A kit for rapidly and quantitatively detecting neutrophil apolipoprotein and a preparation method thereof

Abstract

This invention provides a rapid quantitative detection kit for neutrophil apolipoproteins and its preparation method, belonging to the field of in vitro detection technology. The kit includes a test card, diluent, and ID card. The test card includes a card shell and a test strip. A sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially overlapped on the PVC base of the test strip. The conjugate pad includes conjugate pad A and conjugate pad B. Conjugate pad A overlaps between the sample pad and the nitrocellulose membrane, while conjugate pad B is located below conjugate pad A and does not contact the sample pad. Different fluorescent microsphere-labeled antibody solutions are sprayed onto conjugate pad A and conjugate pad B, respectively. This invention employs a double-layer conjugate pad structure, controlling the time difference of labeled antibody release, significantly broadening the detection linear range while maintaining good sensitivity, accuracy, and precision. It can be used for the rapid quantitative detection of neutrophil apolipoproteins in human serum.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a rapid quantitative detection kit for neutrophil apolipoproteins and its preparation method. Background Technology

[0002] Human neutrophil lipocalin (HNL) is a major component of secondary granules in human neutrophils, with a molecular weight of approximately 45 kDa. Studies have shown that in healthy individuals, HNL levels in peripheral blood are typically maintained at a low level. During bacterial infection, neutrophils release HNL into the peripheral blood, causing a significant increase in HNL levels. However, this increase is not observed during viral infections. Detecting HNL levels in the serum of patients with acute infectious diseases can help differentiate between acute bacterial and viral infections. Furthermore, a rapid increase in HNL levels is observed in the early stages of infection, with a relatively quick peak. Studies have found that HNL peaks earlier than C-reactive protein and serum procalcitonin, which has significant clinical value in the early diagnosis of bacterial infections. After effective antibacterial treatment, serum HNL levels in patients with bacterial infections gradually decrease. Therefore, HNL plays an important role in the auxiliary diagnosis and efficacy evaluation of bacterial infections.

[0003] Currently, there are few methods for quantitative detection of neutrophil apolipoproteins. Measurement can be performed using radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). Radioimmunoassay has high sensitivity and specificity, but it has drawbacks such as radioactive contamination and short reagent shelf life. The most commonly used method for neutrophil apolipoprotein detection in China is still ELISA, but the enzyme-linked immunosorbent assay (ELISA) involves many steps, resulting in poor reproducibility and a long testing time, as detailed below: The operation is complicated, involving multiple steps such as sample addition, incubation, washing, and color development, which takes a long time (usually 1 to 3 hours), and errors in each step will affect the results. High washing requirements: Incomplete washing will leave unbound enzyme-labeled reagent, leading to increased background values ​​and distorted results. Strict requirements for standardized operation are necessary. Narrow detection range: The linear range of the standard curve is limited. When the sample concentration exceeds the range, it needs to be diluted and retested, which can easily introduce dilution error. Absolute concentration cannot be quantified: it is mostly relative quantification, which requires calibration with standard products. Differences in standard products from different batches of reagents may lead to deviations in results. Susceptible to interference from the sample matrix: Impurities such as proteins and lipids in serum may affect antigen-antibody binding, requiring sample pretreatment (such as dilution and extraction). In recent years, time-resolved fluorescence immunochromatography (TRFIG) has attracted much attention due to its ease of operation, speed, and high sensitivity. However, there are relatively few commercially available neutrophil apolipoprotein kits based on TRFIG. Nevertheless, traditional immunochromatographic test strips based on the "sandwich method" generally suffer from a "hook effect" when analyzing high concentrations of analytes. This means that as the analyte concentration increases, the signal value decreases instead of increasing, leading to false negatives or inaccurate quantification, severely limiting the linear range of detection.

[0004] Therefore, developing a time-resolved fluorescence immunochromatography-based quantitative detection kit for neutrophil apolipoproteins with both high sensitivity and a wide linear range has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid quantitative detection kit for neutrophil apolipoproteins and its preparation method, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A rapid quantitative detection kit for neutrophil apolipoproteins includes a test card, a diluent, and an ID card. The test card includes a card shell and a test strip. The test strip includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped and pasted onto the PVC base plate. The conjugate pad includes conjugate pad A and conjugate pad B. Conjugate pad A overlaps between the sample pad and the nitrocellulose membrane, and conjugate pad B is located below conjugate pad A and does not contact the sample pad. The conjugate pad A is coated with a fluorescent microsphere-labeled antibody solution diluted with diluent C; The conjugate pad B is coated with a fluorescent microsphere-labeled antibody solution diluted with diluent D; The fluorescent microspheres are time-resolved fluorescent microspheres with a particle size of 200~400nm.

[0007] Preferably, the diluent C comprises the following components: 30g BSA, 2g Brij 35, 50g sucrose, 3g Proclin 300, and 1000mL of 20mmol / L Tris buffer at pH 8.0.

[0008] Preferably, the diluent D comprises the following components: 30g BSA, 3g PVP8000, 50g sucrose, 50g trehalose, 3g Proclin300, and 1000mL of 20mmol / L Tris buffer at pH 8.0.

[0009] Preferably, the method for preparing the fluorescent microsphere-labeled antibody solution includes the following steps: A1. Activate the fluorescent microspheres with EDC and NHS in MES buffer; A2. Add neutrophil apolipoprotein antibody to the fluorescent microsphere activation solution, mix thoroughly, and centrifuge to remove the supernatant after reaction; A3. Resuspend the precipitate using a 1% BSA solution, react, centrifuge, wash, and centrifuge to remove the supernatant after washing; A4. Resuspend the precipitate to obtain a fluorescent microsphere-labeled antibody solution; The resuspension consists of the following components: 5g BSA, 2g Tween 20, 50g sucrose, 5g trehalose, 3g Proclin 300, and 1000mL of 50mmol / L Tris buffer at pH 8.0.

[0010] Preferably, in step A2, the mass ratio of the fluorescent microsphere activation solution to the neutrophil apolipoprotein antibody is 10:1.

[0011] Another aspect of the present invention discloses a method for preparing the reagent kit according to any of the above-mentioned technical solutions, comprising the following steps for preparing test strips: S1. Neutrophil apolipoprotein antibody and goat anti-mouse IgG were coated onto the detection line and control line of a nitrocellulose membrane, respectively, and dried at 37°C for 16 hours. S2. Dilute the fluorescent microsphere-labeled antibody solution 15-30 times with diluent C and spray it onto blank conjugate pad 1. Place it at 37°C and dry for 16 hours to prepare conjugate pad A. S3. Dilute the fluorescent microsphere-labeled antibody solution 10-15 times with diluent D and spray it onto blank conjugate pad 2. Place it at 37°C and dry for 16 hours to prepare conjugate pad B. S4. Spray blank sample pads with sample pad treatment solution, place them at 37℃ and dry for 16 hours to make sample pads; S5. The above-treated sample pad, conjugate pad A, conjugate pad B, nitrocellulose membrane and absorbent paper are sequentially attached to the PVC board to assemble the test strip.

[0012] Preferably, in step S1, the detection line coating concentration is 1.2~1.8 mg / mL, and the quality control line coating concentration is 1.5~1.8 mg / mL.

[0013] Preferably, in step S2, the spraying volume of the diluted labeled antibody solution is 4 μL / cm to 10 μL / cm, and the capillary flow rate of the blank binding pad 1 is 16 to 26 s / 4cm.

[0014] Preferably, in step S3, the spraying volume of the diluted labeled antibody solution is 2 μL / cm to 5 μL / cm, and the capillary flow rate of the blank binding pad 2 is 30 to 40 s / 4 cm.

[0015] Preferably, in step S4, the sample pad treatment solution comprises the following components: 10g Tween 80, 1g Proclin 300, and 1000mL of 20mmol / L Tris buffer at pH 8.0.

[0016] The beneficial effects of the above-described technical solution of the present invention are as follows: The HNL kit provided by this invention uses mouse anti-HNL monoclonal antibody and goat anti-mouse IgG polyclonal antibody as coating antibodies for the detection line and control line, and uses time-resolved fluorescent microspheres as markers to label mouse anti-HNL monoclonal antibody. It uses a double antibody sandwich method to quantitatively detect the content of HNL in human serum, and has the advantages of simple operation, fast speed, low price, high detection sensitivity and wide linear range.

[0017] This invention employs a dual-layer binding pad. By adjusting the labeled antibody solution, diluent, and dilution factor, the ratio and release time of the labeled antibody on binding pad A and binding pad B can be controlled. Furthermore, by structurally setting the interval between binding pad B and the sample pad and selecting blank binding pads with different flow rates, the chromatographic time difference between the labeled antibody in binding pad B and the analyte in the sample is effectively increased, thus broadening the linear range of the kit.

[0018] The dual-layer binding pad design of this invention also takes into account the detection sensitivity of the kit. When the concentration of the analyte in the sample is low, the analyte can fully bind to the labeled antibody in binding pad A, and after reaching the detection line, it is captured by the capture antibody to form a fluorescent complex. Compared with the labeled antibody binding to the analyte captured on the detection line during flow chromatography, this invention has higher detection sensitivity for low concentrations of analytes. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the test strip of the reagent kit of the present invention.

[0021] Figure 2 The linear relationship between the measured concentration and the theoretical concentration of HNL was determined using the test strip prepared in Example 1.

[0022] Figure 3To test the linear relationship between the measured concentration and the theoretical concentration of HNL using the test strip prepared in Comparative Example 1.

[0023] Figure 4 To test the linear relationship between the measured concentration and the theoretical concentration of HNL using the test strip prepared in Comparative Example 2.

[0024] Figure 5 To investigate the linear relationship between the measured concentration and the theoretical concentration of HNL using the test strip prepared in Comparative Example 3.

[0025] Figure 6 To investigate the linear relationship between the measured concentration and the theoretical concentration of HNL using the test strips prepared in Comparative Example 4. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0027] For experiments not specified in this protocol, the procedures and conditions described in the literature in this field should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Example

[0028] A rapid quantitative detection kit for neutrophil apolipoproteins includes a test card, a diluent, and an ID card. The test card includes a card shell and a test strip. The test strip includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped and pasted onto the PVC base plate. The conjugate pad includes conjugate pad A and conjugate pad B. Conjugate pad A overlaps between the sample pad and the nitrocellulose membrane, and conjugate pad B is located below conjugate pad A and does not contact the sample pad. The conjugate pad A is sprayed with a fluorescent microsphere-labeled antibody solution diluted with diluent C, which includes the following components: 30g BSA, 2g Brij 35, 50g sucrose, 3g Proclin 300 and 1000mL of 20mmol / L Tris buffer at pH 8.0. The conjugate pad B is coated with a fluorescent microsphere-labeled antibody solution diluted with diluent D, which includes the following components: 30g BSA, 3g PVP8000, 50g sucrose, 50g trehalose, 3g Proclin300 and 1000mL of 20mmol / L Tris buffer at pH 8.0. The fluorescent microspheres are time-resolved fluorescent microspheres with a particle size of 200~400nm.

[0029] The preparation method of fluorescent microsphere-labeled antibody solution includes the following steps: A1. Fluorescent microspheres were added to MES buffer at pH 6.2 and a concentration of 50 mmol / L to bring the concentration of fluorescent microspheres to 5.0%. Then, EDC and NHS were added, with the final mass concentration of EDC being 0.0033% and the final mass concentration of NHS being 0.035%. After incubation at room temperature for 20 min, the supernatant was removed by centrifugation, and the microspheres were resuspended in MES buffer at pH 6.2 and a concentration of 50 mmol / L to bring the concentration of fluorescent microspheres to 3.3%. A2. Add 1 mg of neutrophil apolipoprotein antibody to 10 mg of fluorescent microsphere activation solution, mix thoroughly, and centrifuge to remove the supernatant after reaction; A3. Resuspend the precipitate in 1% BSA solution to restore the original volume, incubate at room temperature for 1 hour, centrifuge, and wash. The washing solution consists of 5g BSA, 0.5g Tween 20, 0.3g Proclin 300, and 1000mL of 50mmol / L Tris buffer at pH 8.0. After washing, centrifuge and discard the supernatant. A4. Resuspend the precipitate to restore it to its original volume to obtain the fluorescent microsphere-labeled antibody solution; The resuspension consists of the following components: 5g BSA, 2g Tween 20, 50g sucrose, 5g trehalose, 3g Proclin 300, and 1000mL of 50mmol / L Tris buffer at pH 8.0.

[0030] Another aspect of the present invention discloses a method for preparing the reagent kit according to any of the above-mentioned technical solutions, comprising the following steps for preparing test strips: S1. Neutrophil apolipoprotein antibody and goat anti-mouse IgG were coated onto the detection line and control line of a nitrocellulose membrane, respectively. The coating concentration for the detection line was 1.2~1.8 mg / mL, and the coating concentration for the control line was 1.5~1.8 mg / mL. The membrane was then dried at 37℃ for 16 h. S2. Dilute the fluorescent microsphere-labeled antibody solution 15-30 times with diluent C and spray it onto blank conjugate pad 1. The spraying amount is 4μL / cm-10μL / cm. The capillary flow rate of blank conjugate pad 1 is 16-26s / 4cm. After drying at 37℃ for 16h, conjugate pad A is prepared. S3. Dilute the fluorescent microsphere-labeled antibody solution 10-15 times with diluent D and spray it onto the blank conjugate pad 2. The spraying amount is 2μL / cm-5μL / cm. The capillary flow rate of the blank conjugate pad 2 is 30-40s / 4cm. After drying at 37℃ for 16h, conjugate pad B is prepared. S4. Spray blank sample pads with sample pad treatment solution, place them at 37°C and dry for 16 hours to prepare sample pads. The sample pad treatment solution includes the following components: 10g Tween 80, 1g Proclin 300 and 1000mL of 20mmol / L Tris buffer at pH 8.0. S5. The above-treated sample pad, conjugate pad A, conjugate pad B, nitrocellulose membrane and absorbent paper are sequentially attached to the PVC board to assemble the test strip.

[0031] The kit of this invention uses a time-resolved fluorescence immunoassay with a double antibody sandwich method to quantitatively detect the HNL content in human serum. When the sample is added to the well of the test card, the HNL in the sample binds to the fluorescent microsphere-labeled HNL monoclonal antibody on the conjugate pad to form a reaction complex. During chromatography, the reaction complex is captured by another monoclonal antibody on the nitrocellulose membrane detection line, while the unreacted labeled antibody is captured by the goat anti-mouse IgG polyclonal antibody on the control line. The amount of HNL captured in the sample is positively correlated with the signal intensity of the fluorescent antibody. The HNL content in the sample is quantitatively detected using a matching fluorescence immunoassay analyzer.

[0032] This invention employs a double-layered conjugate pad. Conjugate pad A overlaps between the sample pad and the nitrocellulose membrane, while conjugate pad B is located below conjugate pad A and does not contact the sample pad. The labeled antibody solutions sprayed onto conjugate pads A and B are different, resulting in a faster chromatography speed for the upper conjugate pad A and a relatively slower chromatography speed for the lower conjugate pad B. Furthermore, during chromatography, because conjugate pad B does not contact the sample pad, a portion of the sample is chromatographically directed horizontally towards the nitrocellulose membrane via conjugate pad A, and another portion is chromatographically directed vertically towards conjugate pad B, and then further towards the nitrocellulose membrane via conjugate pad B. When the HNL content in the sample is low, HNL and... The labeled antibody in conjugate pad A binds to the nitrocellulose membrane and is captured on the detection line, forming a complex that is detected by the instrument as a fluorescent signal, ensuring the detection sensitivity for low concentrations of analytes. When the HNL content in the sample is high, HNL preferentially binds to the labeled antibody in conjugate pad A. When the labeled antibody in conjugate pad A becomes saturated, the free HNL that cannot bind to the labeled antibody precipitates in both horizontal and vertical directions. Horizontal chromatography is faster, allowing some free HNL to be captured when it reaches the detection line. Subsequently, the labeled antibody in conjugate pad B is released and can bind to the free HNL on the detection line, effectively broadening the linear range of the kit.

[0033] The present invention will be further described below by way of examples, but the scope of protection of the present invention is not limited to these embodiments. Example 1

[0034] Prepare the test strips for the kit according to the method described above: In step S1, the detection line coating concentration is 1.5 mg / mL, and the quality control line coating concentration is 1.6 mg / mL; In step S2, the fluorescent microsphere-labeled antibody solution is diluted 20 times with dilution buffer C and sprayed onto the blank binding pad 1 at a spraying volume of 6 μL / cm. Dilution buffer C includes the following components: 30g BSA, 2g Brij 35, 50g sucrose, 3g Proclin300 and 1000mL of 20mmol / L Tris buffer at pH 8.0. In step S3, the fluorescent microsphere-labeled antibody solution is diluted 12-fold with diluent D and sprayed onto blank conjugate pad 2 at a spraying volume of 4 μL / cm. Diluent D includes the following components: 30 g BSA, 3 g PVP8000, 50 g sucrose, 50 g trehalose, 3 g Proclin300, and 1000 mL of 20 mmol / L Tris buffer at pH 8.0. Example 2

[0035] The difference between it and Example 1 is that: In step S1, the detection line coating concentration is 1.2 mg / mL, and the quality control line coating concentration is 1.5 mg / mL; In step S2, the fluorescent microsphere-labeled antibody solution is diluted 15 times and sprayed onto the blank binding pad 1 at a spraying volume of 4 μL / cm. In step S3, the fluorescent microsphere-labeled antibody solution is diluted 15 times and sprayed onto the blank binding pad 2 at a spraying volume of 5 μL / cm. Example 3

[0036] The difference between it and Example 1 is that: In step S1, the detection line coating concentration is 1.8 mg / mL, and the quality control line coating concentration is 1.8 mg / mL; In step S2, the fluorescent microsphere-labeled antibody solution is diluted 30 times and sprayed onto the blank binding pad 1 at a spraying volume of 10 μL / cm. In step S3, the fluorescent microsphere-labeled antibody solution is diluted 10 times and sprayed onto the blank binding pad 2 at a spraying volume of 2 μL / cm. Comparative Example 1

[0037] The difference between this and Example 1 is that a single-layer bonding pad A is used, bonding pad B is not added, and step S3 is completely removed. Comparative Example 2

[0038] Compared with Example 1, the difference is that a single-layer conjugate pad B is used, conjugate pad A is not added, step S2 is removed, and the conjugate pad B from step S3 is overlapped between the sample pad and the nitrocellulose membrane (i.e., the original position of conjugate pad A). Comparative Example 3

[0039] The difference between this and Example 1 is that in step S2, only diluent C is sprayed onto the binding pad A, without containing the labeled antibody solution, and the spraying amount is 6 μL / cm. Comparative Example 4

[0040] The difference between this embodiment and Example 1 is that in step S3, the blank conjugate pad 2 of conjugate pad B is replaced with a blank conjugate pad 1. Comparative Example 5

[0041] The difference between this and Example 1 is that, in the preparation of the fluorescent microsphere labeled antibody solution, 10 mg of fluorescent microsphere activation solution and 2 mg of neutrophil apolipoprotein antibody were used. Comparative Example 6

[0042] The difference between this and Example 1 is that, in the preparation of the fluorescent microsphere labeled antibody solution, 10 mg of fluorescent microsphere activation solution and 0.5 mg of neutrophil apolipoprotein antibody were used. Comparative Example 7

[0043] The difference between this and Example 1 is that the coating concentration of the detection line is 2 mg / mL and the coating concentration of the control line is 2 mg / mL. Comparative Example 8

[0044] The difference between this and Example 1 is that the coating concentration of the detection line is 0.5 mg / mL and the coating concentration of the control line is 0.5 mg / mL.

[0045] The method of using the reagent kit prepared in this application is as follows: (1) Preparation: Before testing, equilibrate the test card, diluent and sample to be tested to 15~30℃, and turn on the matching fluorescence immunoassay analyzer; (2) Card reading: Insert the ID card into the marked position on the matching fluorescence immunoassay analyzer and read the information; (3) Serum sample addition: Take 100 μL of serum sample and add it to a tube of 900 μL sample diluent. Mix thoroughly and add 120 μL of the mixture vertically to the sample addition point on the test card. (4) Detection: Select the sample mode on the analyzer according to the sample type. The standard test or the instant test can be used for detection. Standard test: Insert the test card with the sample added into the card holder of the matching fluorescence immunoassay analyzer, press the test button, and the instrument will automatically count down according to the reaction time of the test. After completion, scan the test card and calculate the result. Immediate testing: After the test card with the sample added reacts at room temperature (15~30℃) for 15 minutes, insert it into the card holder of the matching fluorescence immunoassay analyzer, press the test button, and the test scan and result calculation will be performed immediately. (5) Read / print results: Read / print the test results from the display screen of the matching fluorescence immunoassay analyzer.

[0046] Performance Test 1: Detection Limit Limit of detection test: The test strips prepared in Examples 1-3 and Comparative Example 3 were assembled into test cards and tested on 60 blank matrix samples without HNL. Each sample was tested once to obtain 60 test results. The blank limit (LOB) was calculated by parametric analysis. Five low-concentration samples were set near the estimated LOB and tested. Each sample was tested 5 times. The number of test results below LOB was counted. The lowest concentration with ≤3 results was taken as the limit of detection (LOD). The data are shown in Table 1. Table 1 LOB and LOD data

[0047] Performance Testing II: Accuracy, Inter-batch Precision, and Intra-batch Precision Testing The test strips prepared in Examples 1-3 and Comparative Examples 1-7 were subjected to the following performance tests. Accuracy test: Sample A with a concentration of approximately 800 ng / mL (±20%) and sample B with a concentration of less than 118.46 ng / mL were mixed at a volume ratio of 1:9 to obtain mixed sample C. Samples B and C were tested three times, and the average value was taken. The recovery rate (R) was calculated according to formula (1). The data are shown in Table 2. Formula (1) In the formula: R - Recovery rate; V - Volume of liquid A added; V0 - Volume of sample B; C - The average concentration detected after adding solution A to solution B; The average concentration of the CO-B solution was detected; C S - The concentration of solution A.

[0048] Intra-batch precision test: Using the same batch of kits, test the quality control samples at two concentration levels, low (90 ng / mL) and high (400 ng / mL), 10 times for each level, and calculate the average of the 10 test results. The coefficient of variation (CV) of intra-batch precision was calculated using formula (2) and standard deviation SD. The data are shown in Table 2. Formula (2) Inter-batch precision test: Using kits from three different batches, two concentration levels of quality control samples were tested, one at low (90 ng / mL) and the other at high (400 ng / mL), with each test repeated 10 times. The average value of the 30 tests for each quality control sample was calculated. The coefficient of variation (CV) of the inter-batch precision was calculated using formula (2) and the standard deviation SD. The data are shown in Table 2. Table 2 Recovery rate and coefficient of variation data

[0049] As can be seen from Tables 1 and 2, the detection limit of Comparative Example 3 is significantly higher than that of Examples 1-3. This is because the test strip prepared in Comparative Example 3 does not contain labeled antibodies in conjugate pad A. When detecting low-concentration samples, the free analyte first passes through conjugate pad A and is chromatographically deposited to the detection line. Then, the labeled antibody in conjugate pad B is released and binds to the analyte captured at the detection line during flow chromatography, forming a fluorescent complex. In contrast, in Examples 1-3, the analyte in the sample can bind to the labeled antibody on conjugate pad A and is captured by chromatography to the detection line, resulting in a more complete reaction and thus a lower detection limit.

[0050] Changing the formulations of conjugate pad A and conjugate pad B, the position of conjugate pad B, and the flow rate of the blank conjugate pad has a relatively small impact on LOD, accuracy, and precision. However, the amount of labeled antibody and the coating concentration of the detection line and control line significantly affect LOD, accuracy, and precision. This is because excessively high amounts of labeled antibody or coating concentrations can easily lead to non-specific adsorption, resulting in increased background signal and false positives. Conversely, excessively low amounts of labeled antibody or coating concentrations can lead to decreased capture rate, decreased signal intensity, and a weak signal, which in turn amplifies the relative error of the instrument readings and reduces precision.

[0051] Performance Test 3: Movement Speed ​​Test Group 1: In Example 1, when preparing binding pad A, green dye was added to diluent C, and then the diluted labeled antibody solution was sprayed on. Group 2: In Example 1, when preparing the binding pad B, green dye was added to the diluent D, and then the diluted labeled antibody solution was sprayed on. Group 3: In the preparation of binding pad B in Comparative Example 2, green dye was added to diluent D, and then the diluted labeled antibody solution was sprayed on. Group 4: In the preparation of binding pad B in Comparative Example 4, green dye was added to diluent D, and then the diluted labeled antibody solution was sprayed on. Five test strips were tested in each group. The time was started from the time the sample was added and ended when the green liquid reached the end of the reaction zone. The time taken was the average value t. The average distance from the bottom of the sample to the end of the reaction zone was measured with vernier calipers and recorded as L. The migration speed was calculated by L / t. The data are shown in Table 3. Table 3. Travel Speed ​​Data Table

[0052] As shown in Table 3, by adjusting the formulation of the labeled antibody solution dilution buffer, setting the conjugate pad structure, and selecting the flow rate of the blank conjugate pad, the release of labeled antibody from conjugate pad B can be slowed down, thereby increasing the chromatography time difference between the labeled antibody and the free analyte in the sample, delaying the HOOK effect, and broadening the linear range of the kit.

[0053] Performance Test 4: Linear Range Determination Linear range determination: Clinical serum samples with a concentration of 2000 ng / mL were taken and diluted with low-value serum samples close to the lower limit of the linear range to obtain concentrations of 80 ng / mL, 120 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, 800 ng / mL, 1200 ng / mL, and 1500 ng / mL, respectively. The test strips prepared in Example 1 and Comparative Examples 1-4 were used to test each concentration of the sample three times, and the average value was calculated. The data are summarized in Table 4. Table 4 Average Measured Concentration

[0054] The average measured concentration was fitted to the theoretical concentration using the least squares method to obtain a linear regression equation and a linear correlation coefficient r, as shown below. Figures 2-6 As shown, in Example 1 and Comparative Examples 1-4, r 2 The values ​​were 0.9989, 0.9635, 0.9703, 0.9846 and 0.9684, respectively. Therefore, the linear range of the technical solution in the embodiment is 80~1500ng / mL.

[0055] In summary, this invention, through the design of a dual-layer binding pad structure (binding pad A and binding pad B) and the spraying of different labeled antibody solutions, combined with adjustments to the flow rate of the blank binding pad, successfully achieved effective staggering of the labeled antibody release time, delaying the hook effect of high-concentration analytes and broadening the linear range to 80–1500 ng / mL (correlation coefficient r² = 0.9989). Simultaneously, the kit maintained good detection sensitivity (limit of detection 53 ng / mL) in low-concentration samples, with recoveries between 98.8% and 102.5%, and intra- and inter-assay coefficients of variation both below 6.5%, indicating excellent accuracy and precision. Compared with existing technologies, this invention is simple to operate, rapid in detection (15 min), and free from radioactive contamination, overcoming the shortcomings of ELISA methods such as cumbersome procedures, long processing times, and poor repeatability, and has significant application and promotion value.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A rapid quantitative detection kit for neutrophil apolipoproteins, comprising a test card, diluent, and ID card, wherein the test card comprises a card shell and a test strip, and the test strip comprises a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped and adhered to the PVC base plate, characterized in that, The conjugate pad includes conjugate pad A and conjugate pad B. Conjugate pad A overlaps between the sample pad and the nitrocellulose membrane, and conjugate pad B is located below conjugate pad A and does not contact the sample pad. The conjugate pad A is coated with a fluorescent microsphere-labeled antibody solution diluted with diluent C; The conjugate pad B is coated with a fluorescent microsphere-labeled antibody solution diluted with diluent D; The fluorescent microspheres are time-resolved fluorescent microspheres with a particle size of 200~400nm.

2. The reagent kit according to claim 1, characterized in that, The diluent C comprises the following components: 30g BSA, 2g Brij 35, 50g sucrose, 3g Proclin 300, and 1000mL of 20mmol / L Tris buffer at pH 8.

0.

3. The reagent kit according to claim 1, characterized in that, The diluent D comprises the following components: 30g BSA, 3g PVP8000, 50g sucrose, 50g trehalose, 3g Proclin300, and 1000mL of 20mmol / L Tris buffer at pH 8.

0.

4. The reagent kit according to claim 1, characterized in that, The preparation method of fluorescent microsphere-labeled antibody solution includes the following steps: A1. Activate the fluorescent microspheres with EDC and NHS in MES buffer; A2. Add neutrophil apolipoprotein antibody to the fluorescent microsphere activation solution, mix thoroughly, and centrifuge to remove the supernatant after reaction; A3. Resuspend the precipitate using a 1% BSA solution, react, centrifuge, wash, and centrifuge to remove the supernatant after washing; A4. Resuspend the precipitate to obtain a fluorescent microsphere-labeled antibody solution; The resuspension consists of the following components: 5g BSA, 2g Tween 20, 50g sucrose, 5g trehalose, 3g Proclin 300, and 1000mL of 50mmol / L Tris buffer at pH 8.

0.

5. The reagent kit according to claim 4, characterized in that, In step A2, the mass ratio of the fluorescent microsphere activation solution to the neutrophil apolipoprotein antibody is 10:

1.

6. The method for preparing the reagent kit according to any one of claims 1 to 5, characterized in that, The following steps are included in preparing the test strip: S1. Neutrophil apolipoprotein antibody and goat anti-mouse IgG were coated onto the detection line and control line of a nitrocellulose membrane, respectively, and dried at 37°C for 16 hours. S2. Dilute the fluorescent microsphere-labeled antibody solution 15-30 times with diluent C and spray it onto blank conjugate pad 1. Place it at 37°C and dry for 16 hours to prepare conjugate pad A. S3. Dilute the fluorescent microsphere-labeled antibody solution 10-15 times with diluent D and spray it onto blank conjugate pad 2. Place it at 37°C and dry for 16 hours to prepare conjugate pad B. S4. Spray blank sample pads with sample pad treatment solution, place them at 37℃ and dry for 16 hours to make sample pads; S5. The above-treated sample pad, conjugate pad A, conjugate pad B, nitrocellulose membrane and absorbent paper are sequentially attached to the PVC board to assemble the test strip.

7. The preparation method according to claim 6, characterized in that, In step S1, the detection line coating concentration is 1.2~1.8 mg / mL, and the quality control line coating concentration is 1.5~1.8 mg / mL.

8. The preparation method according to claim 6, characterized in that, In step S2, the spraying volume of the diluted labeled antibody solution is 4 μL / cm to 10 μL / cm, and the capillary flow rate of the blank binding pad 1 is 16 to 26 s / 4cm.

9. The preparation method according to claim 6, characterized in that, In step S3, the spraying volume of the diluted labeled antibody solution is 2 μL / cm to 5 μL / cm, and the capillary flow rate of the blank binding pad 2 is 30 to 40 s / 4cm.

10. The preparation method according to claim 6, characterized in that, In step S4, the sample pad treatment solution includes the following components: 10g Tween 80, 1g Proclin 300, and 1000mL of 20mmol / L Tris buffer at pH 8.0.

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