Serum COVID-19 NAb quantitative detection reagent and preparation and application thereof

Abstract

The invention discloses a serum COVID-19 NAb quantitative detection reagent as well as preparation and application thereof, and belongs to the technical field of biomedical detection. The latex microsphere reagent contained in the quantitative detection reagent is produced by adopting a continuous process, so that complicated purification processes such as centrifugation and washing are avoided, the preparation time is remarkably shortened, and the complexity of the production process and the difference between batches are reduced. Meanwhile, by optimizing a buffer solution system and reaction conditions, the detection sensitivity and the linear range are improved, the linear range is 122.46 ng / mL to 10000 ng / mL, the correlation coefficient R2 is equal to 0.9999, the absolute relative deviation does not exceed + / -1.88%, the detection requirements of neutralizing antibodies (NAb) with different concentrations in clinical samples can be met, and the defect that the linear range of a traditional method is narrow is overcome. The reagent is based on an immunoturbidimetry principle, the detection process can be carried out on a full-automatic biochemical analyzer, the operation is simple and convenient, and the detection is efficient.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a serum COVID-19 NAb quantitative detection reagent and its preparation and application. Background Technology

[0002] Neutralizing Antibodies (NAb) against the novel coronavirus (SARS-CoV-2) are antibodies that neutralize viral activity by blocking the binding of the virus to host cell receptors, thereby inhibiting viral infection.

[0003] Currently, the mainstream methods for detecting neutralizing antibodies against the novel coronavirus COVID-19 include the virus neutralization assay (VNT) and pseudovirus-based neutralization assays. Although the results are accurate and reliable, these methods are cumbersome, time-consuming, and require a biosafety level 3 laboratory, making them difficult to implement in clinical practice. While enzyme-linked immunosorbent assays (ELISA) and chemiluminescence immunoassays have improved detection efficiency, they still suffer from limited sensitivity and narrow linear ranges. Furthermore, the microspheres often require multiple purification steps such as centrifugation and washing during reagent preparation, significantly increasing the complexity of the production process and batch-to-batch variability.

[0004] Immunoturbidimetry is a method for determining antibody levels by detecting the scattering or absorption of light by immune complexes formed from antigen-antibody reactions. Compared to traditional methods, immunoturbidimetry offers advantages such as ease of operation and rapid detection, providing results in a shorter time and making it suitable for large-scale clinical screening. However, current immunoturbidimetric microsphere preparation generally relies on complex centrifugation purification processes, including repeated centrifugation-resuspending steps to remove unreacted conjugated reagents and free antigens. These cumbersome operations not only prolong preparation time but may also lead to microsphere aggregation and loss of activity. For example, the method for preparing latex microparticles for novel coronavirus antibody detection disclosed in Chinese patent document CN112730827A involves multiple complex steps in actual operation, such as using high-speed centrifugation to separate latex microparticles, replacing the original buffer solution to adjust the system, and removing excessively large or aggregated particles through centrifugation. These steps severely restrict the efficiency and application scope of this detection reagent in large-scale industrial production, hindering its rapid promotion and widespread application in clinical testing. For example, the reagent and its preparation method for quantitative determination of SARS-CoV-2 neutralizing antibodies disclosed in Chinese patent document CN114487400A using latex immunoturbidimetry involve relatively complex operations such as precipitation and centrifugation in the preparation process, making the entire process cumbersome and time-consuming. Furthermore, its linear detection range is relatively narrow, with a maximum value of only 1000 ng / mL. Therefore, developing a serum COVID-19 NAb quantitative detection reagent and its preparation method that is easy to operate, highly efficient in preparation, has a wide linear range, and is suitable for large-scale industrial production and rapid clinical application is of significant practical importance.

[0005] Currently, although research has focused on developing continuous synthesis processes to circumvent separation and purification steps, current methods still have significant shortcomings. For example, the microspheres coupled with SARS-CoV-2 RBD disclosed in Chinese patent document CN112394180A cannot generate a sufficiently strong turbidity signal when used alone, making them unsuitable for effective observation using conventional turbidity detection methods. These microspheres must be used in combination with another type of latex microsphere coupled with hACE2. Although this patent document utilizes a continuous method for preparing microspheres, additional microspheres are still required in practical applications to achieve the detection function, further increasing the complexity and cost of reagent preparation.

[0006] Therefore, there is an urgent need to develop a novel microsphere preparation technology that can generate strong turbidity signals and has a wide linear range without centrifugation purification, so as to give full play to the advantages of immunoturbidimetry in rapid detection. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, the present invention aims to provide a serum COVID-19 NAb quantitative detection reagent, its preparation method and application. This reagent can be prepared without centrifugation purification and has a strong turbidity signal and a wide linear detection range.

[0008] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a serum COVID-19 NAb quantitative detection kit, comprising a buffer system, latex microsphere reagents, and calibrators; The buffer system includes a buffer solution, salt, surfactant, and coagulant. The latex microsphere reagent includes solution A, solution B, solution C, solution D, a blocking agent, and sugar; wherein, solution A is a buffer ① containing latex microspheres, solution B is a buffer ① containing NHS, solution C is a buffer ① containing EDC, and solution D is a buffer ② containing RBD recombinant protein. Buffer ① is MES-NaOH buffer, and buffer ② is borate-borax buffer. The preparation process of the latex microsphere reagent includes: Preparation of working microspheres: Take solution A and add solution B and solution C in sequence while stirring. After the addition is complete, react the solution under shaking. After the reaction is complete, obtain working microsphere solution. Antibody crosslinking: Buffer ② is added to the working microsphere solution under stirring, followed by solution D. The mixture is reacted under sealed and oscillating conditions. After the reaction is completed, the mixture is removed to obtain the final mixture. Blocking: Buffer ①, blocking agent and sugar are added sequentially to the mixed solution under stirring. After adjusting the volume, the solution is continued to react under shaking conditions. After the reaction is completed, the latex microsphere reagent is obtained.

[0009] Preferably, in the working microsphere preparation step, before adding solution B and solution C sequentially to solution A while stirring, solution A is first shaken at 37°C.

[0010] Preferably, in the working microsphere preparation step, before adding solution B and solution C sequentially to solution A under stirring, solution A is first shaken at 37°C for 2 minutes.

[0011] Preferably, in the sealing step, after adjusting the volume, the solution is reacted for 1 hour under shaking conditions at 37°C to obtain the latex microsphere reagent.

[0012] Preferably, in the sealing step, after adjusting the volume, the solution is reacted at 37°C with shaking for 1 hour, and then aged at 37°C for 24-48 hours to obtain the latex microsphere reagent.

[0013] Preferably, in solution A, the volume ratio of the latex microspheres is 7.78 ± 0.03% of the total volume of solution A; In solution B, the concentration of NHS is 24.30 ± 0.30 g / L; In solution C, the concentration of EDC is 7.30 ± 0.30 g / L; In solution D, the concentration of recombinant RBD protein is 1.0 ± 0.5 g / L; The blocking agent is a 0.10 mol / L phosphate buffer containing 12.5 g / L glycine and a 0.10 mol / L phosphate buffer containing 200 g / L BSA, and the volume ratio of glycine to BSA is (0.9-1):(0.9-1).

[0014] Preferably, the volume ratio of solution A, solution B, solution C, solution D, buffer ② in the antibody crosslinking step, and buffer ① in the blocking step is (1.4-1.5):(0.23-0.24):(0.23-0.24):(2.3-2.4):(17-17.5):(36-37).

[0015] Preferably, the latex microspheres have a particle size of 100±20nm.

[0016] This invention ensures the surface activation efficiency of latex microspheres, NHS, EDC, and RBD recombinant protein during the preparation of latex microsphere reagents by strictly controlling the dosage and addition order. This guarantees the directional cross-linking and blocking effect of the RBD recombinant protein, avoiding problems such as microsphere aggregation, loss of activity, or poor cross-linking efficiency. Strict control over the dosage and addition order of NHS and EDC avoids the impact of unreacted coupling reagents and free antigen residues on the sensitivity and stability of the detection reagents in traditional immunoturbidimetric methods, ensuring the feasibility of continuous reactions. Furthermore, the present invention also provides a buffer system that can be used in conjunction with the latex microsphere reagent prepared by the above method.

[0017] Preferably, the buffer solution is MES; the salt is NaCl; the surfactant is TWEEN20; and the coagulant is PEG8000.

[0018] Preferably, in the buffer system, the concentration of the buffer solution is 0.10±0.03 mol / L; the concentration of the salt is 0.83±0.30 mol / L; the concentration of the surfactant is 10.00±0.20 ml / L; and the concentration of the coagulant is 4.50±0.70 g / L.

[0019] Preferably, the pH of the buffer system is 6.50 ± 0.20.

[0020] Preferably, the preparation process of the buffer system includes: dissolving the buffer, salt, surfactant and coagulant in a solvent, and adjusting the pH of the solution to 6.50±0.20 using an alkaline solution to obtain the buffer system.

[0021] Secondly, the present invention provides a method for quantitative detection of serum COVID-19 NAb, the principle of which is based on immunoturbidimetry, and includes the following steps: After mixing and incubating the serum sample with the above buffer system, add the above latex microsphere reagent, mix well to form a reaction system, and continue incubation. Monitor the turbidity changes of the reaction system during incubation and obtain the turbidity signal value of the serum sample; The turbidity signal value of the serum sample was substituted into a pre-established standard curve to calculate the COVID-19 NAb concentration in the serum sample.

[0022] Preferably, the volume ratio of the serum sample, buffer system, and latex microsphere reagent is (3-4):240:60.

[0023] The present invention has the following beneficial effects: This invention prepares latex particles that meet subsequent testing standards through a specific material addition sequence and precise dosage ratio. While simplifying the preparation process, it achieves comprehensive optimization of detection performance, providing an efficient, stable, and convenient solution for the quantitative detection of COVID-19 neutralizing antibodies. Specifically: This invention optimizes the preparation method of latex microsphere reagents by employing a continuous synthesis process. In the preparation process of this invention, complex purification steps such as centrifugation and washing are unnecessary; the reaction is completed simply by sequentially adding solution, buffer, blocking agent, and sugar. This method significantly shortens the preparation time, reduces the complexity of the production process and batch-to-batch variability, while avoiding microsphere aggregation and activity loss that may occur during centrifugation.

[0024] The latex microsphere reagent prepared in this invention, combined with the buffer system of this invention, can generate a strong turbidity signal in immunoturbidimetric detection, with a wide linear range of 122.46 ng / mL to 10000 ng / mL and a correlation coefficient R. 2 = 0.9999, with absolute and relative deviations both ≤ ±1.88%, which can meet the detection needs of different concentrations of neutralizing antibodies (NAb) in clinical samples and solve the problem of narrow linear range of traditional methods.

[0025] In addition, this invention is based on the principle of immunoturbidimetry, and the detection process can be completed on a fully automated biochemical analyzer. It is characterized by simple operation and fast detection speed, and is suitable for large-scale clinical screening. Attached Figure Description Figure 1 This is a calibration curve diagram for the present invention; Figure 2 This is a dose-response curve of the HOOK effect of the present invention; Figure 3 To prepare reagent calibration curves for four different buffer solutions in Comparative Example 1, where a is MES, b is HEPES, c is Tris-HCl, and d is KH2PO4. Figure 4 The calibration curves for the effect of different pH values ​​on the detection sensitivity of neutralizing antibodies in Comparative Example 2 are shown, where a is pH 6.50, b is pH 7.25, c is pH 7.70, and d is pH 8.15. Figure 5 Calibration curves for different TWEEN20 concentrations in Comparative Example 3 are shown, where a is 2.50 ml / L, b is 5.00 ml / L, c is 10.00 ml / L, and d is 20.00 ml / L. Figure 6 Calibration curves for different PEG8000 concentrations in Comparative Example 4, where a is 3 g / L, b is 4 g / L, c is 5 g / L, and d is 6 g / L; Figure 7 The calibration curves for different MES buffer concentrations in Comparative Example 5 are shown, where a is 0.025 mol / L, b is 0.050 mol / L, c is 0.075 mol / L, and d is 0.100 mol / L. Figure 8 The calibration curves for different NaCl concentrations in Comparative Example 6 are shown, where a is 0.25 mol / L, b is 0.45 mol / L, c is 0.65 mol / L, and d is 0.85 mol / L. Figure 9 The graph shows the linearity results for different sample reagent ratios in Comparative Example 7, where a represents the sample volume of 3 μL, b represents the sample volume of 4 μL, c represents the sample volume of 5 μL, and d represents the sample volume of 6 μL. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] Example 1 1. Reagent preparation (1) Reagent 1 0.488 g of MES was added to the container to achieve a final concentration of 0.100 mol / L. Then, 0.658 g of NaCl was added to bring the final NaCl concentration to 0.85 mol / L, followed by 0.250 mL of TWEEN20 to achieve a final concentration of 10.00 mL / L. Next, 0.125 g of PEG8000 was added to achieve a final concentration of 5.00 g / L. The pH of the solution was adjusted to 6.50 ± 0.20 using a 20% NaOH solution. Finally, the solution was brought to a final volume of 25.0 mL with deionized water to obtain Reagent 1, the buffer system.

[0029] (2) Reagent 2 Preparation of working microspheres: 1.5282 mL of Buffer ① (pH 6.0±0.10) containing 7.78% latex (100 nm, purchased from JSR Japan) was measured and placed in a constant-temperature shaker set at 37℃ and 225 r / min, and shaken for 2 minutes. After removal, it was placed on a magnetic stirrer, and while stirring, 0.2376 mL of Buffer ① (pH 6.0±0.10) containing 24.50 g / L NHS and 0.2376 mL of Buffer ① (pH 6.0±0.10) containing 7.50 g / L EDC were added sequentially. After the addition was complete, the solution was returned to the shaker and the reaction continued for 15 minutes. After the shaker reaction was completed, the solution was removed to obtain the working microsphere solution.

[0030] Antibody crosslinking: 36.48 mL of Buffer ② (pH 7.90 ± 0.20) was added to the working microsphere solution obtained in the working microsphere preparation step under stirring. Then, 2.376 mL of Buffer ② containing 1.03 g / L RBD recombinant protein (pH 8.0 ± 0.10) was slowly added dropwise. The mixture was transferred to a 60 mL R2 flask, sealed, and placed in a shaker at 225 r / min for 3 hours. After the 3-hour reaction, the mixture was removed and the solution was obtained.

[0031] Blocking: The mixed solution obtained in the antibody crosslinking step was placed on a magnetic stirrer. During stirring, 17.28 mL of Buffer ①, 0.51 mL of blocking agent 1 (0.10 mol / L phosphate buffer containing 12.5 g / L glycine, pH 8.0), 0.51 mL of blocking agent 2 (0.10 mol / L phosphate buffer containing 200 g / L BSA, pH 8.0), and 4.08 g of sucrose were added sequentially. The volume was then adjusted to 60.0 mL. The solution was placed in a shaker at 37°C and 225 r / min and the reaction was continued for 1 hour to obtain reagent 2, i.e., the latex microsphere reagent.

[0032] Buffer ① is a 20 mmol / L MES-NaOH buffer solution with a pH of 6.0±0.05; Buffer ② is a 50 mmol / L borate-borax buffer solution with a pH of 8.0±0.05.

[0033] 2. Evaluation of key performance indicators (1) Limit of detection (LOD), limit of quantitation (LOQ) and calibration curve The testing conditions for the fully automated biochemical analyzer are shown in Table 1.

[0034] Table 1. Testing and determination conditions for a fully automated biochemical analyzer.

[0035] The assay procedure includes: First, add 240 μL of reagent 1 (R1) to the reaction vessel, followed by 3 μL of calibrator or sample. Mix thoroughly and incubate the reaction vessel at 37°C for 3 minutes. Then, add 60 μL of reagent 2 (R2) to the reaction vessel and mix thoroughly again. After mixing, incubate at 37°C for 0.5 minutes. At this time, use a suitable detection device to read the absorbance value of each tube relative to the blank tube, and record it as A1. Then, allow the reaction to continue for 4.5 minutes, and read the absorbance value of each tube relative to the blank tube again, and record it as A2. Finally, calculate ΔA = A2 - A1.

[0036] Using the above method, the limit of detection (LOD) and the limit of quantitation (LOQ) were estimated by repeatedly testing the blank solution 10 times and obtaining the blank value b and standard deviation s. The calculation formulas are: LOD = b + 3s, LOQ = b + 10s. Table 2 shows the results of the blank solution determination.

[0037] Table 2 Results of blank solution determination

[0038] The blank solution was tested 10 times to obtain the mean blank value (b) = 2.205 ng / mL and the standard deviation (s) = 12.02544 ng / mL. Based on this, the limit of detection (LOD = b + 3s) was calculated to be 38.28 ng / mL and the limit of quantitation (LOQ = b + 10s) was calculated to be 122.46 ng / mL.

[0039] To construct a calibration curve, high-concentration mixed serum (H) was taken and diluted with deionized water at ratios of 1, 1 / 2, 1 / 4, and 1 / 8 to prepare gradient samples with expected concentrations of 10000, 5000, 2500, and 1250 ng / mL (with a blank control set up). Each sample was measured three times in ascending order of concentration. Data were checked according to the CLSI EP6-A document (CLSI. Evaluation of the linearity of quantitative measurement procedures: A Statistical Approach; Approved Guideline [S]. CLSI EP6-A. Wayne, PA: CLSI, 2003.). A calibration curve was plotted with the mean measured concentration as Y and the expected concentration as X. The linear equation and correlation coefficient were calculated. The linearity test results are detailed in Table 3 and [Table data would be inserted here]. Figure 1 . Table 3 Linearity Test Results

[0040] From Table 3 and Figure 1 As can be seen, the calibration curve exhibits good linearity. The mean measured concentrations at each dilution factor are close to the expected concentrations, with both the absolute and relative linear deviations within acceptable ranges. Specifically, at a dilution factor of 1, the absolute linear deviation is only -0.01 ng / mL, and the relative linear deviation is 0%, almost perfectly consistent with the expected concentration; at a dilution factor of 1 / 2, the relative linear deviation is -1.27%; at a dilution factor of 1 / 4, the relative linear deviation is 0.12%; and at a dilution factor of 1 / 8, the relative linear deviation is -1.88%. These data indicate that within the concentration range set in this experiment, the detection method used in this embodiment can accurately reflect the actual concentration of the sample and exhibits good linearity.

[0041] The calibration curve results showed that the linearity was good in the range of 122.46 ng / mL to 10000 ng / mL (R²=0.9999), and the absolute and relative deviations were all ≤±1.88%. Compared with enzyme-linked immunoturbidimetric assay, the linear range was wider, which met the requirements for method validation.

[0042] Five-point concentration calibration is performed by sequentially inputting the concentration data from each point into the instrument and using a nonlinear spline fitting method to conduct multi-point calibration. During the calibration process, the instrument automatically generates a dose-response curve (i.e., a calibration curve), from which the sample concentration value can be calculated. (2) Repeatability evaluation Following the protocol outlined in the Clinical and Laboratory Standards Institute (CLSI) EP15-A2 document (CLSI. User demonstration of performance for precision and accuracy, Approved Guideline. CLSI document EP15-A. Wayne, PA: CLSI, 2001.), reagents 1 and 2 of this embodiment were used to measure three mixed serum samples of low, medium, and high levels of precision. Twenty consecutive measurements were performed within one day to calculate intra-assay imprecision (CV); measurements were then performed once daily for 12 consecutive days to calculate inter-assay imprecision (CV). The repeatability evaluation (imprecision) results of Example 1 are shown in Tables 4 and 5, where Table 4 shows the intra-batch imprecision results and Table 5 shows the inter-batch imprecision results.

[0043] Table 4. Intra-batch imprecision results

[0044] Table 5. Results of inter-batch imprecision

[0045] The results in Tables 4 and 5 show that the intra-batch imprecision coefficient of variation (CV) (each sample was measured in parallel 20 times using this method): the CV values ​​for low, medium, and high concentration samples were 1.04%, 1.61%, and 2.15%, respectively, all of which meet the requirements of the Clinical and Laboratory Standards Institute (CLSI) EP15-A document regarding CV ≤ 5% for clinical testing methods; the inter-batch imprecision CV (each sample was measured continuously for 12 days using this method): the CV values ​​for low, medium, and high concentration samples were 8.74%, 5.11%, and 4.76%, respectively, which shows that the method has good stability under different time and operating conditions.

[0046] (3) Recovery rate verification In a mixed serum sample (sample background) with a measured COVID-19 neutralizing antibody (NAb) concentration of 1010 ng / mL, low, medium, and high concentrations of NAb standard solutions were added to prepare a standard mixed serum working solution. Separately, mixed serum samples and calibrators were taken, and deionized water was added to the same volume as the standard mixed serum working solution. The recovery rate was then tested. The recovery rate verification results of this embodiment are shown in Table 6.

[0047] Recovery rate = [(measured concentration – sample background concentration) / concentration of added NAb standard solution] × 100%.

[0048] Table 6. Recovery rate verification results

[0049] Table 6 shows that the average recovery rate was 108.90%, indicating that the NAb detection method has high accuracy. A recovery rate exceeding 100% means that under the experimental conditions, the amount of antigen added was likely within an appropriate range, resulting in a relatively sufficient antigen-antibody reaction. There may have been a slight antibody overdose, leading to a slightly higher recovery rate. (4) Interference experiment A mixed serum sample was selected, and different concentrations of vitamin C, conjugated bilirubin, hemoglobin, and fat emulsion, among other major interfering substances, were added. Using an equal volume of deionized water as a baseline control, the NAb concentration in each interfering serum sample was measured using the reagents described in this method. The measurements were repeated three times, and the average value was taken to calculate the percentage difference (Diff%). The interference experiment results of this embodiment are shown in Table 7.

[0050] Diff% = [(Interference serum test value − baseline control test value) / baseline control test value] × 100%.

[0051] Table 7 Results of the interference experiment

[0052] In the detection of NAb concentrations at 5250 ng / mL in serum, the maximum permissible concentrations of the main interfering substances are as follows: hemoglobin ≤ 650 mg / dL, vitamin C ≤ 80 mg / dL, conjugated bilirubin ≤ 35 mg / dL, and fat emulsion ≤ 550 mg / dL. This result indicates that the interference resistance of this detection method meets clinical requirements.

[0053] When the interfering substance is at the maximum permissible concentration, the Diff% tends to be close to the ±10% critical value (e.g., the Diff% for bound bilirubin is -9.35%). This phenomenon can be interpreted as follows: (1) the effectiveness of the anti-interference design: the stabilizer in the reagent (such as BSA) may reduce the impact of the interfering substance on the detection results by competitively binding with it; (2) the risk of critical value: in clinical samples, when multiple interfering substances are present at the same time, their cumulative effect may cause the detection deviation to exceed the specified limit.

[0054] (5) Validation of the HOOK effect Antigen samples with a concentration 10 times the upper limit of the linear range of the detection method were prepared. These high-concentration samples were then diluted geometrically (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, and 1 / 512) to cover the range from low to ultra-high concentrations. The absorbance of each sample was measured using an instrument, with each group of samples tested three times, and the average value was calculated. A dose-response curve was plotted with antigen concentration on the x-axis and absorbance value on the y-axis to verify the existence of a hook effect. The results of the HOOK effect verification in Example 1 are shown in Table 8, and the dose-response curves are shown in the figure. Figure 2 As shown.

[0055] Table 8 Validation results of the HOOK effect

[0056] Experimental data show that the absorbance (OD value) of the undiluted sample (10000 ng / mL) is lower than that of the 2-fold diluted sample (50000 ng / mL), a phenomenon consistent with the typical characteristics of the HOOK effect. The mechanism is as follows: when the antigen concentration is too high, the antibody binding sites are completely occupied, leading to the formation of small molecule soluble immune complexes rather than large molecule cross-linked aggregates, ultimately resulting in a decrease in absorbance. The detection signal reaches its peak after a 2-fold dilution, indicating that this concentration is the optimal antibody-antigen ratio. Therefore, within the linear range of this detection method, for individual high-concentration samples, pre-dilution is necessary before detection.

[0057] (6) Accelerated stability test To determine the shelf life of the reagents in a short time, reagents 1 and 2 were subjected to accelerated stability testing in a 37°C water bath. The accelerated test duration was set at 12 days. On days 0, 7, and 12 of the test, the high-value control sample (H) and the low-value control sample (L) of NAb were measured, with each sample measured three times. Calibration was performed on day 0, and the result on day 0 was set as the target value. The relative deviation between the measured value and the target value was determined by calculating the mean. The accelerated stability test results of Example 1 are shown in Table 9.

[0058] Table 9 Accelerated Stability Test Results

[0059] The relative deviation of the mean values ​​measured over 12 days for the high-value quality control (H) was -4.75%; the relative deviation of the mean values ​​measured over 12 days for the low-value quality control (L) was 1.24%. These results indicate that the stability of the reagents used in this method, compared to the values ​​measured on day 0, does not deviate by more than ±5%. Based on the Arrhenius accelerated stability equation, in this accelerated stability study: Predicted stability = Accelerated stability × 2 △T / 5 Where △T is the difference between the accelerated stability storage temperature (37℃) and the upper limit of the storage temperature (8℃).

[0060] In this invention, a sample that is stable for 12 days at 37°C will be stable for 21 months (12 × 25.8 = 668 days ≈ 21 months at 4~8°C).

[0061] (7) Opening stability test The freshly prepared NAb assay reagent was stored in a refrigerator at 2–8°C with the cap open for 30 days. The concentrations of high- and low-value NAb control samples were measured, with each sample measured three times. The mean value was calculated to determine the reagent stability. The results of the open-cap stability test in Example 1 are shown in Table 10.

[0062] Table 10 Results of Lid-Opening Stability Test

[0063] When the NAb test reagent is stored at 2–8°C after opening, the relative deviation of the measured values ​​of the quality control sample (maximum value -8.60%) within 28 days meets the preset standard (±10%), indicating that the reagent has good stability after opening. It is recommended that the test be completed within 28 days of opening the reagent in clinical applications to ensure the accuracy of the results.

[0064] (8) Selection of sample dilution factor The dilution factor of the samples was adjusted as follows: one mixed serum sample was diluted with deionized water at 1 (original solution), 1 / 2 (2-fold dilution), 1 / 4 (4-fold dilution), 1 / 8 (8-fold dilution), 1 / 16 (16-fold dilution), and 1 / 32 (32-fold dilution). Each diluted sample was measured 3 times, and the mean value was calculated by multiplying the result by the dilution factor, and the result was compared with the original serum sample. The linearity results of samples at different dilution factors are shown in Table 11.

[0065] Table 11 Linearity Measurement Results for Samples at Different Dilution Factors

[0066] The above data shows that within the tested dilution range, as the dilution factor increases, the deviation between the diluted concentration and the original concentration ranges from -6.31% to +8.55%, which meets the ±10% deviation limit specified in the CLSI EP6-A guideline. Specifically, the deviation at 8-fold dilution is 8.55%, close to the critical value; while at 32-fold dilution, the deviation value decreases. This may be because the calibration curve of latex immunoturbidimetric assay is not perfectly linear, especially in the low and high concentration regions where nonlinear changes are prone to occur. For example, at 1-fold and 2-fold dilutions, the sample concentration is in the higher concentration region of the reagent's linear range, and the measurement results are more accurate with a smaller relative deviation. However, at 8-fold dilution, the sample concentration may be at the edge of the reagent's linear range or slightly beyond it, at which point the reagent's response to concentration changes is not linear enough, leading to a larger deviation in the detection results. As for 32-fold dilution, although the sample concentration is lower, it may be below the reagent's detection limit. At this point, the detection results are mainly affected by the reagent blank signal, and the relative deviation may actually decrease.

[0067] In summary, within the 1-32 fold dilution range, the deviation between the diluted concentration and the original concentration does not exceed ±10%, indicating that the optimized NAb detection method is suitable for 1 to 32 fold dilution of clinical samples and can meet routine testing needs.

[0068] (9) Selection of sample storage period at 2~8℃ Nine serum samples, ranging from high to low concentrations, were collected from individuals of all genders. The NAb concentrations of the samples were measured at 0, 1, 3, 5, and 7 days at 2–8°C according to the established storage conditions. The relative deviations of each group's measured values ​​from the 0-day measured value were calculated (the mean value of the 0-day measured value was the target value, and the linear absolute deviation = mean value of a certain day - target value). The results are shown in Table 12.

[0069] Table 12 Sample Storage Measurement Results

[0070] Storing at 2-8℃ can maintain protein stability by inhibiting enzyme activity and slowing down oxidation. However, prolonged refrigeration may still induce slow degradation, especially in low-concentration samples (e.g., samples 2 and 8), where the deviation is slightly higher (Table 12). The above experimental results show that the relative deviation of samples stored at 2-8℃ for up to 7 days compared to the results stored for 0 days does not exceed ±5.65%, which meets the judgment criteria. This means that within this storage period, the test results still have reference value. Comparative Example 1 This comparative example included four experimental groups, one of which consisted of reagents 1 and 2 from Example 1. The preparation methods for reagents 1 and 2 in each experimental group were the same as in Example 1, except that the MES in reagent 1 was replaced with 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and potassium dihydrogen phosphate (KH2PO4), respectively. The aim was to compare the effects of four buffer solutions (MES, HEPES, Tris-HCl, and KH2PO4) on the detection sensitivity of neutralizing antibodies (NAb), and to evaluate the antibody-antigen binding efficiency under different buffer systems. The measurement results are shown below. Figure 3 As shown in Table 13, Figure 3 In the diagram, a represents MES, b represents HEPES, c represents Tris-HCl, and d represents KH2PO4.

[0071] Table 13. Results of determination for different buffer solutions

[0072] As shown in Table 13, the absorbance values ​​of the MES, HEPES, and KH2PO4 buffer systems all exhibited good curve fitting with NAb concentrations. However, the reaction sensitivity of the HEPES and KH2PO4 buffer groups decreased, and the absorbance changes in the calibration curves were relatively small. The dispersion stability of latex particles is highly dependent on the ionic strength of the buffer. The ionic strength (0.10 mol / L) of the MES buffer (pH 6.50±0.20) matches the pKa (~6.5) of the carboxyl groups on the latex particle surface, giving the particle surface an appropriate amount of negative charge, which maintains dispersion stability through electrostatic repulsion. Therefore, the 0.10 mol / L MES buffer (pH=6.50±0.20) was selected as the optimal buffer system.

[0073] Comparative Example 2 This comparative example included four experimental groups. One group consisted of reagents 1 and 2 from Example 1 (pH = 6.5 ± 0.20). The preparation methods for reagents 1 and 2 in the other three groups were the same as in Example 1, except that the pH value of reagent 1 was adjusted to 7.25, 7.70, and 8.15, respectively. This was to investigate the effect of different pH values ​​on the detection sensitivity of neutralizing antibodies (NAb) and to evaluate the effect of pH on latex particle dispersibility by analyzing the changes in absorbance values ​​of calibration curves under different pH conditions. The experimental results are as follows: Figure 4 As shown in Table 14, Figure 4 In the table, a represents pH 6.50, b represents pH 7.25, c represents pH 7.70, and d represents pH 8.15.

[0074] Table 14 Results of measurements at different pH values

[0075] Experimental data showed that the pH 6.50 group in Example 1 exhibited high sensitivity even at a low concentration (1250.00 ng / mL) (ΔA=0.0192), followed by the pH 7.70 group. At pH 8.15, the antibody carried a negative charge, increasing electrostatic repulsion with the antigen and leading to a decrease in binding efficiency (ΔA=0.0185). Compared to the three groups in Comparative Example 2, the MES buffer system at pH 6.50 in Example 1 performed best, with its absorbance value showing a significant gradient change with increasing NAb concentration (ΔA=0.1697 at 10000.00 ng / mL).

[0076] Comparative Example 3 This comparative example included four experimental groups. One group used reagents 1 and 2 from Example 1. The preparation methods for reagents 1 and 2 in the other three groups were the same as in Example 1, except that the concentration of TWEEN20 in reagent 1 was adjusted to 2.50 ml / L, 5.00 ml / L, and 20.00 ml / L, respectively. The experimental results are as follows: Figure 5 As shown in Table 15, Figure 5 In the above, a is 2.50 ml / L, b is 5.00 ml / L, c is 10.00 ml / L, and d is 20.00 ml / L.

[0077] Table 15. Results of determination of different TWEEN20 concentrations

[0078] In Example 1, the absorbance value of 10.0 ml / L TWEEN20 showed a significant gradient change with increasing NAb concentration (ΔA = 0.1990 at 10000.00 ng / mL). Higher concentrations of TWEEN20 (10.0 ml / L) effectively reduced nonspecific binding, decreased background signal, and improved the dispersibility of latex particles. As a surfactant, TWEEN20 can reduce nonspecific adsorption and improve the dispersibility of latex particles.

[0079] According to Figure 5 The data in Table 15 show that the signal value of the 5.00 ml / L TWEEN20 group decreased (ΔA=0.1962 at 10000.00 ng / mL), indicating that a lower concentration of TWEEN20 is insufficient to effectively reduce nonspecific binding. Conversely, the abnormally high signal value of the calibration curve in the 2.50 ml / L TWEEN20 group indicates that excessively low concentrations of TWEEN20 can lead to nonspecific adsorption and artificially inflated signal values. While the signal value of the 20.00 ml / L TWEEN20 group was relatively close to that of the 10.0 ml / L TWEEN20 group (ΔA=0.1968 at 10000.00 ng / mL), the overall fluctuation of the calibration curve data showed some differences compared to the 10.0 ml / L group.

[0080] In summary, the experimental results show that a TWEEN20 concentration of 10.0 ml / L can achieve a balance between reducing the measurement background and maintaining specificity.

[0081] Comparative Example 4 This comparative example included four experimental groups. One group used reagents 1 and 2 from Example 1. The preparation methods for reagents 1 and 2 in the other three groups were the same as in Example 1, except that the concentration of PEG8000 in reagent 1 was adjusted to 3 g / L, 4 g / L, and 6 g / L, respectively. The experimental results are as follows: Figure 6 As shown in Table 16. Figure 6 In the above, a is 3 g / L, b is 4 g / L, c is 5 g / L, and d is 6 g / L.

[0082] Table 16. Determination results of different PEG8000 concentrations

[0083] As a coagulant, the concentration of PEG8000 has a relatively small impact on the slope of the calibration curve and the background signal. The experimental results above show that increasing the concentration of PEG8000 within a certain range (4-5 g / L) can improve detection sensitivity, but excessively high concentrations (6 g / L) may lead to increased background signal and decreased specificity. Therefore, 5 g / L was selected as the optimal PEG8000 concentration after optimization.

[0084] Comparative Example 5 This comparative example included four experimental groups. One group used reagents 1 and 2 from Example 1. The preparation methods for reagents 1 and 2 in the other three groups were the same as in Example 1, except that the concentration of the MES buffer in reagent 1 was adjusted to 0.025 mol / L, 0.050 mol / L, and 0.075 mol / L, respectively. The experimental results are as follows: Figure 7 As shown in Table 17. Figure 7 In the equation, a is 0.025 mol / L, b is 0.050 mol / L, c is 0.075 mol / L, and d is 0.100 mol / L.

[0085] Table 17 Results of concentration determination for different MES buffer systems

[0086] Depend on Figure 7 As shown in Table 17, higher concentrations of buffer solution can better maintain the pH stability of the reaction system, reduce the interference of pH fluctuations on the reaction, and thus improve detection sensitivity. Experimental results indicate that the absorbance value of the 0.100 mol / L MES buffer system exhibits a significant gradient change with increasing NAb concentration (ΔA = 0.1967 at 10000.00 ng / mL); while low concentration buffer solution (0.025 mol / L) may lead to an increase in reagent blank signal, higher background turbidity detected by the instrument, and an artificially high overall signal (ΔA = 0.2013 at 10000.00 ng / mL).

[0087] Comparative Example 6 This comparative example included four experimental groups. One group used reagents 1 and 2 from Example 1. The preparation methods for reagents 1 and 2 in the other three groups were the same as in Example 1, except that the NaCl concentration of reagent 1 was adjusted to 0.25 mol / L, 0.45 mol / L, and 0.65 mol / L, respectively. The experimental results are as follows: Figure 8 As shown in Table 18. Figure 8 In the given values, a is 0.25 mol / L, b is 0.45 mol / L, c is 0.65 mol / L, and d is 0.85 mol / L.

[0088] Table 18. Determination results of different NaCl concentrations

[0089] NaCl concentration significantly affects the stability of antigen-antibody binding. In the 0.85 mol / L experimental group, the calibration curve showed the best fit across the entire concentration range (ΔA = 0.2033). In contrast, lower NaCl concentrations (0.25 mol / L, 0.45 mol / L, and 0.65 mol / L) caused signal attenuation in the calibration curve at high concentrations (10000.00 ng / mL). This phenomenon may be attributed to the negative impact of excessively high ionic strength on antigen-antibody binding efficiency.

[0090] Comparative Example 7 This comparative example consists of four experimental groups. The preparation methods for reagents 1 and 2 in these four experimental groups are the same as in Example 1, except that the amount of sample added is adjusted during the sample testing. Specifically, a high-value sample with a NAb concentration of 10000 ng / mL is taken and serially diluted with deionized water to obtain five gradient concentrations: linear 1 (0 ng / mL), linear 2 (1250 ng / mL), linear 3 (2500 ng / mL), linear 4 (5000 ng / mL), and linear 5 (10000 ng / mL). The volume of reagent 1 (R1) is fixed at 240 μL and the volume of reagent 2 (R2) is fixed at 60 μL. Only the sample addition volume (groups 1 to 4) is changed to test the five gradient concentrations.

[0091] The linearity test results for different sample reagent ratios are as follows: Figure 9 As shown in Table 19. Figure 9 In the figures, a represents a sample volume of 3 μL, b represents a sample volume of 4 μL, c represents a sample volume of 5 μL, and d represents a sample volume of 6 μL.

[0092] Table 19 Results of linearity measurements for different sample reagent ratios

[0093] The results show that the curve fitting effect is better when the sample loading volume is 3 μL and 4 μL. The small sample volume (3 μL) reduces the introduction of interfering substances (such as hemoglobin and bilirubin) in the serum matrix. Simultaneously, the high-sensitivity reagent design (experimentally optimized PEG8000 5 g / L) compensates for signal loss, achieving accurate detection at low concentrations (1250.00 ng / mL) (99% recovery). A sample-to-reagent ratio of 1:100 results in a total reaction volume of 303 μL, which appropriately dilutes the NAb antigen, avoiding the "prozone phenomenon" caused by antigen overdose. In the 5-6 μL group, the increased sample ratio (1:60 to 1:50) leads to a higher final antigen concentration, approaching the antibody binding site saturation threshold and triggering a nonlinear response. Therefore, 3 μL is selected as the optimal loading volume because it exhibits optimal linearity (0.9999) and a 100.0% recovery rate at high concentrations (5000 ng / mL), meeting clinical testing requirements (90%-110% recovery).

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A serum COVID-19 NAb quantitative detection kit, characterized in that, The buffer system, the latex microsphere reagent and the calibrant are included. The buffer system includes a buffer, a salt, a surfactant and a coagulant. The latex microsphere reagent includes solution A, solution B, solution C, solution D, a blocking agent and sugar; wherein, solution A is Buffer ① containing latex microspheres, solution B is Buffer ① containing NHS, solution C is Buffer ① containing EDC, solution D is Buffer ② containing RBD recombinant protein, the Buffer ① is MES-NaOH buffer, and the Buffer ② is borate-borax buffer. The preparation process of the latex microsphere reagent includes: Working microsphere preparation: solution B and solution C are sequentially added to solution A under stirring, after the addition is completed, the solution is reacted under oscillation, and the working microsphere solution is obtained after the reaction is completed; Antibody cross-linking: the working microsphere solution is added to Buffer ② under stirring, then solution D is added to the solution, and the mixed solution is reacted under sealed and oscillation conditions, and the mixed solution is obtained after the reaction is completed; Blocking: the mixed solution is sequentially added with Buffer ①, a blocking agent and sugar under stirring, and the solution is continuously reacted under oscillation after being constant volume, and the latex microsphere reagent is obtained after the reaction is completed.

2. The serum COVID-19 NAb quantitative detection kit according to claim 1, characterized in that, In the solution A, the volume ratio of the latex microspheres accounts for 7.78±0.03% of the total volume of the solution A; In the solution B, the concentration of NHS is 24.30±0.30 g / L; In the solution C, the concentration of EDC is 7.30±0.30 g / L; In the solution D, the concentration of RBD recombinant protein is 1.0±0.5 g / L; The blocking agent is 0.10 mol / L phosphate buffer containing 12.5 g / L glycine and 0.10 mol / L phosphate buffer containing 200 g / L BSA, and the volume ratio of the glycine and BSA is (0.9-1):(0.9-1).

3. The serum COVID-19 NAb quantitative detection kit according to claim 1, characterized in that, The volume ratio of the solution A, the solution B, the solution C, the solution D, Buffer ② in the antibody cross-linking step and Buffer ① in the blocking step is (1.4-1.5):(0.23-0.24):(0.23-0.24):(2.3-2.4):(17-17.5):(36-37).

4. The serum COVID-19 NAb quantitative detection kit of claim 1, wherein, The particle size of the latex microspheres is 100±20 nm.

5. The serum COVID-19 NAb quantitative detection kit according to claim 1, characterized in that, The buffer is MES; the salt is NaCl; the surfactant is TWEEN20; and the coagulant is PEG8000.

6. The serum COVID-19 NAb quantitative detection kit of claim 1, wherein, In the buffer system, the concentration of the buffer is 0.10±0.03 mol / L; the concentration of the salt is 0.83±0.30 mol / L; the concentration of the surfactant is 10.00±0.20 ml / L; and the concentration of the coagulant is 4.50±0.70 g / L.

7. The serum COVID-19 NAb quantitative detection kit according to claim 1, characterized in that, The pH of the buffer system is 6.50±0.

20.

8. A method for quantitatively detecting serum COVID-19 NAb, characterized in that, The principle of the serum COVID-19 NAb quantitative detection method is based on the immunoturbidimetry, and the method includes the following steps: After the serum sample is mixed with the buffer system as claimed in claim 1 and incubated, the latex microsphere reagent as claimed in claim 1 is added and mixed to form a reaction system and continue to incubate; The change in turbidity of the reaction system during incubation is monitored to obtain the turbidity signal value of the serum sample; The turbidity signal value of the serum sample is substituted into the pre-established standard curve to calculate the concentration of COVID-19 NAb in the serum sample.

9. The method of claim 8, wherein the serum COVID-19 NAb quantitation assay is characterized by, The volume ratio of the serum sample, buffer system and latex microsphere reagent is (3-4): 240: 60.

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