Kit and method for determining S100A8 / 9 based on latex immunoturbidimetry

Abstract

The embodiment of the present invention discloses a kit and method for determining S100A8 / 9 based on latex immunoturbidimetry. The kit for determining S100A8 / 9 based on latex immunoturbidimetry provided by the present invention can effectively improve the sensitivity and specificity of detecting S100A8 / 9, has better repeatability, strong anti-interference ability, and good reagent stability, realizes automatic detection on a biochemical analyzer, is simple to operate, and is convenient and fast to detect, which is convenient for clinical promotion. In addition, the kit provided by the present invention is also more convenient for detecting lung cancer and fibrosis progression, and is conducive to the detection of tumor markers and prognosis markers.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of bioanalysis technology, and specifically to a kit and method for determining S100A8 / 9 based on latex immunoturbidimetry. Background Art

[0002] S100A8 (MRP8) and S100A9 (MRP14) are calcium ion binding proteins that belong to the S100 family of binding proteins. Members of this family contain a common structure consisting of two "EF-hand" motifs, which themselves consist of two α chains separated by a peptide loop, in which the ion Ca 2+ Can bind. Between the two "EF-hand" motifs, there is a hinge region variable between S100 proteins, which usually gives different S100 proteins biological activity and specificity. They usually exist in the form of heterodimers, while homodimers rarely exist due to stability. They are constitutively expressed in the cytoplasm of myeloid cells, including myeloid precursor cells, multinuclear neutrophils (PNN) and monocytes, but not in lymphocytes. They appear in the promyelocyte stage and reach their maximum in mature granulocytes, accounting for 40% of the total cytoplasmic protein. Under physiological conditions, they disappear when monocytes differentiate into macrophages. S100A8 / A9 is constitutively expressed as Ca in neutrophils and monocytes. 2+ S100A8 / A9 is a sensor involved in cytoskeletal rearrangement and arachidonic acid metabolism. During inflammation, S100A8 / A9 is actively released and plays a key role in regulating the inflammatory response by stimulating leukocyte recruitment and inducing cytokine secretion. S100A8 / A9 is a candidate biomarker for diagnosis and follow-up, as well as a predictor of therapeutic response in inflammatory diseases. Since blocking S100A8 / A9 activity with small molecule inhibitors or antibodies ameliorates pathological conditions in mouse models, the heterodimer has potential as a therapeutic target. S100A8 / A9 has been found to play an important role in many diseases, such as inflammation and cancer, and can even be used as a typical or atypical marker for diagnosis or prediction of disease progression. These findings have prompted researchers to explore whether S100A8 / A9 can be used as a biomarker or therapeutic target for diseases beyond inflammation and cancer. It could serve as a powerful amplifier of inflammation in autoimmunity, as well as in cancer development and tumor spread. The iNOS-S100A8 / A9 transnitrosylase complex directs the selective inflammatory stimulus-dependent S-nitrosylation of GAPDH by recognizing the [IL]-xCxx-[DE] motif, potentially targeting multiple targets such as ANXA5, EZR, MSN, and VIM; S100A8 appears to contribute to the selectivity of S-nitrosylation sites.

[0003] Collectively, S100A8 and A9 proteins are considered alarmins, otherwise known as damage-associated molecular patterns (DAMPs). Under inflammatory conditions, S100A8 and S100A9 protein expression is induced in a variety of cell types, such as endothelial cells, macrophages, osteoclasts, keratinocytes, and dendritic cells. Consequently, elevated serum S100A8 and / or S100A9 levels are found in numerous inflammatory pathologies (psoriasis, rheumatoid arthritis, chronic inflammatory bowel disease, etc.). S100A8 and S100A9 can also amplify the inflammatory response by ligating to the TLR4 receptor and inducing the NF-κB and mitogen-activated protein kinase (MAPK) pathways, leading to the secretion of proinflammatory cytokines (TNF, IL-6, etc.). They are also involved in ROS-dependent activation of the NLRP3 inflammasome. In addition to their activity in cytokine secretion, they function as chemoattractants, allowing for the recruitment and adhesion of leukocytes. The cell adhesion process occurs through the binding of S100A8 and / or A9 to neutrophil TLR4, which increases endothelial cell expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1), enabling leukocyte adhesion to the endothelial wall. The crucial role of inflammation in tumorigenesis and progression largely explains the role of S100A8 and A9 in tumorigenesis and progression. There is ample evidence supporting the tumor-promoting role of S100A8 and A9 in many solid cancers. First, amplification of the S100 gene locus 1q21 is common in cancer. Furthermore, S100A8 and S100A9 proteins promote tumor growth, metastasis, angiogenesis, and immune evasion in various solid tumors. Indeed, S100A8 and S100A9 protein expression is upregulated in several tumor types, including breast, prostate, colon, and melanoma. S100A8 has been shown to be overexpressed in patients with an aggressive phenotype and poor overall survival, suggesting that regulation of S100A8 gene expression is a mechanism involved in cancer cell control. Proteins S100A8, S100A9, or hetero-oligomers S100A8 / A9 may also promote cancer cell migration and dissemination by modulating the tumor microenvironment. Similarly, data are accumulating on the pathological roles of S100A8 and S100A9 in the development and progression of hematological malignancies, particularly acute leukemia (AL).

[0004] Ebbing et al. studied temporal changes in calprotectin levels in patients with renal tumors who underwent iatrogenic renal ischemia-reperfusion injury due to transient renal artery clamping. Calprotectin concentrations began to increase significantly at the end of surgery (approximately 2 hours after ischemia) and reached maximal levels 48 hours after surgery, with a 69-fold increase in calprotectin levels compared to baseline. Calprotectin levels remained significantly elevated 5 days after surgery. Elevations in calprotectin have been described in several other diseases, such as rheumatoid arthritis, inflammatory bowel disease, myocardial infarction, cancer, and several other conditions. In the clinical interpretation of urinary calprotectin findings, one should be aware that, in addition to AKI, there are two clinical settings that can lead to increased calprotectin levels: Because calprotectin is primarily derived from neutrophils and monocytes, pyuria can significantly increase urinary calprotectin. Furthermore, urothelial carcinoma is associated with increased concentrations.

[0005] Currently, the main method for detecting S100A8 / 9 is enzyme-linked immunosorbent assay (ELISA). The ELISA method is relatively cumbersome to operate, takes a long time to detect, and has many interfering factors. In contrast, the latex immunoturbidimetric method for determining S100A8 / 9 on a biochemical analyzer is simple to operate, convenient and fast to detect, highly sensitive, specific, and reproducible. Moreover, this test method can be used on various biochemical analyzers and has strong applicability. Summary of the Invention

[0006] To this end, embodiments of the present invention provide a kit and method for determining S100A8 / 9 based on latex immunoturbidimetry.

[0007] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of the embodiments of the present invention, the present invention provides a method for preparing a latex microsphere solution coated with S100A8 / 9 antibody, comprising the following steps:

[0009] 1) adding latex microspheres to a buffer solution to obtain an activated latex microsphere system;

[0010] 2) adding S100A8 / 9 antibody to the activated latex microsphere system, stirring, and then adding EDC·HCl solution and NHS solution to carry out coupling reaction to obtain a coupling system;

[0011] 3) adding a blocking solution to the coupling system for blocking, then centrifuging, and collecting the supernatant to obtain a mixed system;

[0012] 4) adding a storage solution to the mixed system and storing the mixture overnight to obtain the latex microsphere solution coated with the S100A8 / 9 antibody;

[0013] The buffer solution is composed of an MES buffer solution and a boric acid buffer solution in a volume ratio of 1:3-5. The MES buffer solution uses water as a solvent, includes 6.0-7.0 g / L of MES·H2O, 4.5-5.5 g / L of NaOH, and has a pH of 6.0-6.5. The boric acid buffer solution uses water as a solvent, includes 3.0-3.2 g / L of boric acid, 1.4-1.6 g / L of sodium tetraborate, and has a pH of 7.2-7.5.

[0014] The present invention discovered that, when using a two-step coupling reaction, the method of adding the buffer and materials significantly affects the binding of the S100A8 / 9 antibody to the latex microspheres. Activating the latex microspheres in the aforementioned specific buffer, followed by the addition of the S100A8 / 9 antibody, and then EDC and NHS, can improve the coupling efficiency between the S100A8 / 9 antibody and the latex microspheres. Preferably, the buffer comprises MES buffer and boric acid buffer in a volume ratio of 1:4. The MES buffer, with a water solvent, contains 6.52 g / L MES·H2O and 5 g / L NaOH, with a pH of 6.2. The boric acid buffer, with a water solvent, contains 3.12 g / L boric acid and 1.58 g / L sodium tetraborate, with a pH of 7.4.

[0015] Furthermore, the latex microspheres are carboxylated polystyrene latex particles with a particle size of 140 to 160 nm; the volume ratio of the latex microspheres to the buffer is 1:3.8 to 4.2. The present invention further discovered that the use of carboxylated polystyrene latex particles with smaller particle sizes, under the aforementioned coupling conditions, is more conducive to improving the stability of the conjugate of the S100A8 / 9 antibody and the latex microspheres, thereby improving the detection sensitivity and accuracy of the kit.

[0016] The S100A8 / 9 antibodies used in the present invention can be commercially available. Preferably, the present invention employs single-cell B cell sequencing technology to perform single-cell sequencing on the spleens of immunized mice. The genes with the highest expression frequency are screened based on data analysis, and then validated by antigen ELISA to screen for specific S100A8 / 9 antibodies, thereby further improving the detection sensitivity and accuracy of the kit.

[0017] Specifically, the S100A8 / 9 antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of CDR1 of the heavy chain variable region is shown as amino acids 26-33 of SEQ ID No.1; the amino acid sequence of CDR2 of the heavy chain variable region is shown as amino acids 51-58 of SEQ ID No.1; the amino acid sequence of CDR3 of the heavy chain variable region is shown as amino acids 97-110 of SEQ ID No.1; the amino acid sequence of CDR1 of the light chain variable region is shown as amino acids 26-31 of SEQ ID No.2; the amino acid sequence of CDR2 of the light chain variable region is shown as amino acids 49-51 of SEQ ID No.2; and the amino acid sequence of CDR3 of the light chain variable region is shown as amino acids 88-97 of SEQ ID No.2.

[0018] Furthermore, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2.

[0019] Furthermore, in step 2), the final concentration of the S100A8 / 9 antibody is 0.04-0.06 mg / ml; the final concentration of the EDC·HCl is 1.8-2.2 mM; and the final concentration of the NHS is 4.8-5.2 mM;

[0020] In step 3), the blocking solution uses water as a solvent, includes Tween 20 with a final concentration of 1.8 to 2.2 mL / L, BSA with a final concentration of 0.8 to 1.2 g / L, and a pH of 7.2 to 7.5;

[0021] In step 4), the storage solution uses water as a solvent, includes Tween 20 with a final concentration of 1.8 to 2.2 ml / L, BSA with a final concentration of 0.8 to 1.2 g / L, and Proclin 300 with a final concentration of 0.3 to 0.6 ml / L, and has a pH of 7.2 to 7.5; the volume concentration of the latex microspheres in the latex microsphere solution is 2% to 3%.

[0022] According to a second aspect of the embodiments of the present invention, the present invention provides a latex microsphere solution coated with S100A8 / 9 antibody, which is prepared by the method described in any one of the above items.

[0023] According to a third aspect of the embodiments of the present invention, the present invention provides a kit for determining S100A8 / 9 based on latex immunoturbidimetry, comprising a reagent R1 and a latex reagent R2.

[0024] The composition and content of the reagent R1 are: buffer solution 50-150 mmol / L, inorganic salt 0.05-0.2 mol / L, preservative 0.01-3%, and the solvent is pure water;

[0025] The components and contents of the latex reagent R2 are: 50-350 mmol / L buffer, 0.5-5% stabilizer, 0.2-2% surfactant, 0.02-5% preservative, 2-3% of the latex microsphere solution coated with S100A8 / 9 antibody as described above, and pure water as the solvent.

[0026] Furthermore, in the reagent R1, the buffer is Tris-HCl buffer, the inorganic salt is sodium chloride, and the preservative is Proclin 300;

[0027] In the latex reagent R2, the buffer is Tris-HCl buffer, the stabilizer is BSA, the surfactant is Tween 20, and the preservative is Proclin 300.

[0028] Furthermore, the composition and content of the reagent R1 are: Tris-HCl buffer 100mM, sodium chloride 0.15mol / L, Proclin3000.03%, and the solvent is pure water;

[0029] The components and contents of the latex reagent R2 are: Tris-HCl buffer 100 mM, BSA 1%, Tween 20 1%, Proclin 3000 0.03% and the latex particle solution 3%.

[0030] According to a fourth aspect of the embodiments of the present invention, the present invention provides a method for determining S100A8 / 9 using the above-mentioned kit, comprising the following steps:

[0031] 2 μL of the sample to be tested was drawn up, 200 μL of the reagent R1 was added, and the mixture was incubated at 37°C for 5 min.

[0032] Add 40 μL of the latex reagent R2, incubate at 37°C for 30 seconds, and read the absorbance A1;

[0033] Incubate at 37°C for 5 min and read the absorbance A2;

[0034] ΔA=A2-A1 is calculated, and the content of S100A8 / 9 in the sample to be tested is measured based on ΔA.

[0035] The reaction principle of the kit of the present invention:

[0036] S100A8 / 9 in the test sample binds to the specific mouse monoclonal antibody S100A8 / 9 latex particles in latex reagent R2, triggering an antigen-antibody reaction and forming an antigen-antibody complex, which in turn causes a change in turbidity. This turbidity change is measured by an automated biochemical analyzer, and a linear curve is plotted using a standard to determine the S100A8 / 9 content in the sample.

[0037] The embodiments of the present invention have the following advantages:

[0038] The latex immunoturbidimetric assay kit provided by the present invention effectively improves the sensitivity and specificity of S100A8 / 9 detection, offers improved reproducibility, strong anti-interference capabilities, and excellent reagent stability. It also enables automated detection on a biochemical analyzer, simplifies operation, and facilitates rapid and convenient testing, facilitating clinical application. Furthermore, the kit provided by the present invention is more convenient for detecting lung cancer and fibrosis progression, facilitating the detection of tumor markers and prognostic markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0040] Figure 1 Schematic diagram of the fitting curve of the S100A8 / 9 kit provided in an embodiment of the present invention;

[0041] Figure 2 Comparison of S100A8 / 9 levels in sputum between normal subjects and lung cancer patients provided in the embodiments of the present invention;

[0042] Figure 3 The present invention provides a comparison of the S100A8 / 9 levels in the serum of normal subjects and patients with different grades of liver fibrosis. DETAILED DESCRIPTION

[0043] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0044] Example 1 Preparation of S100A8 / 9 Antibody

[0045] The S100A8 / 9 antibody was obtained by screening antibodies with high gene expression frequency through antibody sequencing. The preparation process is as follows:

[0046] Animal immunization, B cell collection and enrichment, single B cell introduction, single B cell analysis: specificity, functionality and affinity, single cell export, single cell sequencing, data analysis and recombinant antibody expression verification.

[0047] The statistical results of antigen immunity are shown in Table 1.

[0048] Table 1

[0049] immunity Immunization date Antigen amount / μl Pure water volume / μl Adjuvant dose / μl immune site One Free 20210618 1132.9 0 1133 (complete) Belly skin+ Second exemption 20210702 571 129 700 (unfinished) Belly skin+ Three exemptions 20210716 571 129 700 (unfinished) Belly skin+

[0050] Note: Antigen concentration is 1.05 mg / ml. S100A8 / 9 antigen: The S100A8 and S100A9 sequences were constructed on the pcDNA3.1 vector to express the heterodimeric S100A8 / 9 protein.

[0051] One month after immunization, spleen cells were harvested from mice for single-cell sequencing. B cells were enriched and purified using a SORPFACS Aria 5L18c flow cytometer, followed by analysis of the antibody VDJ library and 5' expression library using the 10X genomics 5'HT Reagent Kits v2. The constructed libraries were sequenced using an Illumina second-generation sequencer. Based on the sequencing results, antibody genes with high expression frequencies were sorted and synthesized. Antibody expression was then expressed in 293T cells and assayed using ELISA.

[0052] After sequencing, the amino acid sequence of the H chain variable region of the screened specific S100A8 / A9 antibody is shown in SEQ ID No. 1, and the amino acid sequence of the L chain variable region is shown in SEQ ID No. 2.

[0053] The amino acid sequences of CDR1, CDR2, and CDR3 in the H chain variable region are: GFSFTSYA; LTGSGAFT; GKPPRGYNTAPSDY. The amino acid sequences of CDR1, CDR2, and CDR3 in the L chain variable region are: NIASTS; PDS; QVWDHSNEDL.

[0054] Example 2 Preparation of Latex Microsphere Solution Coated with S100A8 / 9 Antibody

[0055] The method for preparing a latex microsphere solution coated with S100A8 / 9 antibody comprises the following steps:

[0056] Step 1: Suspend 300 μL of latex microspheres (carboxyl polystyrene latex particles, particle size of 140 nm) in 5 ml of reaction buffer (1 ml of the first reaction buffer + 4 mL of the second reaction buffer). Keep the entire reaction system at a constant temperature of 28° C. for 90 min to obtain an activated latex microsphere solution.

[0057] In this example, the first reaction buffer was an MES buffer containing 6.52 g / L MES·H2O and 5 g / L NaOH, with a pH of 6.2. The second reaction buffer was a borate buffer containing 3.12 g / L boric acid and 1.58 g / L sodium tetraborate, with a pH of 7.4. The volume ratio of the MES buffer to the borate buffer was 1:4.

[0058] Step 2: Add the S100A8 / 9 antibody of Example 1 to the reaction system of Step 1 to a final concentration of 0.05 mg / ml.

[0059] Step 3: Stir at 28°C for 90 minutes.

[0060] Step 4: Add EDC·HCl solution and NHS solution sequentially to the reaction system in Step 3. The final concentration of EDC·HCl is 2 mM, and the final concentration of NHS is 5 mM. Prepare the NHS solution immediately before use.

[0061] Step 5: Add Tween 20 and BSA to the reaction system in step 4 for blocking. The final concentration of Tween 20 is 2 mL / L; the final concentration of BSA is 1 g / L. The pH is 7.4. Centrifuge after the reaction.

[0062] Step 6: Add the stock solutions of Tween 20, BSA, and Proclin 300 to the reaction system of step 5. The final concentrations of Tween 20, BSA, and Proclin 300 are 2 mL / L, 1 g / L, and 0.5 mL / L, respectively, and the pH is 7.2.

[0063] Step 7: Store overnight to obtain a latex microsphere solution coated with the S100A8 / 9 antibody, wherein the volume concentration of the latex microspheres is 3%.

[0064] Example 3 Preparation of the kit

[0065] The preparation of the kit is shown in Table 2.

[0066] Table 2 Kit

[0067]

[0068]

[0069] 1) Preparation of R1 and R2

[0070] Prepare reagent 1 and reagent 2 according to Table 1.

[0071] 2) Preparation of calibrators

[0072] S100A8 / 9 was prepared into a series of calibrators using calibrator diluent, with concentrations of 0 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0073] 3) Establishment of calibration curve

[0074] S100A8 / 9 calibrators, ranging in concentration from 0 to 10,000 pg / mL, were reacted with the prepared S100A8 / 9 latex reagent in a reaction cup. The reaction was measured using a biochemical analyzer, and the degree of reactivity was recorded. The detection wavelength was 570 nm, and the intensity of transmitted light was measured using the endpoint method.

[0075] Example 4 Detection method of the kit

[0076] 1. Reagent Preparation

[0077] Liquid R1 and R2 reagents are ready for use upon opening. Before use, reconstitute the calibrator with 1.00 mL of purified water. Place at 10°C to 30°C for 30 minutes to allow the lyophilized material to completely dissolve. Gently mix and use as the original calibrator solution. Dilute the original calibrator solution with purified water (volume ratio) as shown in the table below to create six concentration gradients. The dilution method is shown in Table 3.

[0078] Table 3 Dilution ratios of original calibrators

[0079] Serial number 1 2 3 4 5 6 Purified water: stock solution 1:0 15:1 7:1 3:1 1:1 0:1

[0080] 2. Parameter settings

[0081] Test method: End point method Detection temperature: 37℃ Detection wavelength: 570nm Reaction direction: ascending reaction Colorimetric cup light diameter: 1cm Blank control; deionized water

[0082] 3. Detection steps

[0083]

[0084] 4. Calibration Procedure

[0085] Multi-point calibration is used.

[0086] 5. Calculation

[0087]

[0088] The test kit of Example 3 was tested for various indicators.

[0089] 1. Linear Correlation Verification

[0090] In the range of [0, 2000] pg / mL, dilute a high-value sample near the lower limit of the linear interval with a low-value sample near the lower limit of the linear interval to form at least 5 samples of different concentrations. Measure each sample 3 times and take the average value. Perform linear regression analysis on the measured values. The correlation coefficient of the calculated linear regression should be ≥0.9900.

[0091] The specific data comparison values ​​are shown in Table 4.

[0092] Table 4 Linearity test results

[0093]

[0094] Figure 1 is a linear regression graph, such as Figure 1 As shown, the straight line fitting curve is y = 0.9983x + 0.3682, R 2 =1.0>0.9900 and P<0.05. The requirements are met.

[0095] 2. Precision Verification

[0096] The kit of Example 3 was tested for intra-batch precision and inter-day precision. Two levels of quality control serum, low value (50) and high value (5000), were selected as evaluation samples. For intra-batch precision, the test was repeated 20 times in a short period of time and under stable conditions. The results of each test were recorded and the mean (Mean), standard deviation (SD), and coefficient of variation (CV) were calculated. For inter-day precision, the test sample was tested once a day, and the results of 20 tests were accumulated. The mean (Mean), standard deviation (SD), and coefficient of variation (CV) were calculated. The experimental results are shown in Tables 5-1 and 5-2.

[0097] Table 5-1 Results of inter-batch repeatability test

[0098]

[0099] Table 5-2 Daily repeatability test results

[0100]

[0101] It can be concluded from Tables 5-1 and 5-2 that the maximum intra-batch CV of the kit is 1.2%, and the maximum inter-day CV is 0.53%, which meets the requirement of ≤4%.

[0102] Example 5 is applied to the detection of S100A8 / 9 content in sputum of lung cancer patients

[0103] Because S100A8 / 9 is associated with tumor growth and escape, the S100A8 / A9 levels in sputum from lung cancer patients were measured. Using the kit described in Example 3, the assay was performed on healthy controls (n=20) and lung cancer patients (n=20). The results are shown in Table 6. These results demonstrate that S100A8 / 9 plays a promoting role in solid tumor development.

[0104] Table 6 Statistical graph of test results

[0105]

[0106] Example 6 Detection of S100A8 / 9 Levels in Serum of Patients with Liver Fibrosis, Cirrhosis and Liver Cancer

[0107] The kit of Example 3 was used to detect the S100A8 / 9 levels in the serum of 60 subjects including normal subjects (n=15), subjects with liver fibrosis (n=15), subjects with liver cirrhosis (n=15) and subjects with liver cancer (n=15). The results are shown in Table 7.

[0108] Table 7 Test results statistics

[0109]

[0110]

[0111] The results show that the increase in S100A8 / 9 content in the process of fibrosis may be related to participation in inflammatory response. In cirrhosis, high expression of S100A8 / 9 may be related to promoting the formation of liver cancer. The increased content in the liver cancer stage may be related to tumor formation and tumor metastasis.

[0112] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0113]

[0114]

[0115]

[0116]

Claims

1. A method for preparing a latex microsphere solution coated with S100A8 / 9 antibody, characterized in that: The steps include: 1) adding latex microspheres to a buffer solution to obtain an activated latex microsphere system; 2) adding S100A8 / 9 antibody to the activated latex microsphere system, stirring, and then adding EDC·HCl solution and NHS solution to carry out coupling reaction to obtain a coupling system; 3) adding a blocking solution to the coupling system for blocking, then centrifuging, taking the supernatant, and obtaining a mixed system; 4) adding storage solution to the mixed system and storing it overnight to obtain the latex microsphere solution coated with S100A8 / 9 antibody; The buffer solution is composed of a MES buffer solution and a boric acid buffer solution in a volume ratio of 1:3-5. The MES buffer solution uses water as a solvent and includes 6.0-7.0 g / L MES·H2O and 4.5-5.5 g / L NaOH, with a pH of 6.0-6.

5. The boric acid buffer solution uses water as a solvent and includes 3.0-3.2 g / L boric acid and 1.4-1.6 g / L sodium tetraborate, with a pH of 7.2-7.

5. The S100A8 / 9 antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of CDR1 of the heavy chain variable region is shown as amino acids 26-33 of SEQ ID No. 1; The amino acid sequence of CDR2 of the heavy chain variable region is shown in amino acids 51 to 58 of SEQ ID No. 1; The amino acid sequence of CDR3 of the heavy chain variable region is shown in amino acids 97 to 110 of SEQ ID No. 1; The amino acid sequence of CDR1 of the light chain variable region is shown in amino acids 26 to 31 of SEQ ID No. 2; The amino acid sequence of CDR2 of the light chain variable region is shown in amino acids 49 to 51 of SEQ ID No. 2; The amino acid sequence of CDR3 of the light chain variable region is shown in amino acids 88 to 97 of SEQ ID No.

2.

2. The preparation method according to claim 1, characterized in that: The latex microspheres are carboxyl polystyrene latex particles with a particle size of 140-160 nm; the volume ratio of the latex microspheres to the buffer solution is 1:3.8-4.

2.

3. The preparation method according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

4. The preparation method according to claim 1, characterized in that: In step 2), the final concentration of the S100A8 / 9 antibody is 0.04-0.06 mg / ml; the final concentration of the EDC·HCl is 1.8-2.2 mM; and the final concentration of the NHS is 4.8-5.2 mM; In step 3), the blocking solution uses water as solvent, includes Tween 20 with a final concentration of 1.8-2.2 mL / L, BSA with a final concentration of 0.8-1.2 g / L, and a pH of 7.2-7.5; In step 4), the storage solution uses water as solvent, includes Tween 20 with a final concentration of 1.8-2.2 ml / L, BSA with a final concentration of 0.8-1.2 g / L, and Proclin 300 with a final concentration of 0.3-0.6 ml / L, and the pH is 7.2-7.5; the volume concentration of latex microspheres in the latex microsphere solution is 2%-3%.

5. A latex microsphere solution coated with S100A8 / 9 antibody, characterized in that: The method is prepared by the method described in any one of claims 1 to 3.

6. A kit for determining S100A8 / 9 based on latex immunoturbidimetry, characterized in that: Comprising reagent R1 and latex reagent R2, The composition and content of the reagent R1 are: buffer solution 50-150 mmol / L, inorganic salt 0.05-0.2 mol / L, preservative 0.01-3%, and the solvent is pure water; The composition and content of the latex reagent R2 are: buffer 50-350 mmol / L, stabilizer 0.5-5%, surfactant 0.2-2%, preservative 0.02-5%, latex microsphere solution coated with S100A8 / 9 antibody as claimed in claim 5 2-3%, and the solvent is pure water.

7. The kit according to claim 6, characterized in that In the reagent R1, the buffer is Tris-HCl buffer, the inorganic salt is sodium chloride, and the preservative is Proclin300; In the latex reagent R2, the buffer is Tris-HCl buffer, the stabilizer is BSA, the surfactant is Tween 20, and the preservative is Proclin 300.

8. The kit according to claim 6, characterized in that The composition and content of the reagent R1 are: Tris-HCl buffer 100mM, sodium chloride 0.15mol / L, Proclin3000.03%, and the solvent is pure water; The composition and content of the latex reagent R2 are: Tris-HCl buffer 100mM, BSA 1%, Tween 20 1%, Proclin 300 0.03% and the latex microsphere solution 3%, and the solvent is pure water.

9. A method for determining S100A8 / 9 using the kit according to claim 6 for non-diagnostic purposes, characterized in that: The steps include: Pipette 2 μL of the sample to be tested, add 200 μL of the reagent R1, and incubate at 37°C for 5 min; Add 40 μL of the latex reagent R2, incubate at 37° C. for 30 s, and read the absorbance A1; Incubate at 37°C for 5 min and read the absorbance A2; ΔA= A2-A1 is calculated, and the content of S100A8 / 9 in the sample to be tested is measured based on ΔA.

10. A S100A8 / 9 antibody, characterized in that The S100A8 / 9 antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of CDR1 of the heavy chain variable region is shown as amino acids 26-33 of SEQ ID No. 1; The amino acid sequence of CDR2 of the heavy chain variable region is shown in amino acids 51 to 58 of SEQ ID No. 1; The amino acid sequence of CDR3 of the heavy chain variable region is shown in amino acids 97 to 110 of SEQ ID No. 1; The amino acid sequence of CDR1 of the light chain variable region is shown in amino acids 26 to 31 of SEQ ID No. 2; The amino acid sequence of CDR2 of the light chain variable region is shown in amino acids 49 to 51 of SEQ ID No. 2; The amino acid sequence of CDR3 of the light chain variable region is shown in amino acids 88 to 97 of SEQ ID No.2.

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