A method for detecting oxidized low density lipoprotein and a detection kit

Abstract

This invention belongs to the field of bioassay technology, and specifically relates to a method and kit for detecting oxidized low-density lipoprotein (ODL). This invention provides a novel application of quaternized chitosan in the kit, specifically using quaternized chitosan of different molecular weights in the immunoturbidimetric detection of ODL. Results show that when high molecular weight quaternized chitosan (100-200 kDa) and low molecular weight quaternized chitosan (30-50 kDa) are used as components of the ODL detection kit, the different molecular weights of quaternized chitosan react with citrate to form gel particles of varying mechanical strengths. This allows for the detection of ODL content via immunoturbidimetry. Furthermore, the use of quaternized chitosan of different molecular weights significantly improves the sensitivity and linear range of the ODL detection kit.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a method and kit for detecting oxidized low-density lipoprotein. Background Technology

[0002] Oxidized low-density lipoprotein (ox LDL (ox) is an important marker of atherosclerosis, and its level is closely related to the occurrence and development of cardiovascular diseases. Accurate and rapid detection of ox... LDL (Lower-Low Disorder) is of great significance for the early diagnosis and prevention of cardiovascular diseases.

[0003] Existing ox LDL detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and latex-enhanced immunoturbidimetric assay (LEIA). Among them, LEIA, which uses latex microspheres labeled with anti-ox-LDL antibodies and achieves ox-LDL quantification by increasing the particle size of the complex formed after antigen-antibody binding and observing changes in turbidity, has received widespread attention due to its advantages of simple operation and fast detection speed. However, traditional LEIA with single-size latex microspheres has problems such as weak signal response, insufficient sensitivity, and narrow linear range, making it difficult to meet the requirements for accurate detection of low and high concentrations of ox-LDL. Therefore, detection methods with dual-size latex microspheres have emerged.

[0004] However, existing ox-based latex microsphere labeling based on dual particle size... LDL detection methods still have some technical shortcomings, such as:

[0005] Firstly, the preparation process of the dual-size latex microsphere kit is relatively complex. As disclosed in patent CN119667179A, the two types of latex microspheres involved in the preparation of the kit require two-step labeling. The surface modification conditions and antibody labeling parameters of the two types of latex microspheres need to be optimized independently, and the process parameters are difficult to unify, resulting in a complicated and time-consuming production process for the kit.

[0006] Secondly, because the sensitivity and linear range of the detection concentration of immunoturbidimetric assay are affected by the ratio of antigen and labeled antibody concentrations, the detection sensitivity is low and the linear range is narrow, and it cannot fundamentally change the problem of the imbalance between labeled antibody and antigen.

[0007] Therefore, there is an urgent need to improve existing technologies and provide a new approach to further enhance the sensitivity of immunoturbidimetry and expand its linear detection range. Summary of the Invention

[0008] To address the above technical problems, this invention proposes a method and kit for detecting oxidized low-density lipoprotein.

[0009] This invention controls the reaction ratio of antigen and labeled antibody by controlling the release rates of latex microsphere-labeled antibody (i.e., latex microsphere-labeled oxidized low-density lipoprotein antibody, hereinafter referred to as labeled antibody) and free antigen in the test sample in the reaction system. This allows the labeled antibody and the test antigen to remain within the optimal reaction ratio of antigen and labeled antibody for an extended period during the reaction process, i.e., the equivalence band, to form a strong turbidity signal. Although the ratio may change after the labeled antibody and the test antigen are completely released, the reversible reaction to reduce the complexes, which is a lengthy process, results in a large number of immune complexes already formed. Therefore, the above operation can achieve high sensitivity when the antigen is insufficient in the pre-band stage of the detection kit and low hook effect when the labeled antibody is insufficient in the post-band stage, which is beneficial to further improving the sensitivity and linear range of the detection concentration of the kit.

[0010] The technical solution provided by this invention is as follows:

[0011] The first aspect of the present invention is to provide a novel use of quaternized chitosan, specifically the application of quaternized chitosan in the detection of oxidized low-density lipoprotein.

[0012] Preferably, the application specifically involves using quaternized chitosan with different molecular weights (including high molecular weight quaternized chitosan and low molecular weight quaternized chitosan) and a soluble salt containing citrate in the detection of oxidized low-density lipoprotein. The reaction of quaternized chitosan with different molecular weights with citrate forms gel particles of varying mechanical strengths. These gel particles, with their different mechanical strengths, exhibit different diffusion resistance to antigens and labeled antibodies, thereby regulating the diffusion rate of antigens and labeled antibodies. This allows the antigens and labeled antibodies to contact in an appropriate ratio for an immune reaction, forming immune complexes. The content of oxidized low-density lipoprotein is then detected using immunoturbidimetry. The high molecular weight quaternized chitosan has a molecular weight of 100-200 kDa, and the low molecular weight quaternized chitosan has a molecular weight of 30-50 kDa.

[0013] A second aspect of the present invention is to provide a method for detecting oxidized low-density lipoprotein, specifically:

[0014] The reagent containing high molecular weight quaternized chitosan is mixed with the sample to be tested, and citrate buffer is added to allow it to fully react with the high molecular weight quaternized chitosan to form gel particles with high mechanical strength. Then, the reagent containing low molecular weight quaternized chitosan and latex microsphere-labeled oxidized low-density lipoprotein antibody is dispersed in the aforementioned high mechanical strength gel system to react and form gel particles with lower mechanical strength. The two types of gel particles respectively encapsulate the antigen and the labeled antibody. The antigen and the labeled antibody are removed from the gel particles through diffusion and react with each other to form immune complexes. The content of oxidized low-density lipoprotein is calculated by measuring the change in absorbance of the system. The molecular weight of the high molecular weight quaternized chitosan is 100~200 kDa, and the molecular weight of the low molecular weight quaternized chitosan is 30~50 kDa.

[0015] In the detection method provided by this invention, citrate ions in the citrate buffer react with high-molecular-weight quaternized chitosan of a specific concentration during incubation to form gel particles with high mechanical strength. These particles encapsulate the antigen in the test sample, thereby limiting the movement speed of the antigen. After incubation for 2-5 minutes, the high-mechanical-strength gel particle system becomes basically stable. Based on this, the added oxidized low-density lipoprotein antibody containing low-molecular-weight quaternized chitosan and latex microspheres disperses among the previously formed, high-mechanical-strength gel particles. Simultaneously, the remaining citrate ions react with uniformly distributed low-molecular-weight quaternized chitosan to form lower-mechanical-strength gel particles, which disperse among the aforementioned high-mechanical-strength gel particles that encapsulate the test antigen. By adjusting the concentration and molecular weight of the quaternized chitosan, the mechanical strength of the two types of gel particles can be controlled, thereby controlling the release rate of the test antigen and latex microsphere-labeled antibody from the two types of gel particles, controlling the antigen-antibody reaction ratio, and ultimately ensuring that the antigen-antibody complex forms the strongest turbidity signal in the equivalence band.

[0016] A third aspect of the present invention is that, based on the reaction principle and detection method provided above, a detection kit for oxidized low-density lipoprotein is provided. The detection kit includes: reagent R1, reagent R2, and reagent R3. Each reagent, in terms of mass-volume concentration, comprises the following components: Reagent R1 includes: 0.03%~0.6% (w / v) of high molecular weight quaternized chitosan, 0.6%~0.9% (w / v) of KCl, 0.5%~3% (w / v) of polyethylene glycol 6000, and 50~100 mM of buffer solution.

[0017] The reagent R2 is a citrate buffer solution of 2.0%–10.0% (w / v) with a pH of 7.0–7.2.

[0018] The reagent R3 comprises: 0.02%~0.3% (w / v) low molecular weight quaternized chitosan, 0.01%~0.05% (w / v) latex microsphere-labeled oxidized low-density lipoprotein antibody, 0.1%~0.5% (w / v) ethanolamine, 1%~5% (w / v) trehalose, and 50~100 mM buffer.

[0019] The buffers described in reagents R1 and R3 are each independently selected from any one of the following: HEPES buffer, Tris-HCl buffer, MES buffer, MOPS buffer, TAPS buffer, and glycine buffer.

[0020] The buffers selected in reagents R1 and R3 of this invention are buffers without secondary or higher negative charges. The basis for this selection is to prevent them from reacting with the quaternized chitosan in the reagents to form a gel and lose the sustained release effect on the antigen. Furthermore, the buffers are preferably Tris-HCl buffers.

[0021] Furthermore, unless otherwise specified, the concentrations of all components involved in the kit provided by this invention are expressed as mass-volume concentrations (w / v, %).

[0022] Preferably, the molecular weight of the high molecular weight quaternary ammonium salted chitosan in reagent R1 is 100~200 kDa, more preferably 150~180 kDa.

[0023] Preferably, the molecular weight of the low molecular weight quaternary ammonium salted chitosan in reagent R3 is 30~50 kDa, more preferably 30~45 kDa.

[0024] Preferably, in the kit, the citrate buffer in reagent R2 is selected from any one or a combination of several of sodium dihydrogen citrate, potassium dihydrogen citrate, disodium hydrogen citrate, dipotassium hydrogen citrate, trisodium citrate, and tripotassium citrate buffer, and the pH is adjusted to 7.0-7.2 with 1 M hydrochloric acid and sodium hydroxide solution, more preferably pH 7.0.

[0025] Preferably, reagents R1 and R3 further contain an antibacterial agent with a mass-volume concentration of 0.05% to 0.15% (w / v), and the antibacterial agent includes, but is not limited to, the following: parabens, isothiazolinones, quaternary ammonium salts, potassium sorbate, and proclin 300.

[0026] Preferably, the pH of both reagent R1 and reagent R3 is 7.0 ± 0.5.

[0027] Preferably, the method for detecting oxidized low-density lipoprotein using the kit described above is as follows:

[0028] Mix the sample to be tested with reagent R1, then add reagent R2 and incubate for 2-5 minutes. After incubation, add reagent R3 and mix well. Immediately read the absorbance value A1. React at 35-38℃ for 5-8 minutes, then read the absorbance value A2 again and calculate the degree of reactivity. A = A2 - A1, which is converted into the corresponding content of oxidized low-density lipoprotein according to the standard curve.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] (1) A new use of quaternized chitosan is provided, namely, its application in the detection of oxidized low-density lipoprotein content. At the same time, a method for detecting oxidized low-density lipoprotein content is also provided. That is, by using quaternized chitosan of different molecular weights (100~200 kDa high molecular weight quaternized chitosan, 30~50 kDa low molecular weight quaternized chitosan) to combine with citrate to form gel particles of different mechanical strengths, the diffusion rate of antigen and labeled antibody is controlled by the different diffusion resistance of gels of different mechanical strengths, so that antigen and labeled antibody can contact each other in an appropriate ratio for a longer period of time to carry out immune reaction, form immune complexes, improve their equivalence band period, and finally realize the detection of oxidized low-density lipoprotein content by immunoturbidimetry.

[0031] (2) Based on the above principles, the present invention provides a detection kit for oxidized low-density lipoprotein. By adjusting the concentration of quaternized chitosan with different molecular weights, the release rates of labeled antibody and free antigen in the test sample in the reaction system are controlled, so that the labeled antibody and the test antigen can be in the optimal reaction ratio for a long time during the reaction process, so as to form a strong turbidity signal, which greatly improves the detection sensitivity of the oxidized low-density lipoprotein detection kit and reduces the detection concentration of oxidized low-density lipoprotein to 3.0 U / L, which is far below the detection limit of the prior art (9.0 U / L).

[0032] Furthermore, the aforementioned improvements also reduce the occurrence of the hook effect when there is insufficient antibody in the later stage, which is beneficial to further improve the sensitivity of the kit and the linear range of detection concentration. The results show that after the improvement treatment of the present invention, the linear range of detection of oxidized low-density lipoprotein of the kit is increased to 155 U / L, and the concentration at which the hook effect occurs is increased to 190 U / L, while the linear range of detection of the prior art kit is below 115 U / L, and the hook effect begins to appear when the concentration is higher than 155 U / L. Attached Figure Description

[0033] Figure 1This is a schematic diagram illustrating the detection principle of the kit provided by the present invention for detecting oxidized low-density lipoprotein. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0035] Example 1

[0036] A kit for detecting oxidized low-density lipoprotein includes reagent R1, reagent R2, and reagent R3, wherein the components of each reagent are as follows:

[0037] Reagent R1: 180 kDa quaternized chitosan 0.2% (w / v), KCl 0.8% (w / v), polyethylene glycol 6000 2% (w / v), Tris-HCl buffer 50 mM;

[0038] Reagent R2: Disodium citrate buffer 8.0% (w / v), adjusted to pH 7.0 with 1 M sodium hydroxide or hydrochloric acid solution;

[0039] Reagent R3: 0.09% (w / v) quaternized chitosan (50 kDa), 0.03% (w / v) oxidized low-density lipoprotein antibody labeled with latex microspheres, 0.5% (w / v) ethanolamine, 2% (w / v) trehalose, and 50 mM Tris-HCl buffer.

[0040] Example 2

[0041] The only difference from Example 1 is that the molecular weight of the quaternized chitosan in reagent R1 is 150 kDa.

[0042] Example 3

[0043] The only difference from Example 1 is that the molecular weight of the quaternized chitosan in reagent R3 is 30 kDa.

[0044] Example 4

[0045] The only difference from Example 1 is that the concentration of quaternized chitosan in reagent R1 is 0.3%.

[0046] Comparative Example 1

[0047] An oxidized low-density lipoprotein detection kit (immunoturbidimetric method) manufactured by a commercially available company was used as a control.

[0048] Unlike Example 1, reagent R2 is absent, and neither reagents R1 nor R3 contain quaternized chitosan. The specific formulation is as follows:

[0049] Reagent R1: Tris buffer 50 mmol / L, sodium chloride 9 g / L, polyethylene glycol 6000 10 g / L, disodium ethylenediaminetetraacetate 80 mmol / L;

[0050] Reagent R2: 50 mmol / L phosphate buffer, 0.15 g / L latex microsphere-labeled oxidized low-density lipoprotein antibody, and 2 g / L bovine serum albumin.

[0051] Comparative Example 2

[0052] A kit for detecting oxidized low-density lipoprotein, comprising reagents R1, R2, and R3, differs from Example 1 only in that:

[0053] In reagent R1, 500 kDa quaternized chitosan was used instead of 180 kDa quaternized chitosan, and the formulations of the remaining components and other reagents were exactly the same as in Example 1.

[0054] When using the comparative test kit to detect oxidized low-density lipoprotein, the large molecular weight of quaternized chitosan results in a large gel particle volume and high overall gel strength when it reacts with citrate. This leads to high resistance to antigen movement during diffusion and a low antigen-immunoreactivity ratio. In contrast, reagent R3, with the addition of low molecular weight quaternized chitosan, forms a gel with lower mechanical strength, causing the latex microsphere-labeled antibody dispersed within it to move faster relative to the antigen. This results in a significant difference in the antigen-labeled antibody reaction ratio, reduced detection sensitivity, and larger errors in immunoturbidimetry.

[0055] Comparative Example 3

[0056] A kit for detecting oxidized low-density lipoprotein, comprising reagents R1, R2, and R3, differs from Example 1 only in that:

[0057] The concentration of 180 kDa quaternary ammonium salted chitosan in reagent R1 was increased to 1.0%, and the formulations of the remaining components and other reagents were exactly the same as in Example 1.

[0058] In reagent R1 of this comparative example, a higher concentration of quaternized chitosan was used, which had a similar effect to that of comparative example 2. However, due to the excessively high concentration of quaternized chitosan, the viscosity of the system was too high, making it difficult to disperse the gel into microparticles and hindering antigen diffusion. Reagent R3 was also difficult to disperse evenly in the gel, resulting in the inability to detect the sample normally.

[0059] Comparative Example 4

[0060] A kit for detecting oxidized low-density lipoprotein, comprising reagents R1, R2, and R3, differs from Example 1 only in that:

[0061] Reagent R3 uses 80 kDa quaternary ammonium salted chitosan at a concentration of 0.2%, and the formulations of the remaining components and other reagents are exactly the same as in Example 1.

[0062] In this comparative example, reagent R3 increased the molecular weight and concentration of quaternized chitosan, which reduced the difference between its molecular weight and that of quaternized chitosan in reagent R1. This resulted in slow diffusion of the latex microsphere-labeled antibody, ultimately leading to a significant hook effect at low antigen concentrations and a narrower linear detection range.

[0063] The effects of the molecular weight and concentration of quaternized chitosan on the mechanical strength of the gel and the resulting changes in the performance of the test kits prepared in the various embodiments and comparative examples of this invention are shown in Table 1 below.

[0064] Table 1. Differences between the various reagent kits and an overall comparison of their detection effects.

[0065] ;

[0066] Experimental Example 1

[0067] The kits prepared in Example 1 and Comparative Example 1 were used to detect the content of oxidized low-density lipoprotein in the samples. The specific operating principle is described in [link to example]. Figure 1 As shown, the specific operational details are as follows:

[0068] Samples with three concentration gradients (high, medium, and low) were prepared. The content of oxidized low-density lipoprotein in the samples was detected using the kits prepared in Example 1 and Comparative Example 1. First, the sample to be tested was mixed with reagent R1, then reagent R2 was added, mixed well, and incubated for 3 min. After incubation, reagent R3 was added and mixed well. The absorbance value A1 was immediately read at 570 nm. After reacting at 35°C for 8 min, the absorbance value A2 was read again, and the reactivity was calculated. A = A2 - A1, which is converted to the corresponding content of oxidized low-density lipoprotein according to the standard curve; the detection method of Comparative Example 1 is carried out according to the instructions of the kit, and the detection limit, linear range, coefficient of variation (CV) and HOOK effect after 150 U / L are examined.

[0069] 1.1 Sensitivity Evaluation: The ox-LDL reference standard was accurately diluted to four low-concentration samples with concentrations of 3.0 U / L, 5.0 U / L, 7.0 U / L, and 9.0 U / L. The diluted samples and the matrix solution (0.2% BSA + 10 mM Tris-HCl buffer) were measured using the kits from Example 1 and Comparative Example 1, respectively. Each sample was measured 10 times, and the mean was calculated. ), standard deviation (SD), CV, and matrix solution +3SD value and sample measurement results -3SD value; where the sample measurement results are... -3SD is greater than the matrix solution The minimum value of +3SD is the detection limit, and the CV is less than 10%, which meets the usage requirements. The measurement results are shown in Tables 2 and 3.

[0070] Table 2. Detection results of oxidized low-density lipoprotein content using the kit from Example 1.

[0071] ;

[0072] Table 3. Results of Comparative Example 1 kit for detecting oxidized low-density lipoprotein content.

[0073] ;

[0074] The results in Tables 2 and 3 show that when using the kit from Example 1, the concentrations (3.0 U / L to 9.0 U / L) of the test samples were... The -3SD values ​​were all greater than those of the matrix solution. The +3SD value was obtained, and the CV values ​​for different concentrations of the test samples were all less than 10%; while using the kit of Comparative Example 1, although the test samples showed good CV values ​​at concentrations of 5.0 U / L to 7.0 U / L, the CV values ​​were also less than 10%. The -3SD values ​​(1.738 and 3.632) are both greater than those of the matrix solution. The +3SD value was 0.752, but the CV values ​​were all higher than 10%, while the CV value was less than 10% when the sample concentration was 9.0 U / L.

[0075] The above comparison clearly shows that the reagent kit prepared by this invention has higher detection sensitivity than commercially available reagent kits, and can detect samples at a sample concentration of 3.0 U / L.

[0076] 1.2 Determination of linear range: High ox-LDL samples that are close to the upper limit of the linear range (theoretical value 160 U / L) were serially diluted to at least 6 concentrations. Each concentration was tested 3 times. The linear regression equation was obtained and the correlation coefficient was calculated. The results are shown in Table 4.

[0077] Table 4. Results of Linear Range Measurement

[0078] ;

[0079] The results in Table 4 show that the regression equation for the kit provided by this invention is y = 0.9998x, and the correlation coefficient r is... 2 =0.9998, while the correlation coefficient r of the reagent kit in Comparative Example 1 is 0.9998.2 =0.9819, which shows that the reagent kit prepared by the method of the present invention has a stronger linear correlation.

[0080] 1.3 Test method for HOOK effect: High-value serum samples containing the analyte were selected as high-value ox-LDL samples for hospital testing. If necessary, pure analyte could be added, and the theoretical value was calculated. The high-value test samples were diluted with sample diluent, and the theoretical value was calculated after dilution. Then, the test kits of Example 1 and Comparative Example 1 were used to repeat the measurement 3 times, and the relative deviation of the average value from the theoretical concentration was calculated.

[0081] Table 5. Detection results of the HOOK effect of the kit in Example 1

[0082] ;

[0083] Table 6. Detection results of the HOOK effect using the kit in Comparative Example 1

[0084] ;

[0085] As can be seen from Tables 5 and 6, the kit provided by this invention exhibits a linear range with relatively small deviation in the concentration range of oxidized low-density lipoprotein (ODL) of 5–155 U / L. It shows a slow increase in the concentration range of 165–175 U / L and reaches a plateau in the concentration range of 185–190 U / L, which is the HOOK effect point of the kit. In contrast, the kit of Comparative Example 1 shows a linear range in the concentration range of 5–115 U / L, begins to show a slow increase at 125 U / L, and begins to exhibit a HOOK effect at a sample concentration of 165 U / L. The signal weakens as the concentration continues to increase. Therefore, the kit provided by this invention has a wider linear detection range, can increase the concentration at which the HOOK effect occurs, and has higher detection accuracy and sensitivity.

[0086] In summary, the above analysis shows that this invention optimizes the components of the immunoturbidimetric assay kit for oxidized low-density lipoprotein by using quaternized chitosan of different molecular weights to react with citrate to form gels with different mechanical strengths. By controlling the gel strength, the release rate of the antigen and labeled antibody can be adjusted, allowing the labeled antibody and the antigen to remain within the optimal reaction ratio for a longer period during the reaction process, thus generating a stronger turbidity signal. This further broadens the linear detection range of the kit, improves the concentration limit of the hook effect, and greatly enhances the detection sensitivity and accuracy of the kit.

[0087] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. The application of quaternized chitosan in the detection of oxidized low-density lipoprotein, characterized in that, High molecular weight quaternized chitosan, low molecular weight quaternized chitosan, and soluble salts containing citrate were used in the detection of oxidized low-density lipoprotein (LDL). Different molecular weight quaternized chitosan reacts with citrate to form gel particles of varying mechanical strengths. The content of oxidized LDL was then detected by immunoturbidimetry. The high molecular weight quaternized chitosan had a molecular weight of 100-200 kDa, and the low molecular weight quaternized chitosan had a molecular weight of 30-50 kDa.

2. A detection kit for oxidized low-density lipoprotein, characterized in that, Including reagent R1, reagent R2, and reagent R3; The reagent R1 comprises: high molecular weight quaternized chitosan with a mass-volume concentration of 0.03% to 0.6%, KCl with a mass-volume concentration of 0.6% to 0.9%, polyethylene glycol 6000 with a mass-volume concentration of 0.5% to 3%, and a 50 to 100 mM buffer solution, wherein the high molecular weight quaternized chitosan has a molecular weight of 100 to 200 kDa; The reagent R2 is a citrate buffer solution with a mass-volume concentration of 2.0% to 10.0% and a pH of 7.0 to 7.

2. The reagent R3 comprises: 0.02%~0.3% (w / v) low molecular weight quaternized chitosan, 0.01%~0.05% (w / v) latex microsphere-labeled oxidized low-density lipoprotein antibody, 0.1%~0.5% (w / v) ethanolamine, 1%~5% (w / v) trehalose, and 50~100 mM buffer, wherein the molecular weight of the low molecular weight quaternized chitosan is 30~50 kDa; The buffers described in reagents R1 and R3 are each independently selected from any one of the following: HEPES buffer, Tris-HCl buffer, MES buffer, MOPS buffer, TAPS buffer, and glycine buffer.

3. The detection kit for oxidized low-density lipoprotein as described in claim 2, characterized in that, The citrate buffer solution described in reagent R2 is selected from any one or a combination of several of the following: sodium dihydrogen citrate, potassium dihydrogen citrate, disodium hydrogen citrate, dipotassium hydrogen citrate, trisodium citrate, and tripotassium citrate buffer solution, and the pH is adjusted to 7.0-7.2 with hydrochloric acid and sodium hydroxide solution.

4. The detection kit for oxidized low-density lipoprotein as described in claim 2, characterized in that, The reagents R1 and R3 further contain an antibacterial agent with a mass-volume concentration of 0.05% to 0.15%, and the antibacterial agent includes one or more of parabens, isothiazolinones, quaternary ammonium salts, potassium sorbate, and proclin 300.

5. The detection kit for oxidized low-density lipoprotein as described in claim 2, characterized in that, The pH of both reagents R1 and R3 is 7.0 ± 0.

5.

6. A method for detecting oxidized low-density lipoprotein using the kit as described in any one of claims 2 to 5, characterized in that, Mix the sample to be tested with reagent R1, then add reagent R2 and incubate for 2-5 min. After incubation, add reagent R3 and mix well. Immediately read the absorbance value A1 at 570 nm. After reacting at 35-38℃ for 5-8 min, read the absorbance value A2 again and calculate the degree of reactivity. A = A2 - A1, which is converted into the corresponding content of oxidized low-density lipoprotein according to the standard curve.

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