R2 reagent, microemulsion suspension technology is adopted, and a mixture of glycerol and Tween / Span surfactant is added to form a stable suspension of latex microspheres.

6. The preparation process of the kit for detecting C-reactive protein in whole blood by latex-enhanced immunoturbidimetry as claimed in claim 1, characterized in that: The following steps are involved: (1) Preparation of hemolytic agent R1: Dissolve MOPS buffer, boric acid, alkyl glycoside C16APG, sodium chloride, PEG6000, and proclin300 in deionized water in appropriate proportions and mix well; (2) Preparation of immune latex R2 reagent: a. Take carboxyl polystyrene microspheres of four particle sizes of 50nm, 100nm, 200nm, and 500nm, mix them in a ratio of 0.5:3:6:0.5, dilute them with 10mmol / L MES buffer, add a mixture of glycerol and Tween 20, Tween 80, and Span 80, and mix them at 37°C and 400±10r / min for 30 minutes to form a microemulsion suspension; b. Add 10 mmol / L MES buffer containing 50 g / L EDC and 40 g / L NHS and shake at 37°C, 300 ± 10 rpm for 15 minutes; c. Neutralize with 10 mmol / L boric acid buffer and mix for 2 minutes; d. Add rabbit anti-human CRP antibody and mouse anti-human CRP antibody (volume ratio 2:1) and shake at 37°C, 300 ± 10 rpm for 2 hours; e. Add 50 mmol / L PBS buffer containing 50 g / L bovine serum albumin, shake at 37°C for 0.5 hours, and store at 4°C; f. The prepared multi-size mixed latex microsphere-antibody complex was mixed with boric acid buffer, sucrose, Tween 20, and proclin 300 to prepare the immune latex R2 reagent.

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