A long-chain carboxyl microsphere, its preparation method and reagent kit

Abstract

This invention discloses a long-chain carboxyl microsphere, its preparation method, and a kit. The conventional method for preparing polystyrene (PS) carboxyl microspheres is improved by using long-chain carboxyl functional monomers such as 3-methyl-4-pentenoic acid and 5-methyl-5-hexenoic acid, which have similar structures. Using these functional monomers to prepare microspheres promotes longer carboxyl chains, overcoming steric hindrance. Crosslinking the prepared microspheres with amino-containing small-molecule haptens allows for turbidity reactions with corresponding antibodies, eliminating the need for pre-crosslinking of small-molecule antigens with proteins. A kit constructed using the conjugated small-molecule microspheres exhibits satisfactory calibration curves and can be used to detect the content of corresponding substances in samples.

Description

Technical Field

[0001] This invention relates to a long-chain carboxyl microsphere, its preparation method, and a kit. Background Technology

[0002] The common principle of latex-enhanced turbidimetry is that antigens or antibodies bind to microspheres via adsorption or covalent coupling. When the antigen or antibody of the analyte binds to the antigen or antibody coupled to the microsphere, the microsphere amplifies the signal and generates turbidity in the reaction solution. Within a certain range, the strength of this turbidity signal is linearly related to the concentration of the analyte, thus enabling the determination of the analyte content in the sample. Over the years, immunoturbidimetric detection has evolved from a manual method on a spectrophotometer to a fully automated mode and is widely used in fields such as medical diagnostics.

[0003] Latex-enhanced turbidimetry generally employs two reaction modes: a direct method, where the antibody / antigen on the microspheres reacts directly with the analyte, typically used for the detection of biomolecules; and an indirect method, also known as immunosuppression, where the antibody and antigen pre-generate turbidity. Upon addition of the sample, the analyte competes with the antigen / antibody for turbidity, inhibiting the turbidity; the degree of inhibition is proportional to the concentration of the analyte. Generally, indirect analysis is suitable when the sample contains only one epitope, such as haptens, peptides, and small molecules.

[0004] Currently, most microspheres produced using chemical coupling methods are carboxylated polystyrene (PS) microspheres. Generally, during the PS microsphere preparation process, monomers containing carboxyl groups are introduced for copolymerization to achieve carboxylation modification of the PS microsphere surface. The surface structure of PS microspheres is as follows: Figure 1 Part A. When using ordinary carboxyl microspheres for chemical coupling, the carboxyl groups on the surface of the microspheres are generally cross-linked with the amino groups of the antigen and antibody using a coupling agent, forming a cross-linked structure like... Figure 1 The structure of part B. Since the carboxyl groups of PS microspheres are usually located on the side chains of the polystyrene chain, only a small number of carboxyl groups are located on the surface of the PS microspheres, with most carboxyl groups "embedded" within the styrene groups. When the carboxyl groups cross-link with amino-containing antigens or antibodies to form sensitized microspheres, if the cross-linked antigens or antibodies are large molecular proteins (antibodies are all large molecules), the antigenic epitopes are often far from the surface of the microspheres. The steric hindrance effect has little impact on the capture ability, and the antibody or antigen in the test sample can capture the sensitized microspheres and generate a turbidity signal.

[0005] However, when the carboxyl groups of microspheres are coupled with small-molecule haptens, a significant steric hindrance effect occurs due to the short spatial distance between the antigen epitope and the microsphere surface. This drastically reduces the antibody's ability to capture the hapten, preventing the formation of effective turbidity. Current solutions involve pre-coupling the small-molecule antigen to a protein, such as bovine serum albumin (BSA) or hemocyanin (KLH), before coupling this conjugate to the microspheres. It can be seen that the above method suffers from drawbacks such as cumbersome process, low yield, and difficulty in scale-up. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a long-chain carboxyl microsphere, its preparation method, and a kit.

[0007] The technical solution for achieving the objective of this invention is as follows:

[0008] A method for preparing long-chain carboxyl microspheres includes the following steps:

[0009] 1) Aqueous phase preparation;

[0010] 2) Monomer formulation;

[0011] 3) Polymerization reaction;

[0012] 4) Add functional units;

[0013] The functional monomers described herein have or are one of the following three structures, and when n=1, their names are: 3-methyl-4-pentenoic acid, ethyl 2-methyl-4-pentenoate, and 2,2-dimethyl-4-pentenoic acid.

[0014] In the formula, n is an integer from 1 to 9;

[0015] 5) End the filtering process;

[0016] 6) Remove impurities from the latex to obtain the finished monodisperse polymer latex.

[0017] The method for preparing long-chain carboxyl microspheres,

[0018] 1) When preparing the aqueous phase, the hydrophilic substance in the reaction is pre-dissolved in an appropriate amount of water to obtain a well-mixed solution;

[0019] 2) When preparing the monomer, the nucleated polymer monomer is added to the aqueous phase and stirred to ensure uniform dispersion;

[0020] As mentioned in 3), the polymerization reaction will be carried out by raising the temperature and adding an initiator to start the polymerization reaction of the monomers;

[0021] The fourth step involves adding functional monomers to promote the formation of functional groups on the surface of the spheres.

[0022] As stated in step 5), the filtration process ends, the reaction is complete, and large particles are removed by filtration.

[0023] The process described in step 6) involves dialysis of the latex to remove impurities, resulting in a monodisperse polymer latex product.

[0024] The method for preparing long-chain carboxyl microspheres includes the following steps:

[0025] 1) Aqueous phase preparation: Add 0.3~0.5 g sodium dodecyl sulfate, 0.5 g sodium hydroxide, and 0~2 g polyethylene glycol 4000 to 820-1200 mL of deionized water, then add to a 2 L glass reactor and stir at 300-400 RPM to dissolve.

[0026] 2) Monomer preparation: Take 23-60 mL of styrene purified in step 1 and 0-0.6 mL of divinylbenzene (DVB) and slowly add them to the aqueous phase while stirring at a speed of 300-400 RPM.

[0027] 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction.

[0028] 4) Add 2 mL of the functional monomer, dissolved in 20 mL of ethanol; after reacting for 0.5–2 hours, add it to the reaction vessel;

[0029] 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper.

[0030] 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

[0031] The method for preparing long-chain carboxyl microspheres,

[0032] 1) Aqueous phase preparation: Add 0.3 g sodium dodecyl sulfate, 0.5 g sodium hydroxide, and 2 g polyethylene glycol 4000 to 820 mL of deionized water, then add to a 2 L glass reactor and stir at 400 RPM to dissolve.

[0033] 2) Monomer preparation: Take 23 mL of purified styrene and 0.6 mL of divinylbenzene (DVB) and slowly add them to the aqueous phase while stirring at a speed of 400 RPM.

[0034] 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction.

[0035] 4) Add 2 mL of the functional monomer 3-methyl-4-pentenoic acid, dissolved in 20 mL of ethanol; after reacting for 2 hours, add it to the reaction vessel;

[0036] 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper.

[0037] 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

[0038] The method for preparing long-chain carboxyl microspheres,

[0039] 1) Aqueous phase preparation: Add 0.5 g sodium dodecyl sulfate and 0.5 g NaOH to 1200 mL of deionized water, then add to a 2 L glass reactor and stir at 300 RPM to dissolve.

[0040] 2) Monomer preparation: Add 60 mL of purified styrene to the aqueous phase, stir, and maintain a stirring speed of 300 RPM.

[0041] 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction.

[0042] 4) Add 2 mL of the functional monomer 5-methyl-5-hexenoic acid, dissolved in 20 mL of ethanol. After reacting for 2 hours, add it to the reaction vessel;

[0043] 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper.

[0044] 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

[0045] A long-chain carboxyl microsphere was prepared according to the method described above.

[0046] A kit employing long-chain carboxyl microspheres, wherein the long-chain carboxyl microspheres are directly coupled with small molecule antigens for immunoturbidimetric detection.

[0047] Another kit uses long-chain carboxyl microspheres, which employ direct small molecule antigen conjugation of the aforementioned long-chain carboxyl microspheres and use immunoturbidimetry for the detection of urinary creatinine and serum anti-cyclic citrulline antibodies.

[0048] The beneficial effects of this invention are:

[0049] This invention improves upon the shortcomings of PS microspheres by incorporating long-chain carboxyl groups onto the polystyrene microspheres. Using functional monomers to prepare the microspheres promotes longer carboxyl chains, overcoming steric hindrance. Crosslinking the prepared microspheres with amino-containing small-molecule haptens allows for turbidity reactions with corresponding antibodies, eliminating the need for pre-crosslinking of the small-molecule antigen with the protein. A kit constructed using the conjugated small-molecule microspheres exhibits satisfactory calibration curves and can be used to detect the content of corresponding substances in samples. Attached Figure Description

[0050] Figure 1 This is a diagram showing the surface structure and cross-linking pattern of carboxyl microspheres.

[0051] Figure 2 These are structural diagrams of three different carboxyl compounds.

[0052] Figure 3A This is a diagram showing the particle size and distribution of microspheres after Example 2.

[0053] Figure 3B This is a PS-A electron microscope image of microspheres.

[0054] Figure 3C This is an electron microscope image of PS-B microspheres.

[0055] Figure 4 This is a diagram of the creatinine structure.

[0056] Figure 5 This is the calibration curve for Example 3.

[0057] Figure 6 This is the correlation between the reagents in Example 3 and the control protocol in detecting the specimens.

[0058] Figure 7 This is the correlation between the reagent and the control protocol used in Example 4 for detecting specimens. Detailed Implementation

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0060] The carboxyl microspheres prepared in this invention have long-chain carboxyl groups on their surface that, after direct coupling with haptens, form a structure like... Figure 1 The extended carboxyl group in the structure shown in Figure C effectively reduces steric hindrance. Using this method, the step of pre-coupling small molecule antigens to proteins can be eliminated; they can be directly coupled to microspheres. The microspheres coupled with small molecules can then react with antibodies to produce turbidity, which can be used for quantitative detection.

[0061] In the preparation of carboxylated microspheres, this invention incorporates functionally modified compounds, such as acrylic acid and its derivatives, that are carboxylated during traditional microsphere preparation. Figure 2 1), improved to a long-chain olefinic acid, has the following properties: Figure 2Three similar structures of A, B, and C in Chinese. Figure 2 In the formula n = (1-9), the methyl group is placed in a reasonable position on the long chain.

[0062] In the preparation of carboxyl microspheres, compounds with the above-mentioned structure copolymerize with styrene, incorporating long-chain carboxyl groups. The distance between the carboxyl terminus and the microsphere surface can be significantly greater than that of carboxyl microspheres prepared by conventional methods. When a compound (peptide or small molecule) with an amino group is coupled to the long-chain carboxyl group, the antibody can effectively avoid steric hindrance, recognize the antigen epitope, and bind effectively, further enhancing turbidity through the microspheres.

[0063] The above-mentioned method not only eliminates the step of small molecule conjugated proteins, simplifying the process and increasing the yield, but also improves the batch stability of the product. This invention prepared long-chain carboxyl microspheres and used these microspheres to conjugate small molecules and peptides, respectively. Then, a detection reagent was constructed using the conjugated microspheres, and the detection reagent was successfully calibrated. Samples were tested, and the test results were consistent with those obtained using conventional methods.

[0064] I. Reagents

[0065] Table 1 lists the relevant reagents involved in the invention patent.

[0066] Table 1. Reagents required for the experiment

[0067] Serial Number name source Serial Number name source 1 disodium hydrogen phosphate Sinopharm Reagent 13 3-Methyl-4-pentenoic acid Shanghai Yuanye 2 Sodium hydroxide Sinopharm Reagent 14 Potassium dihydrogen phosphate Sinopharm Reagent 3 Anhydrous calcium chloride Sinopharm Reagent 15 5-Methyl-5-hexenoic acid Shanghai Yuanye 4 Polyethylene glycol 4000 Sinopharm Reagent 16 Creatinine Sinopharm Reagent 5 Polyethylene glycol 8000 Sinopharm Reagent 17 Hemocyanin (KLH) Shanghai Yuanye 6 Sodium chloride Sinopharm Reagent 18 Microsphere PC02N Bangs Laboratories 7 ethanol Sinopharm Reagent 19 Microsphere P0323 Japan JSR 8 styrene Hangzhou Huipu 20 NHS Aladdin 9 Potassium persulfate Sinopharm Reagent 21 EDC Aladdin 10 Divinylbenzene Hangzhou Shengli 22 Tween-20 Sinopharm Reagent 11 MES Aladdin 23 Creatinine polyclonal antibody Hangzhou Lian Biotechnology 12 HEPES Aladdin 24 Cyclic citrullinated peptides Hangzhou Peptide

[0068] II. Microsphere Preparation Methods

[0069] Example 1: Preparation of long-chain carboxyl microspheres PS-A from type A substances

[0070] like Figure 2 The structure of substance A, n=1, corresponds to the compound 3-methyl-4-pentenoic acid. In the later stages of preparing carboxyl microspheres, this compound is added to the reactor as a carboxyl monomer, forming long carboxyl chains on the surface.

[0071] 1. Purification of styrene

[0072] Styrene was first washed with 10% sodium hydroxide solution to remove polymerization inhibitors, then washed with deionized water until neutral, and dried with anhydrous CaCl2. In a round-bottom distillation flask, the system was evacuated to below 1 kPa, and 50-55 μL of the solution was collected. o C fraction, stored at low temperature.

[0073] 2. Synthesis of seed emulsion

[0074] 1) Aqueous phase preparation: Add 0.3 g sodium dodecyl sulfate (SDS), 0.5 g sodium hydroxide (NaOH), and 2 g polyethylene glycol 4000 to 820 mL of deionized water, then add to a 2 L glass reactor and stir at 400 RPM to dissolve.

[0075] 2) Monomer preparation: Take 23 mL of styrene purified in step 1 and 0.6 mL of divinylbenzene (DVB) and slowly add them to the aqueous phase while stirring at a speed of 400 RPM.

[0076] 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate initiator (which can be pre-dissolved in 10 times its volume of water), purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction.

[0077] 4) Add 2 mL of the functional monomer 3-methyl-4-pentenoic acid, dissolved in 20 mL of ethanol. After reacting for 2 hours, add it to the reaction vessel.

[0078] 5) End filtration: The reaction ends after 12 hours. Cool to room temperature and filter with 10µm filter paper.

[0079] 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

[0080] Example 2: Preparation of long-chain carboxyl microspheres PS-B from type B substances

[0081] Figure 2 shows the structure of substance B, n=2. In the preparation of carboxyl microspheres, the long-chain compound providing the carboxyl group is 5-methyl-5-hexenoic acid. This compound is added to the reaction vessel in the later stages of carboxyl microsphere preparation.

[0082] 1. Purification of styrene

[0083] Same as Example 1.

[0084] 2. Synthesis of Seed Emulsion

[0085] 1) Aqueous phase preparation: Add 0.5 g sodium dodecyl sulfate (SDS) and 0.5 g NaOH to 1200 mL of deionized water, then add to a 2 L glass reactor and stir at 300 RPM to dissolve.

[0086] 2) Monomer preparation: Take 60 mL of the purified styrene from step 1 and add it to the aqueous phase. Stir and maintain a stirring speed of 300 RPM.

[0087] 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate (pre-dissolved in water) as an initiator. Purge the reactor with nitrogen gas to maintain a constant temperature and start the reaction.

[0088] 4) Add 2 mL of the functional monomer 5-methyl-5-hexenoic acid, dissolved in 20 mL of ethanol. After reacting for 2 hours, add it to the reaction vessel.

[0089] 5) End filtration: The reaction ends after 12 hours. Cool to room temperature and filter with 10µm filter paper.

[0090] 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

[0091] Latex Analysis

[0092] The particle size and distribution of the prepared PS-A latex were tested using a Malvern particle size analyzer (Hydro 2000MU). The particle size distribution and particle size of the latex met the requirements. Figure 3A The microspheres PS-A were further observed using an electron microscope. Figure 3B PS-B Figure 3C The microspheres have a normal particle size and appearance.

[0093] III. Long-chain carboxyl microspheres as a cross-linking scheme for detecting small molecule antigens or peptide antibodies

[0094] Small molecules are haptens, requiring conjugation with proteins to aggregate with antibodies. Therefore, latex-enhanced turbidimetry for detecting small molecules typically employs an indirect method, namely immunosuppression. The polystyrene microspheres prepared in this invention contain long-chain carboxyl groups on their surface, directly conjugating small molecules. This effectively overcomes the steric hindrance of the microspheres and allows them to bind to antibodies via their long arms, significantly enhancing the antibody-antigen agglutination reaction.

[0095] The reagents and buffer solutions required for coupling are shown in Table 2.

[0096] Table 2 Reagents and buffer solutions for coupling preparation

[0097] Serial Number name Require 1 Microspheres Preparation in Example 1, with a concentration calculated at 10%. 2 MES buffer MES pH 5.9, 50mM 3 Coupling buffer PB 50mM, pH 6.9 4 Activator EDC, NHS: 10 mg / mL, prepare before use. 5 Blocking buffer BSA 1%, PB 50mM, pH 7.4 6 Diluent (R2 buffer) 50mM PBS buffer, pH 7.4, 1% NaCl, 0.5% BSA

[0098] Example 3: Determination of urinary creatinine by latex-enhanced turbidimetric assay

[0099] The structural formula of creatinine is shown below. Figure 4 Creatinine contains an amino group that can be directly cross-linked with a carboxyl group.

[0100] Creatinine is a routine biochemical indicator of kidney function. The main methods for detecting creatinine include enzymatic methods and immunoturbidimetric methods. Immunoturbidimetric methods usually employ an immunosuppression method, in which creatinine antibodies are coupled to ordinary carboxyl microspheres. The microspheres and the pre-coupled creatinine-derived conjugates form turbidity. The amount of creatinine in the sample is inversely proportional to the turbidity, thereby detecting the creatinine content in the sample.

[0101] This case uses the microspheres prepared in Example 1, with creatinine coupled to the long-chain carboxyl groups on the microspheres, eliminating the need for prior preparation of creatinine-derived conjugated conjugates. The creatinine on the long-chain microspheres can react with anti-creatinine antibodies to form turbidity, and the turbidity is directly proportional to the creatinine content in the sample.

[0102] 1. Preparation of R2 reagent

[0103] Creatinine-microsphere crosslinking scheme (Example 2)

[0104] step name plan 1 Latex preparation Take 140 μL of 10% PS-B latex prepared in Example 2 above, add 2 ml (50 mM pH 5.2), and the latex concentration is 7 mg / ml. 2 Latex activation Dissolve NHS and EDC in purified water to a concentration of 10 mg / mL before use. Add 5 mg of NHS and 1 mg of EDC dropwise to the latex preparation solution and stir at room temperature for 25 min. 3 Latex washing Transfer to EP tubes (365ul / tube), centrifuge at 18000rpm for 20 minutes, at 6℃, discard the supernatant, resuspend the precipitate in 1.8ml PBS buffer (50mM pH6.9), centrifuge again and resuspend in 2ml PB buffer, sonicate for 3 minutes, pause for 10 minutes, cycle for 40 minutes. 4 Molecular crosslinking Add 0.05 mg creatinine (pre-diluted to 0.25 mg / ml), antigen: latex = 1:280, total volume 2.2 ml, diluent PBS (0.05 M pH 6.9), stir at 35 °C, crosslink for 2 h, and then sonicate for 10 cycles. 5 Closed Add sealing solution, sonicate for 10 cycles, let stand for 30 minutes, then 12000 rpm for 12 minutes. 6 dilution Discard the supernatant, add 2 mL of R2 buffer, sonicate for 10 cycles, and dilute 7 mg / mL to 1.5 mg / mL or 2.5 mg / mL to prepare R2 reagent.

[0105] 2. Preparation of R1 reagent

[0106] HEPES buffer, 50 mM, pH 7.5, polyethylene glycol (PEG) 8000 1%, Tween 20 0.25%, anti-creatinine polyclonal antibody was added while stirring to a final concentration of 20 mg / L, allowed to stand overnight, and filtered through a 0.22 μM filter membrane.

[0107] 3. Calibration data

[0108] The detection reagents constructed above were successfully calibrated on an automated biochemical analyzer, and the calibration curve was normal. Calibration parameters are shown in Table 3, and the calibration curve is shown in [Table 3]. Figure 5 .

[0109] Table 3 Calibration parameters of AU680 automated biochemical analyzer

[0110] Project Name Crea Decimal places 1 unit mg / L Determination methods Endpoint method Reaction direction rise dominant wavelength 600 nm Second wavelength none Sample size 5uL R1 quantity 200uL R2 quantity 50uL Start and end points 12-27 o'clock Calibration method Log-4p

[0111] 4. Comparison

[0112] Crosslinking was performed using commercially available BangS PC02N carboxyl 150nm microspheres according to the above crosslinking scheme. Calibration testing failed (see Table 4). The main reason for the calibration failure was that the reagents failed to form effective turbidity.

[0113] Table 4 AU680 reference calibration OD values

[0114]

[0115] If the standard procedure is used, creatinine is first cross-linked to BSA protein, and then the conjugate is cross-linked to BANGs PC02N carboxyl microspheres. Using this as the main raw material, the test reagent is constructed and the calibration is successful.

[0116] We further compared the detection reagents of Example 3 with the control reagents, and initially tested 24 urine samples. The correlation met the requirements, and the detection data are shown in Table 5. The correlation coefficient was above 0.97. Figure 6 ).

[0117] Table 5 Comparison of sample values ​​detected by the test reagent and the control reagent in Example 3

[0118] Serial Number Control (mg / L) Example 3 (mg / L) Serial Number Control (mg / L) Example 3 (mg / L) 1 5.44 5.94 13 15.79 18.09 2 6.42 7.12 14 18.92 20.18 3 6.87 6.84 15 20.10 20.27 4 7.31 6.59 16 20.62 21.26 5 7.93 9.05 17 23.72 25.37 6 9.05 9.72 18 26.02 26.24 7 9.64 9.56 19 27.30 29.04 8 9.78 10.42 20 29.52 28.45 9 10.26 10.17 21 33.10 30.37 10 11.76 12.18 22 37.89 38.52 11 15.28 14.05 23 64.84 52.75 12 15.68 15.17 24 66.21 53.94

[0119] Example 4: Long-chain carboxyl microspheres for detecting serum anti-cyclic citrulline antibodies

[0120] Cyclic citrullinated peptide (CCP) is a major constitutive antigenic determinant recognized by anti-firaglin-associated antibodies in the serum of rheumatoid arthritis patients. CCP antibodies are autoantibodies that use synthetically cyclic citrullinated polypeptides as antigens. Detecting serum anti-CCP antibody levels is a marker signal for rheumatoid arthritis.

[0121] Because the amino acid sequence of cyclic citrulline peptide is HQCHQESTXGRSRGRCGRSGS, consisting of 21 amino acids, directly cross-linking the cyclic citrulline peptide to traditional carboxyl microspheres for antibody detection results in ineffective turbidity formation due to the steric hindrance of the short-chain carboxyl groups in the serum. The solution is to first cross-link the cyclic citrulline peptide to a suitable carrier protein (such as BSA or KLH), and then further cross-link the conjugate carrier to the microspheres, thus enabling effective turbidity formation with the anti-cyclic citrulline antibody.

[0122] The above methods involve numerous process steps and are difficult to control. In actual reagent production or preparation, multiple processes and variables are involved, including microsphere batches, peptide conjugate preparation, microsphere conjugate preparation, and reagent formulation and adjustment. Batch variations of reagents are difficult to control, and reagent costs are also high due to losses during peptide conjugate preparation.

[0123] This invention directly couples peptides to long-chain carboxyl microspheres, eliminating the peptide coupling step, which can effectively stabilize the process, improve yield, and reduce costs.

[0124] 1. Preparation of R2 reagent

[0125] CCP polypeptide-latex microspheres (Example 1) PS-A crosslinking scheme

[0126] step name plan 1 Latex preparation Take 300 μL of the 10% PS-A latex prepared in Example 1 above, add 2 ml (50 mM pH 5.2), and the latex concentration is 15 mg / ml. 2 Latex activation Dissolve NHS and EDC in purified water to a concentration of 10 mg / mL before use. Add 5 mg of NHS and 1 mg of EDC dropwise to the latex preparation solution and stir at room temperature for 25 min. 3 Latex washing Transfer to EP tubes (365ul / tube), centrifuge at 18000rpm for 20 minutes, at 6℃, discard the supernatant, resuspend the precipitate in 1.8ml PBS buffer (50mM pH 6.9), centrifuge again and resuspend in 2ml PB buffer, sonicate for 3 minutes, stop for 10 minutes, cycle for 40 minutes. 4 Molecular crosslinking Add 0.1 mg CCP peptide (pre-diluted to 0.5 mg / ml), antigen: latex = 1:210, total volume 2.2 ml, diluent PBS (0.05 M pH 6.9), stir at 35 °C, crosslink for 2 h, and then sonicate for 10 cycles. 5 Closed Add the blocking solution, sonicate for 10 cycles, let stand for 30 minutes, and centrifuge at 12,000 rpm for 20 minutes. 6 dilution Discard the supernatant, add 2 mL of R2 buffer, sonicate for 10 cycles, and dilute 15 mg / mL to 3 mg / mL or 5 mg / mL to prepare R2 reagent.

[0127] 2. Preparation of R agent

[0128] HEPES buffer, 50mM, pH 7.5, polyethylene glycol (PEG) 8000 2%, BSA 0.25%, Tween 20 0.25%, 0.22uM filter membrane filtration.

[0129] 3. Calibration data

[0130] The calibration parameters and calibration data of CCP reagent on the Hitachi automated biochemical analyzer are shown in Tables 6 and 7.

[0131] Table 6 Calibration Parameters of Hitachi 7170 Automated Biochemical Instrument

[0132]

[0133] Table 7. Calibration OD Values ​​of Hitachi 7170 Automated Biochemical Analyzer

[0134]

[0135] 4. Result Comparison

[0136] Crosslinking with commercially available JSR carboxylated P0323 microspheres according to the above crosslinking scheme failed the calibration test. However, when CCP peptides and KLH protein were further coupled to these carboxylated microspheres, the calibration passed. Data from 100 serum samples are shown in Table 8, with a correlation coefficient greater than 0.99. Figure 7 ).

[0137] Table 8 Comparison of sample values ​​detected by the test reagent and the control reagent in Example 4

[0138] The technical features of the above embodiments can be further combined. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0139] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing long-chain carboxyl microspheres, characterized in that, Includes the following steps: 1) Aqueous phase preparation: Add 0.3~0.5 g sodium dodecyl sulfate, 0.5 g sodium hydroxide, and 0~2 g polyethylene glycol 4000 to 820-1200 mL of deionized water, then add to a 2 L glass reactor and stir at 300-400 RPM to dissolve. 2) Monomer preparation: Take 23-60 mL of purified styrene and 0-0.6 mL of divinylbenzene (DVB) and slowly add them to the aqueous phase, stirring at a speed of 300-400 RPM. 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction. 4) Dissolve 2 mL of the functional monomer in 20 mL of ethanol; after reacting for 0.5-2 hours, add the functional monomer mixture to the reaction vessel; the functional monomer has or is one of the following three structures. In the formula, n is an integer from 1 to 9; 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper. 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

2. The method for preparing long-chain carboxyl microspheres according to claim 1, characterized in that, Includes the following steps: 1) Aqueous phase preparation: Add 0.3 g sodium dodecyl sulfate, 0.5 g sodium hydroxide, and 2 g polyethylene glycol 4000 to 820 mL of deionized water, then add to a 2 L glass reactor and stir at 400 RPM to dissolve. 2) Monomer preparation: Take 23 mL of purified styrene and 0.6 mL of divinylbenzene (DVB) and slowly add them to the aqueous phase while stirring at a speed of 400 RPM. 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction. 4) Dissolve 2 mL of the functional monomer 3-methyl-4-pentenoic acid in 20 mL of ethanol; after reacting for 2 hours, add the functional monomer mixture to the reaction vessel; 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper. 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

3. A method for preparing long-chain carboxyl microspheres, characterized in that, Includes the following steps: 1) Aqueous phase preparation: Add 0.5 g sodium dodecyl sulfate and 0.5 g NaOH to 1200 mL of deionized water, then add to a 2 L glass reactor and stir at 300 RPM to dissolve. 2) Monomer preparation: Add 60 mL of purified styrene to the aqueous phase, stir, and maintain a stirring speed of 300 RPM; 3) Polymerization reaction: Heat to 70°C o C. Add 0.5 g of potassium persulfate as an initiator, purge the reactor with nitrogen gas for protection, maintain a constant temperature, and start the reaction. 4) Dissolve 2 mL of the functional monomer 5-methyl-5-hexenoic acid in 20 mL of ethanol; after reacting for 2 hours, add the functional monomer mixture to the reaction vessel; 5) End filtration: After 12 hours of reaction, the reaction is completed. Cool to room temperature and filter with 10µm filter paper. 6) Latex impurity removal: Dialyze the product with 50 mM MES buffer using a 50 kD dialysis bag to remove impurities and obtain the monodisperse polymer latex product.

4. A long-chain carboxyl microsphere, characterized in that, The long-chain carboxyl microspheres were prepared according to the method described in claim 1.

5. A reagent kit employing long-chain carboxyl microspheres, characterized in that, The long-chain carboxyl microspheres described in claim 4 are directly coupled with small molecule antigens for immunoturbidimetric detection.

6. A reagent kit employing long-chain carboxyl microspheres, characterized in that, The long-chain carboxyl microspheres described in claim 4 are directly coupled with small molecule antigens, and immunoturbidimetric assay is used to detect urinary creatinine and serum anti-cyclic citrulline antibodies.

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