A kit for determining p2PSA based on latex-enhanced immunoturbidimetry, a preparation method and a detection method

Abstract

This invention relates to the field of immunoassay technology, specifically to a kit, preparation method, and detection method for determining p2PSA based on latex-enhanced immunoturbidimetric assay. The kit includes: a reaction solution and a latex antibody reagent; the reaction solution includes at least one of a buffer, inorganic salt, sensitizer, surfactant, blocking agent, and preservative; the latex antibody reagent includes a mixture of a first latex antibody reagent and a second latex antibody reagent; the first latex antibody reagent is formed by coupling a polyclonal antibody of p2PSA with large-particle latex microspheres; the particle size range of the large-particle latex microspheres is 172–405 nm; the second latex antibody reagent is formed by coupling a monoclonal antibody of p2PSA with small-particle latex microspheres; the particle size range of the small-particle latex microspheres is 71–121 nm; the monoclonal antibody of p2PSA includes three different monoclonal antibodies of p2PSA; the kit of this invention can ensure sensitivity in determining p2PSA while broadening the linearity and enhancing specificity.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to a kit, preparation method, and detection method for determining p2PSA based on latex-enhanced immunoturbidimetric assay. Background Technology

[0002] Prostate cancer is a common malignant tumor that threatens the health of middle-aged and elderly men. Serum prostate-specific antigen (PSA) testing combined with digital rectal examination is a preliminary screening method for early detection of prostate cancer. For suspected cases, prostate biopsy can be performed to aid diagnosis. However, when a patient's total PSA (tPSA) is between 4.0 and 10.0 ng / mL, which is traditionally considered the gray zone, it is difficult to make a definitive differential diagnosis using PSA alone. Although free PSA (fPSA) can be detected simultaneously and the fPSA / tPSA ratio (f / tPSA) can be calculated for differentiation, the results are not significant.

[0003] Serum prostate-specific antigen isoform 2 (p2PSA) is an isoform of PSA precursor, characterized by its non-hydrolyzable stability, tumor specificity, and tissue region specificity. Current research increasingly indicates that, compared to traditional PSA, the detection and calculation of percentage p2PSA (%, p2PSA) and the prostate health index (PHI) can significantly improve the diagnostic specificity of prostate cancer (PCa), reduce unnecessary prostate biopsies, and demonstrate good application value in predicting tumor malignancy and in the active monitoring of low-risk, localized PCa patients.

[0004] Currently, the primary method for detecting prostate-specific antigen (p2PSA) is chemiluminescence immunoassay. However, commercially available p2PSA test kits are mainly developed using large-scale chemiluminescence platforms. Clinical testing of serum p2PSA primarily relies on chemiluminescence assay kits manufactured by Beckman Coulter. Chemiluminescence methods are closed-system assays, and imported reagents are very expensive, requiring specialized equipment and placing a significant financial burden on patients. Furthermore, for a single test, chemiluminescence methods typically require approximately one hour to report results, and their sensitivity, specificity, and linearity are often unsatisfactory. Therefore, clinical practice demands a detection method that is highly sensitive, highly specific, linear, easy to operate, low-cost, and compatible with various instruments. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a kit, preparation method and detection method for determining p2PSA based on latex-enhanced immunoturbidimetry that is highly sensitive, highly specific, linear, easy to operate, low cost and applicable to a variety of instruments.

[0006] Therefore, the present invention provides the following technical solution:

[0007] The embodiments disclosed in this invention relate to a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay, the kit comprising: latex antibody reagent;

[0008] The latex antibody reagent comprises a mixture of a first latex antibody reagent and a second latex antibody reagent; the first latex antibody reagent is formed by coupling a polyclonal antibody of p2PSA with large-particle latex microspheres; the particle size range of the large-particle latex microspheres is 172–405 nm; the second latex antibody reagent is formed by coupling a monoclonal antibody of p2PSA with small-particle latex microspheres; the particle size range of the small-particle latex microspheres is 71–121 nm; the monoclonal antibody of p2PSA comprises three different monoclonal antibodies of p2PSA.

[0009] In a preferred embodiment, the p2PSA polyclonal antibody is p2PSA-pAb;

[0010] And / or, the monoclonal antibodies of p2PSA include p2PSA-1, p2PSA-2 and p2PSA-3 monoclonal antibodies; the p2PSA-1 monoclonal antibody is p2PSA-mAb-1; the p2PSA-2 monoclonal antibody is p2PSA-mAb-2; and the p2PSA-3 monoclonal antibody is p2PSA-mAb-3.

[0011] In a preferred embodiment, the particle size of the large-particle latex microspheres is 405nm, 218nm, 192nm or 172nm, preferably 405nm;

[0012] And / or, the particle size of the small-diameter latex microspheres is 71nm, 89nm, 100nm, 108nm or 121nm, preferably 71nm;

[0013] And / or, the volume ratio of the p2PSA polyclonal antibody to the large-particle latex microspheres is 2 to 5:1, preferably 2:1; the concentration of the p2PSA polyclonal antibody is 2 mg / L to 20 mg / L; and the concentration of the large-particle latex microspheres is 0.5% w / v to 1% w / v.

[0014] And / or, the volume ratio of the p2PSA monoclonal antibody to the small-particle latex microspheres is 2 to 5:1, preferably 2:1; the concentration of the p2PSA monoclonal antibody is 2 mg / L to 20 mg / L; and the concentration of the small-particle latex microspheres is 0.5% w / v to 1% w / v.

[0015] And / or, the mass ratio of p2PSA-1, p2PSA-2 and p2PSA-3 monoclonal antibodies is 1:1:9 to 9:1:1; preferably 1:1:1;

[0016] And / or, the volume ratio of the first latex antibody reagent to the second latex antibody reagent is 1:3 to 3:1, preferably 1:2 to 2:1; preferably, it is 1:1.

[0017] And / or, the concentration of the p2PSA polyclonal antibody conjugate with large-particle latex microspheres in the first latex antibody reagent is 0.1-0.5 w / v, preferably 0.3 w / v%.

[0018] And / or, the concentration of the p2PSA monoclonal antibody conjugate with small-particle latex microspheres in the second latex antibody reagent is 0.05–0.3 w / v, preferably 0.1 w / v.

[0019] In some embodiments, a reaction solution is also included; the reaction solution includes at least one of a buffer solution, an inorganic salt, a sensitizer, a surfactant, a blocking agent, and a preservative.

[0020] The buffer solution includes any one of N(2-acetamido)-2-iminodiacetic acid buffer, piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer.

[0021] And / or, the inorganic salt includes at least one of sodium molybdate, ammonium molybdate, sodium bisulfate, or ammonium bisulfate;

[0022] And / or, the sensitizer includes at least one of Biopidure 103, Biopidure 405, and PEG6000; preferably Biopidure 103 and / or Biopidure 405;

[0023] And / or, the preservative is at least one of sodium azide, gentamicin, and isothiazolinones;

[0024] And / or, the surfactant is at least one of polyoxyethylene fatty alcohol ether, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, and polysorbate;

[0025] And / or, the blocking agent is at least one of THBR-1, THBR-2, and A1101; preferably, it is THBR-1, THBR-2, and A1101 in a volume ratio of 0.5-1.5:0.5-1.5:0.5-1.5, more preferably 1:1:1;

[0026] Optionally, the reaction solution comprises: buffer solution 13–18.2 g / L, inorganic salt 8.5–20 g / L, sensitizer 20–35 g / L, surfactant 0.1–0.2 v / v%, blocking agent 0.002–0.006 v / v%, and preservative 0.5–1.5 g / L; pH is 7.2 ± 0.05–8.2 ± 0.05.

[0027] In some embodiments, a calibrator is also included; the calibrator contains human p2PSA antigen;

[0028] Optionally, the calibrator comprises a buffer solution of 13–18.2 g / L, a protective agent of 0.5–1 g / L, and a preservative of 13–18.2 g / L;

[0029] Optionally, the buffer solution includes any one of phosphate buffer, N(2-acetamido)-2-iminodiacetic acid buffer, piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer.

[0030] Optionally, the protective agent is at least one of bovine serum albumin, casein, gelatin, or NaCl;

[0031] Optionally, the preservative is at least one of sodium azide, gentamicin, and isothiazolinones.

[0032] The embodiments disclosed in this invention relate to a method for preparing a kit for determining p2PSA based on latex-enhanced immunoturbidimetry, comprising the following steps:

[0033] Large-particle-size latex microspheres and small-particle-size latex microspheres were activated with carbodiimide (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) respectively, and then put into use.

[0034] The activated large-particle latex microspheres were conjugated with p2PSA polyclonal antibodies, blocked, centrifuged to remove the supernatant, and resuspended to obtain the first latex antibody reagent.

[0035] The activated small-particle latex microspheres were coupled with the p2PSA monoclonal antibody, blocked, centrifuged to remove the supernatant, and resuspended to obtain the second latex antibody reagent.

[0036] The first latex antibody reagent and the second latex antibody reagent are mixed in a certain proportion to obtain the latex antibody reagent.

[0037] In some embodiments, the large-diameter latex microspheres are washed, centrifuged, and reconstituted prior to coupling;

[0038] And / or, prior to coupling, the small-diameter latex microspheres are washed, centrifuged, and reconstituted;

[0039] And / or, the volume ratio of the carbodiimide and N-hydroxythiosuccinimide is 3-5:6-10, preferably 1:2, the concentration of carbodiimide is 10-30 mg / mL, and the concentration of succinimide is 20-30 mg / mL.

[0040] And / or, the sealing liquid used in the sealing step includes at least one of DB1130, CE510, and CE210;

[0041] And / or, in the resuspension step, a preservation solution is used for resuspension, the preservation solution comprising a buffer, a stabilizer, a surfactant, and a preservative; wherein the buffer comprises any one of piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer; the stabilizer comprises at least one of gelatin or casein; the surfactant is at least one of polyoxyethylene fatty alcohol ether, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, and polysorbate; and the preservative is at least one of sodium azide, gentamicin, and isothiazolinones.

[0042] The embodiments disclosed in this invention relate to the use of a kit for determining p2PSA based on latex-enhanced immunoturbidimetry in the preparation of products for determining p2PSA.

[0043] The embodiments disclosed in this invention relate to a method for determining p2PSA based on latex-enhanced immunoturbidimetric assay. The method involves using a sample to be tested and performing an immunoassay using the kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay according to any one of claims 1-5. The turbidity of the resulting reaction product is then detected.

[0044] In some embodiments, the process includes: mixing the test sample and calibrator with the reaction solution, incubating, then adding latex antibody reagent, and then measuring the absorbance of the test sample and calibrator respectively; plotting a standard curve based on the content of the calibrator and the measured absorbance, and calculating the p2PSA content in the test sample based on the standard curve.

[0045] Optionally, the volume ratio of the test sample, reaction solution, and latex antibody reagent is 10-20:90-270:30-90, preferably 15:180:60.

[0046] The technical solution of this invention has the following advantages:

[0047] 1. This invention provides a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay. The kit includes: a reaction solution and a latex antibody reagent; the reaction solution includes at least one of buffer, inorganic salt, sensitizer, surfactant, blocker, and preservative; the latex antibody reagent includes a mixture of a first latex antibody reagent and a second latex antibody reagent; the first latex antibody reagent is formed by coupling a polyclonal antibody of p2PSA with large-particle latex microspheres; the particle size range of the large-particle latex microspheres is 172–405 nm; the second latex antibody reagent is formed by coupling a monoclonal antibody of p2PSA with small-particle latex microspheres; the particle size range of the small-particle latex microspheres is 71–121 nm; the monoclonal antibody of p2PSA includes three different monoclonal antibodies of p2PSA; this invention has found that for the determination of p2PSA using latex-enhanced immunoturbidimetric assay... SA, the particle size of latex microspheres affects the sensitivity, specificity, and linearity of p2PSA assays. It has been found that larger particle sizes result in higher sensitivity, while smaller particle sizes result in a wider linear range. The latex antibody reagent prepared from latex microspheres within the aforementioned particle size range selected in this invention can ensure sensitivity while simultaneously broadening the linear range and enhancing specificity. The p2PSA monoclonal antibody comprises three different monoclonal antibodies simultaneously coated with small-diameter microspheres. Monoclonal antibodies have high specificity, but a single monoclonal antibody only recognizes a specific antigenic epitope. This extremely high specificity can reduce sensitivity. Therefore, this invention uses three different monoclonal antibodies simultaneously coated with small-diameter microspheres. Compared to the traditional pairwise pairing of monoclonal antibodies, this results in higher specificity and targets more antigen binding sites. In summary, the above kit for detecting p2PSA can simultaneously achieve high sensitivity, high specificity, and a wide linear range.

[0048] 2. This invention provides a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay, wherein the polyclonal antibody for p2PSA is p2PSA-pAb; and / or, the monoclonal antibodies for p2PSA include p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies; p2PSA-1 monoclonal antibody is p2PSA-mAb-1; p2PSA-2 monoclonal antibody is p2PSA-mAb-2; and p2PSA-3 monoclonal antibody is p2PSA-mAb-2. Ab-3; This invention has discovered that for the latex-enhanced immunoturbidimetric assay of p2PSA, a method using large-particle microspheres conjugated with a p2PSA polyclonal antibody with higher affinity, and small-particle microspheres conjugated with three monoclonal antibodies (p2PSA-1, p2PSA-2, and p2PSA-3) with higher specificity, is more effective than traditional monoclonal antibody methods. Because the three different monoclonal antibodies (p2PSA-1, p2PSA-2, and p2PSA-3) are simultaneously coated with small-particle microspheres, this method differs from traditional monoclonal antibody methods. The advantage of latex-enhanced immunoturbidimetric assay lies in the high specificity of monoclonal antibodies. A single monoclonal antibody recognizes only a specific antigenic epitope, and this extremely high specificity can reduce sensitivity. Therefore, this invention employs a detection method that simultaneously coats small-diameter microspheres with three different monoclonal antibodies. Compared to the traditional method of pairing monoclonal antibodies, this method has higher specificity, targets more antigen binding sites, and avoids competition between antibodies for binding sites, thus further improving detection sensitivity. Therefore, this invention uses large-diameter microspheres coupled with selected polyclonal antibodies with strong affinity to ensure reagent sensitivity, and uses small-diameter microspheres coupled with three selected monoclonal antibodies with strong specificity to ensure reagent linearity and specificity. The two are mixed to prepare the latex antibody reagent (R2), perfectly avoiding the problems of low monoclonal antibody sensitivity and poor polyclonal antibody specificity. Combining the two ensures both sensitivity and broadens the linearity of the reagent, while also providing strong specificity.

[0049] 3. This invention provides a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay. The sensitizer used in R1 is a Biolipidure series product, which differs from traditional polyethylene glycol turbidiizers. Biolipidure series sensitizers offer superior efficacy and higher specificity. Mechanism of action: Biolipidure-103 has strong hydrophilicity, occupying a certain physical space in solution, indirectly increasing the concentration of antigen and antibody; additionally, it forms a loose, porous structure in solution, providing a smaller, more efficient reaction space for antigen and antibody. Biolipidure-405 has an anionic functional group and strong dispersibility, greatly improving the dispersion of antigen and antibody and enhancing reaction uniformity; the high molecular structure of Biolipidure-405 also occupies a certain physical space in solution, indirectly increasing the concentration of antigen and antibody and improving reactivity.

[0050] 4. This invention provides a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay, wherein the blocking agent is at least one of THBR-1, THBR-2, and A1101; preferably, it is THBR-1, THBR-2, and A1101 in a volume ratio of 1:1:1. In R1 of this invention, three different blocking agents are used simultaneously, which provides better anti-interference effect compared to the traditional method of adding only one blocking agent. Traditional blocking agents can reduce interference from endogenous substances to a certain extent, but not all immune interference can be eliminated. This invention uses three different blocking agents in combination, which can eliminate false positive results caused by various types of interference, making it highly efficient and convenient. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This refers to the standard curve of the calibrator in Example 18 of the present invention;

[0053] Figure 2 This refers to the correlation between the sample measurement results and the theoretical results in Example 18 of the present invention;

[0054] Figure 3 This is the result of the influence of microspheres of different particle sizes on the signal value in the experimental example of this invention;

[0055] Figure 4 This is the result of the effect of mixing different monoclonal antibodies on the signal value in experimental example 2) of the present invention;

[0056] Figure 5 This is the result of the effect of the mixing ratio of microspheres of different particle sizes on the signal value in experimental example 3) of the present invention;

[0057] Figure 6 This is the result of the effect of different turbidifying agents on the signal value in experimental example 4) of the present invention;

[0058] Figure 7 This is a comparison of the detection results of the kit in Example 1 and the determination results of the Beckman Coulter kit in the accuracy experiment of Experiment 8) of the present invention. Detailed Implementation

[0059] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0060] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0061] This invention has found that in the application of latex-enhanced immunoturbidimetric assays, labeling with polyclonal antibodies can improve sensitivity to some extent, but polyclonal antibodies have poor specificity, leading to decreased accuracy of detection results. Labeling latex with monoclonal antibodies can improve reagent specificity, but sensitivity is affected. To address these issues, this invention combines the characteristics of antigens / antibodies and latex microspheres. The selection of antigens / antibodies mainly involves comparing their isoelectric point, specificity, and affinity. Raw materials with higher affinity have higher sensitivity, but excessively high affinity may affect linearity; raw materials with better specificity have better clinical relevance. The selection of latex microspheres mainly involves comparing particle size: larger particle size results in higher sensitivity; smaller particle size results in a wider linear range. A latex antibody reagent (R2) was prepared by conjugating a highly affinity p2PSA polyclonal antibody with large-particle microspheres and a highly specific p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibody with small-particle microspheres in a 1:1 ratio. This method perfectly avoids the problems of low sensitivity of monoclonal antibodies and poor specificity of polyclonal antibodies. Combining the two ensures both sensitivity and broadens the linearity of the reagent, while also providing strong specificity. Furthermore, the addition of an inhibitor significantly enhances the reagent's anti-interference ability.

[0062] Therefore, this invention discloses a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay and its preparation method. The main steps are as follows: screening out highly specific p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies and highly affinity p2PSA polyclonal antibodies; conjugating polyclonal antibodies with large-particle latex microspheres and monoclonal antibodies with small-particle microspheres; then mixing the two in a 1:1 ratio to form a latex antibody reagent; preparing reagent R1; and finally assembling the latex antibody reagent (R2 reagent), reagent R1, and calibrators into a kit. This method can simultaneously improve the sensitivity, specificity, and linearity of p2PSA determination using latex-enhanced immunoturbidimetric assay, resulting in a kit with higher sensitivity, a wider linear range, and higher accuracy. It has extremely high economic value and broad market application prospects. The reagent kit prepared by the method described in this invention has a detection limit as low as 1.02 pg / mL; it exhibits excellent reagent precision, with a CV controlled within 1%, far below the industry standard requirement of 10%; and its linear range can reach up to 8000 pg / mL, significantly higher than the 5000 pg / mL linearity of conventional reagent kits, with a correlation coefficient R0. 2 Greater than 0.99; the reagent has strong anti-interference ability, bilirubin ≤40mg / dL, fat emulsion ≤0.5%, RF ≤400IU / mL, hemoglobin ≤500mg / dL, high clinical comparison accuracy, and the correlation coefficient R2 is greater than the industry standard requirement of 0.95.

[0063] In the following examples, Biolipidure 103 and Biolipidure 405 were purchased from Beijing Bomei Biotechnology Co., Ltd.

[0064] The inhibitors THBR-1, THBR-2, and A1101 were purchased from Beijing Bomei Biotechnology Co., Ltd.

[0065] The blocking solutions DB1130, CE510, and CE210 were purchased from Beijing Bomei Biotechnology Co., Ltd.

[0066] The polyclonal antibody for p2PSA involved in the following examples is p2PSA-pAb, the monoclonal antibody for p2PSA-1 is p2PSA-mAb-1, the monoclonal antibody for p2PSA-2 is p2PSA-mAb-2, and the monoclonal antibody for p2PSA-3 is p2PSA-mAb-3; all the above antibodies were purchased from Wuhan Aokebotai Biotechnology Co., Ltd.

[0067] Example 1

[0068] This embodiment provides a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay, comprising: p2PSA reagent R1 (reaction solution) and p2PSA reagent R2 (latex antibody reagent).

[0069] I. Preparation of p2PSA reagent R1 (reaction solution):

[0070] Accurately weigh the chemical substances according to the proportions in List 1, place them in a beaker containing 0.8L of pure water, stir and mix well, adjust the pH value to 7.20±0.05 with 3mol / L dilute hydrochloric acid, transfer the solution to a 1L volumetric flask and make up to volume, filter the solution with a 0.22um filter membrane, store the prepared solution in a glass container, label it, and store it at 2~8℃.

[0071]

[0072] II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0073] 1. Washing: Take 100 μL of microspheres (JSR 405 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 20 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 5 min, with sonication for 0.5 s and an interval of 0.5 s. Repeat the same procedure with 100 μL of microspheres (JSR 71 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 30 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 5 min, with sonication for 0.5 s and an interval of 0.5 s.

[0074] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the 405nm (1000uL) and 71nm (1000uL) microspheres dispersed in step 1 for activation (EDC concentration was 20mg / mL, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the microspheres were reconstituted with 50Mm boric acid-borax at pH 8.0 to 1000uL. The microspheres were then dispersed by sonication for 5min, with sonication intervals of 0.5s.

[0075] 3. Coupling: Take p2PSA polyclonal antibody and a mixture of p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies (the three strains are mixed in a mass ratio of 1:1:1). Add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL to obtain polyclonal antibody dilution (concentration of 5mg / ml) and monoclonal antibody mixture dilution (concentration of 10mg / ml). Then add 1000uL of activated microspheres to each. Add 405nm activated microspheres (concentration of 1% w / v) to the polyclonal antibody dilution and 71nm activated microspheres (concentration of 0.5% w / v) to the monoclonal antibody mixture dilution. Place in a shaker for coupling at 30℃ for 4h.

[0076] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 30 minutes and discard the supernatant. The blocking solution is DB1130.

[0077] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0078] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20). The precipitate was dispersed by sonication for 5 min, followed by 0.5 s intervals. The resuspended latex antibody reagent for 405 nm microspheres (first latex antibody reagent) (concentration 0.3 w / v%) and latex antibody reagent for 71 nm microspheres (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8 °C for later use.

[0079] Comparative Example 1

[0080] The difference between this embodiment and Embodiment 1 is as follows: II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0081] 1. Washing: Take 100 μL of microspheres (JSR 405 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G centrifugation force, 20 min), remove the supernatant and reconstitute with 1000 μL of MES. Disperse the microspheres by sonication for 5 min in a cell disruptor, sonicating for 0.5 s with a 0.5 s interval.

[0082] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the dispersed 405nm microspheres (volume 1000uL) in step 1 for activation (EDC concentration was 20mg / mL, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the mixture was reconstituted with 50Mm boric acid-borax at pH 8.0 to 1000uL. The mixture was then dispersed by sonication for 5min, with sonication intervals of 0.5s.

[0083] 3. Coupling: Take the p2PSA polyclonal antibody and monoclonal antibody mixture (p2PSA-1, p2PSA-2, and p2PSA-3 are mixed in a mass ratio of 1:1:1), add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL, to obtain the polyclonal antibody dilution (concentration of 5mg / ml) and the monoclonal antibody mixture dilution (concentration of 10mg / ml). Then add 1000uL of activated 405nm microspheres (concentration of 1% w / v) to each, and place them in a shaker for coupling at 30℃ for 4h.

[0084] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 30 minutes and discard the supernatant. The blocking solution is DB1130.

[0085] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0086] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20), and dispersed by sonication for 5 min in a cell disruptor, with sonication for 0.5 s and an interval of 0.5 s. The resuspended latex antibody reagent (first latex antibody reagent) of 405 nm microspheres conjugated with polyclonal antibodies (concentration 0.3 w / v%) was mixed with the latex antibody reagent (second latex antibody reagent) of 405 nm microspheres conjugated with monoclonal antibodies (concentration 0.1 w / v%) at a volume ratio of 1:1 and stored at 2-8 °C for later use.

[0087] Comparative Example 2

[0088] The difference between this embodiment and Example 1 lies in the preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0089] 1. Washing: Take 100 μL of microspheres (JSR71nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (25000G centrifugation force, 30 min), remove the supernatant and reconstitute with 1000 μL of MES. Disperse the microspheres by sonication for 5 min in a cell disruptor, sonicating for 0.5 s with a 0.5 s interval.

[0090] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the dispersed 71nm microspheres (volume 1000uL) in step 1 for activation (EDC concentration was 20mg / ml, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the mixture was reconstituted with 50mM boric acid-borax at pH 8.0 to 1000uL. The mixture was then dispersed by sonication for 5min, with sonication intervals of 0.5s.

[0091] 3. Coupling: Take the p2PSA polyclonal antibody and monoclonal antibody mixture (p2PSA-1, p2PSA-2, and p2PSA-3 are mixed in a mass ratio of 1:1:1), add 50 Mm boric acid-borax pH 8.0 to make a final volume of 2000 uL, to obtain polyclonal antibody dilution (concentration of 5 mg / ml) and monoclonal antibody mixture dilution (concentration of 10 mg / ml), then add 1000 uL of activated 71 nm microspheres (concentration of 0.5% w / v) to each, and place them in a shaker for coupling at 30℃ for 4 h.

[0092] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 40 minutes and discard the supernatant. The blocking solution is DB1130.

[0093] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0094] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20), and dispersed by sonication for 5 min in a cell disruptor, with sonication for 0.5 s and an interval of 0.5 s. The resuspended latex antibody reagent (first latex antibody reagent) of 71 nm microspheres conjugated with polyclonal antibodies (concentration 0.3 w / v%) was mixed with the latex antibody reagent (second latex antibody reagent) of 71 nm microspheres conjugated with monoclonal antibodies (concentration 0.1 w / v%) at a volume ratio of 1:1 and stored at 2-8 °C for later use.

[0095] Comparative Example 3

[0096] The difference between this embodiment and Example 1 lies in the preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0097] 1. Washing: Take 100 μL of microspheres (JSR272 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G centrifugation force, 30 min), remove the supernatant and reconstitute with 1000 μL of MES. Disperse the microspheres by sonication for 5 min in a cell disruptor, sonicating for 0.5 s with a 0.5 s interval.

[0098] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the dispersed 272nm microspheres (volume 1000uL) in step 1 for activation (EDC concentration was 20mg / ml, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the mixture was reconstituted with 50mM boric acid-borax at pH 8.0 to 1000uL. The mixture was then dispersed by sonication for 5min, with sonication intervals of 0.5s.

[0099] 3. Coupling: Take the p2PSA polyclonal antibody and monoclonal antibody mixture (p2PSA-1, p2PSA-2, and p2PSA-3 are mixed in a mass ratio of 1:1:1), add 50 Mm boric acid-borax pH 8.0 to make a final volume of 2000 uL, and obtain the polyclonal antibody dilution (concentration of 5 mg / ml) and the monoclonal antibody mixture dilution (concentration of 10 mg / ml). Then add 1000 uL of the activated 272 nm microspheres to each, and place them in a shaker for coupling at 30℃ for 4 h.

[0100] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 40 minutes and discard the supernatant. The blocking solution is DB1130.

[0101] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0102] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20), and dispersed by sonication for 5 min in a cell disruptor, with sonication for 0.5 s and an interval of 0.5 s. The resuspended latex antibody reagent (first latex antibody reagent) of 272 nm microspheres conjugated with polyclonal antibodies (concentration 0.3 w / v%) was mixed with the latex antibody reagent (second latex antibody reagent) of 272 nm microspheres conjugated with monoclonal antibodies (concentration 0.1 w / v%) at a volume ratio of 1:1 and stored at 2-8 °C for later use.

[0103] Comparative Example 4

[0104] The difference between this embodiment and Example 1 lies in the preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0105] 1. Washing: Take 100 μL of microspheres (JSR83nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (also known as MES: 20 mM pH 5.5) to wash. Centrifuge (25000G centrifugation force, 30 min), remove the supernatant and reconstitute with 1000 μL of MES. Disperse the microspheres by sonication for 5 min in a cell disruptor, sonicating for 0.5 s with a 0.5 s interval.

[0106] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the dispersed 83nm microspheres (volume 1000uL) in step 1 for activation (EDC concentration was 20mg / ml, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the mixture was reconstituted with 50mM boric acid-borax at pH 8.0 to 1000uL. The mixture was then dispersed by sonication for 5min, with sonication intervals of 0.5s.

[0107] 3. Coupling: Take the p2PSA polyclonal antibody and monoclonal antibody mixture (p2PSA-1, p2PSA-2, and p2PSA-3 are mixed in a mass ratio of 1:1:1), add 50 Mm boric acid-borax pH 8.0 to make a final volume of 2000 uL, and obtain the polyclonal antibody dilution (concentration of 5 mg / ml) and the monoclonal antibody mixture dilution (concentration of 10 mg / ml). Then add 1000 uL of the activated 83 nm microspheres to each, and place them in a shaker for coupling at 30℃ for 4 h.

[0108] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 40 minutes and discard the supernatant. The blocking solution is DB1130.

[0109] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0110] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20), and dispersed by sonication for 5 min in a cell disruptor, with sonication for 0.5 s and an interval of 0.5 s. The resuspended latex antibody reagent (first latex antibody reagent) of 83 nm microspheres conjugated with polyclonal antibodies (concentration 0.3 w / v%) was mixed with the latex antibody reagent (second latex antibody reagent) of 83 nm microspheres conjugated with monoclonal antibodies (concentration 0.1 w / v%) at a volume ratio of 1:1 and stored at 2-8 °C for later use.

[0111] Comparative Example 5

[0112] The difference between this embodiment and Embodiment 1 lies in the preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0113] 1. Washing: Take 100 μL of microspheres (JSR 405 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 20 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 5 min, with a sonication time of 0.5 s and an interval of 0.5 s. Repeat the same procedure with 100 μL of microspheres (JSR 71 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 30 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 5 min, with a sonication time of 0.5 s and an interval of 0.5 s.

[0114] 2. Activation: EDC and sulfo-NHS (4uL:8uL ratio) were added to the dispersed 405nm and 71nm microspheres in step 1 for activation (EDC concentration was 20mg / ml, sulfo-NHS concentration was 30mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, the supernatant was removed, and the mixture was reconstituted with 50Mm boric acid-borax at pH 8.0 to 1000uL. The mixture was then sonicated for 5min using a cell disruptor, with sonication intervals of 0.5s.

[0115] 3. Coupling: Take the mixture of p2PSA polyclonal antibody and monoclonal antibody (p2PSA-1 and p2PSA-2 are mixed in a 1:1 mass ratio), add 50mM boric acid-borax pH8.0 to make a final volume of 2000uL, to obtain polyclonal antibody dilution (concentration of 5mg / ml) and monoclonal antibody mixture dilution (concentration of 10mg / ml), and then add 1000uL of activated microspheres respectively. Add 405nm activated microspheres (concentration of 1% w / v) to the polyclonal antibody dilution and 71nm activated microspheres (concentration of 0.5% w / v) to the monoclonal antibody mixture dilution. Place in a shaker for coupling at 30℃ for 4h.

[0116] 4. Blocking: Add 230 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 30 minutes and discard the supernatant. The blocking solution is DB1130.

[0117] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0118] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.5% TWEEN-20 + 0.1% sodium azide, pH 7.20), and dispersed by sonication for 5 min in a cell disruptor, with sonication for 0.5 s and an interval of 0.5 s. The resuspended 405 nm microsphere latex reagent (first latex antibody reagent) (concentration 0.3 w / v%) and 71 nm microsphere latex reagent (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8 °C for later use.

[0119] Comparative Example 6

[0120] The difference between this embodiment and Comparative Example 5 is that the p2PSA-2 antibody is replaced with the p2PSA-3 antibody.

[0121] Comparative Example 7

[0122] The difference between this embodiment and Comparative Example 5 is that the p2PSA-1 antibody is replaced with the p2PSA-3 antibody.

[0123] Example 2

[0124] The difference between this embodiment and Example 1 is that the reconstituted 405nm microsphere latex reagent (first latex antibody reagent) and 71nm microsphere latex reagent (second latex antibody reagent) are mixed in a 2:1 volume ratio.

[0125] Example 3

[0126] The difference between this embodiment and Example 1 is that the reconstituted 405nm microsphere latex reagent (first latex antibody reagent) and 71nm microsphere latex reagent (second latex antibody reagent) are mixed in a 1:2 volume ratio.

[0127] Example 4

[0128] The difference between this embodiment and Example 1 is that the reconstituted 405nm microsphere latex reagent (first latex antibody reagent) and 71nm microsphere latex reagent (second latex antibody reagent) are mixed in a 3:1 volume ratio.

[0129] Example 5

[0130] The difference between this embodiment and Example 1 is that the reconstituted 405nm microsphere latex reagent (first latex antibody reagent) and 71nm microsphere latex reagent (second latex antibody reagent) are mixed in a volume ratio of 1:3.

[0131] Example 6

[0132] The difference between this embodiment and Example 1 is that the Biolipidure 103 in the reaction solution formulation in Table 1 is replaced with PEG6000 by mass.

[0133] Example 7

[0134] The difference between this embodiment and Example 1 is that the equivalent mass of Biolipidure 103 in the reaction solution formulation in Table 1 is replaced with Biolipidure 405.

[0135] Example 8

[0136] I. Preparation of p2PSA reagent R1 (reaction solution):

[0137] Accurately weigh the chemical substances according to the proportions in the list, place them in a beaker containing 0.8L of pure water, stir and mix well, adjust the pH value to 7.50±0.05 with 3mol / L dilute hydrochloric acid, transfer the solution to a 1L volumetric flask and make up to volume, filter the solution with a 0.22um filter membrane, store the prepared solution in a glass container, label it, and store it at 2~8℃.

[0138]

[0139] II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0140] 1. Washing: Take 100 μL of microspheres (JSR 218 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 30 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 8 min, with sonication for 0.5 s and an interval of 0.5 s. Repeat the same procedure with 100 μL of microspheres (JSR 89 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 35 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 5 min, with sonication for 0.5 s and an interval of 0.5 s.

[0141] 2. Activation: EDC and sulfo-NHS (5uL:10uL ratio) were added to the 218nm (1000uL) and 89nm (1000uL) microspheres dispersed in step 1 for activation (EDC concentration was 30mg / mL, sulfo-NHS concentration was 20mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 30min, and reconstituted with 50Mm boric acid-borax at pH 8.0 to 1000uL. The microspheres were then dispersed by sonication for 8min, with sonication intervals of 0.5s.

[0142] 3. Coupling: Take p2PSA polyclonal antibody and a mixture of p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies (the three strains are mixed in a mass ratio of 1:1:1). Add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL to obtain a polyclonal antibody dilution (concentration of 5mg / ml) and a monoclonal antibody mixture dilution (concentration of 10mg / ml). Then add 1000uL of activated microspheres to each. Add 218nm activated microspheres to the polyclonal antibody dilution and 89nm activated microspheres to the monoclonal antibody mixture dilution. Place in a shaker for coupling at 30℃ for 4h.

[0143] 4. Blocking: Add 280 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 30 minutes and discard the supernatant. The blocking solution is DB1130.

[0144] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0145] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mm PIPES + 0.5% BSA + 0.1% TWEEN-20 + 0.1% sodium azide, pH 7.5), and dispersed by sonication for 8 min in a cell disruptor, with sonication for 0.5 s and 0.5 s intervals. The resuspended latex antibody reagent for 218 nm microspheres (first latex antibody reagent) (concentration 0.3 w / v%) and latex antibody reagent for 89 nm microspheres (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8 °C for later use.

[0146] Example 9

[0147] I. Preparation of p2PSA reagent R1 (reaction solution):

[0148] Accurately weigh the chemical substances according to the proportions in the list, place them in a beaker containing 0.8L of pure water, stir and mix well, adjust the pH value to 7.80±0.05 with 3mol / L dilute hydrochloric acid, transfer the solution to a 1L volumetric flask and make up to volume, filter the solution with a 0.22um filter membrane, store the prepared solution in a glass container, label it, and store it at 2~8℃.

[0149]

[0150] II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0151] 1. Washing: Take 100 μL of microspheres (JSR 218 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 40 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 10 min, with sonication for 0.5 s and an interval of 0.5 s. Repeat the same procedure with 100 μL of microspheres (JSR 100 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 35 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the microspheres using a cell disruptor by sonication for 5 min, with sonication for 0.5 s and an interval of 0.5 s.

[0152] 2. Activation: EDC and sulfo-NHS (3uL:6uL ratio) were added to the 218nm (1000uL) and 100nm (1000uL) microspheres dispersed in step 1 for activation (EDC concentration was 10mg / mL, sulfo-NHS concentration was 25mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 40min, the supernatant was removed, and the microspheres were reconstituted with 50mM boric acid-borax at pH 8.0 to 1000uL. The microspheres were then dispersed by sonication for 8min, with sonication intervals of 0.5s.

[0153] 3. Coupling: Take p2PSA polyclonal antibody and a mixture of p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies (the three strains are mixed in a mass ratio of 1:1:1). Add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL to obtain a polyclonal antibody dilution (concentration of 5mg / ml) and a monoclonal antibody mixture dilution (concentration of 10mg / ml). Then add 1000uL of activated microspheres to each. The polyclonal antibody dilution should contain 218nm activated microspheres, and the monoclonal antibody mixture dilution should contain 100nm activated microspheres. Place them in a shaker for coupling at 30℃ for 3.5h.

[0154] 4. Blocking: Add 310 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 40 minutes and discard the supernatant. The blocking solution is DB1130.

[0155] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0156] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 0.5% BSA + 0.2% TWEEN-20 + 0.1% sodium azide, pH 7.8), and dispersed by sonication for 10 min using a cell disruptor, with sonication for 0.5 s and intervals of 0.5 s. The resuspended latex antibody reagent for 218 nm microspheres (first latex antibody reagent) (concentration 0.3 w / v%) and latex antibody reagent for 100 nm microspheres (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8 °C for later use.

[0157] Example 10

[0158] I. Preparation of p2PSA reagent R1 (reaction solution):

[0159] Accurately weigh the chemical substances according to the proportions in the list, place them in a beaker containing 0.8L of pure water, stir and mix well, adjust the pH value to 8.00±0.05 with 3mol / L dilute hydrochloric acid, transfer the solution to a 1L volumetric flask and make up to volume, filter the solution with a 0.22um filter membrane, store the prepared solution in a glass container, label it, and store it at 2~8℃.

[0160]

[0161]

[0162] II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0163] 1. Washing: Take 100 μL of microspheres (JSR 192 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 50 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 12 min, with sonication for 0.5 s and 0.5 s intervals. Repeat the same procedure with 100 μL of microspheres (JSR 108 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 35 min), remove the supernatant, and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 5 min, with sonication for 0.5 s and 0.5 s intervals.

[0164] 2. Activation: EDC and sulfo-NHS (3uL:6uL ratio) were added to the 192nm and 108nm microspheres dispersed in step 1 for activation (EDC concentration was 25mg / ml, sulfo-NHS concentration was 20mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 50min, the supernatant was removed, and the mixture was reconstituted with 50Mm boric acid-borax at pH 8.0 to 1000uL. The mixture was then sonicated for 10min using a cell disruptor, with sonication intervals of 0.5s.

[0165] 3. Coupling: Take p2PSA polyclonal antibody and a mixture of p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies (p2PSA-1, p2PSA-2, and p2PSA-3 in a mass ratio of 1:1:1), add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL, to obtain polyclonal antibody dilution (concentration of 5mg / ml) and monoclonal antibody mixture dilution (concentration of 10mg / ml), and then add 1000uL of activated microspheres respectively. Add 192nm activated microspheres to the polyclonal antibody dilution and 108nm activated microspheres to the monoclonal antibody mixture dilution. Place in a shaker for coupling at 30℃ for 3h.

[0166] 4. Blocking: Add 310 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 50 minutes and discard the supernatant. The blocking solution is DB1130.

[0167] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0168] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.2% TWEEN-20 + 0.1% Proclin 300, pH 8.0), and dispersed by sonication for 10 min using a cell disruptor, with sonication intervals of 0.5 s. The resuspended latex antibody reagent for 192 nm microspheres (first latex antibody reagent) (concentration 0.3 w / v%) and latex antibody reagent for 108 nm microspheres (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8℃ for later use.

[0169] Example 11

[0170] I. Preparation of p2PSA reagent R1 (reaction solution):

[0171] Accurately weigh the chemical substances according to the proportions in the list, place them in a beaker containing 0.8L of pure water, stir and mix well, adjust the pH value to 8.20±0.05 with 3mol / L dilute hydrochloric acid, transfer the solution to a 1L volumetric flask and make up to volume, filter the solution with a 0.22um filter membrane, store the prepared solution in a glass container, label it, and store it at 2~8℃.

[0172]

[0173] II. Preparation of p2PSA reagent R2, i.e., latex antibody reagent:

[0174] 1. Washing: Take 100 μL of microspheres (JSR 172 nm) (10% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (20000 G, 55 min). Remove the supernatant and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 15 min, sonicating for 0.5 s with a 0.5 s interval. Repeat the same procedure with 100 μL of microspheres (JSR 121 nm) (5% w / v) and add 900 μL of 2-(N-morpholino)-ethanesulfonic acid (MES: 20 mM pH 5.5) to wash. Centrifuge (25000 G, 35 min) to wash. Remove the supernatant and reconstitute with 1000 μL of MES. Disperse the cells using a cell disruptor by sonication for 5 min, sonicating for 0.5 s with a 0.5 s interval.

[0175] 2. Activation: EDC and sulfo-NHS (3uL:6uL ratio) were added to the 172nm (1000uL) and 121nm (1000uL) microspheres dispersed in step 1 for activation (EDC concentration was 30mg / mL, sulfo-NHS concentration was 25mg / mL). EDC was added first, followed by sulfo-NHS. Activation was carried out at 30℃ for 20min, centrifuged at 20000G for 55min, the supernatant was removed, and the mixture was reconstituted with 50mM boric acid-borax at pH 8.0 to 1000uL. The mixture was then sonicated for 15min using a cell disruptor, with sonication intervals of 0.5s.

[0176] 3. Coupling: Take p2PSA polyclonal antibody and a mixture of p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies (the three strains are mixed in a mass ratio of 1:1:1). Add 50mM boric acid-borax pH 8.0 to make a final volume of 2000uL to obtain a polyclonal antibody dilution (concentration of 5mg / ml) and a monoclonal antibody mixture dilution (concentration of 10mg / ml). Then add 1000uL of activated microspheres to each. Add 172nm activated microspheres to the polyclonal antibody dilution and 121nm activated microspheres to the monoclonal antibody mixture dilution. Place in a shaker for coupling at 30℃ for 2.5h.

[0177] 4. Blocking: Add 310 μL of blocking solution and block on a shaker for 1 hour. Centrifuge at 20,000 G for 50 minutes and discard the supernatant. The blocking solution is DB1130.

[0178] 5. Washing: Wash the precipitate twice with washing solution, and centrifuge to remove the supernatant.

[0179] 6. Resuspension: The precipitate was resuspended in 2.5 mL of latex diluent (50 mM PIPES + 1% BSA + 0.2% TWEEN-20 + 0.1% Proclin 300, pH 8.5), and dispersed by sonication for 15 min in a cell disruptor, with sonication for 0.5 s and 0.5 s intervals. The resuspended latex antibody reagent for 172 nm microspheres (first latex antibody reagent) (concentration 0.3 w / v%) and latex antibody reagent for 121 nm microspheres (second latex antibody reagent) (concentration 0.1 w / v%) were mixed at a 1:1 volume ratio and stored at 2-8 °C for later use.

[0180] Example 12

[0181] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is only THBR-1, 60 μL.

[0182] Example 13

[0183] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is only THBR-2 at 60 μL.

[0184] Example 14

[0185] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is only A1101 at 60 μL.

[0186] Example 15

[0187] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is replaced with THBR-1 at 30 μL and THBR-2 at 30 μL.

[0188] Example 16

[0189] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is replaced with THBR-1 at 30 μL and A1101 at 30 μL.

[0190] Example 17

[0191] The difference between this embodiment and Example 1 is that the blocking agent in the reaction solution formulation is replaced with THBR-2 at 30 μL and A1101 at 30 μL.

[0192] Example 18

[0193] This embodiment provides a method for determining p2PSA based on latex-enhanced immunoturbidimetry, including the following steps:

[0194] Testing instrument: Hitachi 7180 fully automated biochemical analyzer;

[0195] Temperature: 37℃; Cuvette optical path: 1cm;

[0196] Analysis method: Two-point endpoint method (absorbance at point 16 and point 34), main wavelength: 570nm;

[0197] Reagent volume: S:R1:R2 = 15uL:180uL:60uL (where S is the sample, R1 is the reaction solution, and R2 is the latex antibody reagent);

[0198] Calibrator formulation: 20 mM PB (phosphate buffer); 0.9 wt% NaCl; 0.1 v / v% P300; human p2PSA antigen concentrations of 0, 506.3, 1012.5, 2025, 4050, and 8100 pg / mL;

[0199] The steps for using the kit described in this invention are as follows: Mix reaction solution R1 and sample (or purified water, or bridging standard), incubate at 37°C for 5 minutes, then add latex antibody reagent R2, mix well, incubate at 37°C for 30 seconds, read the absorbance A1 of each tube, incubate for another 4.5 minutes, and read the absorbance A2 of each tube. See the table below for reference:

[0200] Table 6

[0201]

[0202] The calibration method is a multi-point nonlinear spline. A multi-point calibration mode is used, with purified water as the zero point, to calibrate the calibrators and establish a working curve.

[0203] Calculation method: A calibration curve is fitted to the corresponding ΔA using the calibrator concentration. Taking the kit from Example 1 as an example, the calibration curve is as follows: Figure 1 As shown, the sample concentration was obtained through a calibration curve. The correlation between the measured value and the theoretical concentration was analyzed, and the results are as follows: Figure 2 As shown.

[0204] Experimental kit performance evaluation

[0205] 1) The effect of microspheres of different sizes on signal values

[0206] Comparing Example 1 and Comparative Examples 1-4, the calibrator solution was fitted with a standard curve according to Example 18. The study found that Example 1 (a mixture of microspheres of varying sizes) had higher sensitivity and a wider linear range than single 405nm microspheres (Comparative Example 1), 71nm microspheres (Comparative Example 2), 272nm microspheres (Comparative Example 3), and 83nm microspheres (Comparative Example 4). See the table below for details. Figure 3While single large-diameter microspheres exhibit high sensitivity at 405 nm, their linearity is poor, making it impossible to distinguish the signal values ​​at 4050 pg / mL from those at 8100 pg / mL. Single small-diameter microspheres, while showing broad linearity at 71 nm, also suffer from poor sensitivity, with a very weak signal value at 506.3 pg / mL. Mixing microspheres of varying sizes ensures both sensitivity and linearity. Therefore, Example 1 (mixing microspheres of varying sizes) represents the optimal conditions.

[0207] Table 7. Comparison of signal values ​​for microspheres of different particle sizes

[0208]

[0209] 2) Effect of mixing different monoclonal antibodies on signal values

[0210] Comparing Example 1 of this invention with Comparative Examples 5-7, the calibrator solution was fitted with a standard curve according to Example 18. The study found that Example 1 (p2PSA-1:p2PSA-2:p2PSA-3 = 1:1:1) had higher sensitivity and a wider linear range than the paired monoclonal antibodies p2PSA-1:p2PSA-2 = 1:1 (Comparative Example 5), p2PSA-1:p2PSA-3 = 1:1 (Comparative Example 6), and p2PSA-2:p2PSA-3 = 1:1 (Comparative Example 7). See the table below for details. Figure 4 .

[0211] Table 8. Comparison of mixed signal values ​​of different monoclonal antibody strains

[0212]

[0213] 3) The effect of the mixing ratio of microspheres of different particle sizes on the signal value

[0214] Comparing Examples 1, 2-5 of this invention, and fitting a standard curve using the calibrator solution according to Example 18, the study found that Example 1 (mixing ratio of large and small particle sizes of microspheres of 1:1) exhibited the best linearity, followed by Examples 2 (mixing ratio of large and small particle sizes of microspheres of 2:1) and 3 (mixing ratio of large and small particle sizes of microspheres of 1:2), while Examples 4 (mixing ratio of large and small particle sizes of microspheres of 3:1) and 5 (mixing ratio of large and small particle sizes of microspheres of 1:3) showed the worst linearity. See the table below for details. Figure 5 In summary, the optimal mixing ratio of microspheres of different particle sizes is between 1:2 and 2:1, with a ratio of 1:1 being the most suitable. Beyond this range, linearity deteriorates.

[0215] Table 9 Comparison of mixing ratios of microspheres with different particle sizes

[0216]

[0217] 4) The effect of different sensitizers on signal values

[0218] Comparing Examples 1, 6-7 of this invention, and using calibrator solutions, a standard curve was fitted according to Example 18. The study found that Examples 1 (turbidity enhancer: Biolipidure 103) and 7 (sensitizer: Biolipidure 405) showed good linearity and similar signal values. The signal intensity of Example 6 (sensitizer: PEG6000) was weaker than that of Examples 1 (turbidity enhancer: Biolipidure 103) and 7 (sensitizer: Biolipidure 405). See the table below for details. Figure 6 In conclusion, the Biolipidure series of turbidity enhancers are more effective.

[0219] Table 10. Comparison of different sensitizers

[0220]

[0221] 5) Blank Limit

[0222] The blank sample (physiological saline containing 5% bovine serum) was repeatedly measured 20 times using the kit described in this invention, following the procedure in Example 18. The blank limit was the mean of the blank plus 2 SD, and the specific blank limit is shown in Table 11. The results show that the blank limit of Example 1 was 1.05, which is significantly better than that of the other examples and comparative examples.

[0223] Table 11 Results of Blank Limit Determination of the Reagent Kit of the Present Invention

[0224]

[0225]

[0226] 6) Precision

[0227] The p2PSA sample was tested according to Example 18, repeated 10 times. The mean (X) and standard deviation (s) of the measured values ​​were calculated, and the coefficient of variation (CV) of the measurement results was calculated using the following formula: The results are as follows:

[0228] Table 12 Precision determination results of the kit of the present invention

[0229] Precision Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 1 510.2 522.4 503.5 552.3 561.2 2 513.8 501.3 543.2 501.3 511.3 3 511.1 531.5 551.3 515.3 515.4 4 509.8 509.8 508.1 508.9 509.3 5 510.1 512.3 514.2 518.5 512.3 6 511.2 511.2 513.2 509.3 503.5 7 511.1 509.5 507.5 508.4 511.5 8 509.1 509.1 511.3 543.5 501.2 9 510.2 513.2 510.5 512.4 510.3 10 511.2 541.3 511.5 506.8 513.2 mean 510.78 516.16 517.43 517.67 514.92 SD 1.27 12.06 16.12 16.73 16.83 CV 0.25％ 2.34％ 3.12％ 3.23％ 3.27％

[0230] The test results show that Example 1 has the lowest CV value for precision, which is the best and significantly better than the other comparative examples.

[0231] 7) Linear

[0232] Dilute high-concentration samples (approaching the upper limit of the linear interval) with low-concentration samples (approaching the lower limit of the linear interval) to create at least seven dilution concentrations (xi). Test each of these samples using the kit, following Example 18, with each dilution concentration tested three times. Calculate the mean (yi) of the test results for each dilution concentration. Develop a linear regression equation with dilution concentration (xi) as the independent variable and the mean (yi) of the test results as the dependent variable. This represents the mean of x. Let represent the mean of y. Calculate the correlation coefficient r for linear regression using the following formula: Absolute deviation = Mean - Estimated value. Relative deviation = (Mean - Estimated value) / Estimated value. A linear equation is plotted using theoretical values ​​and the mean. Using Excel, with all theoretical values ​​on the X-axis and all means on the Y-axis, the linear equation is Y = kX + b, where k is the slope and b is the intercept. Then, specific theoretical values ​​are substituted into the linear equation as X-values ​​to calculate the corresponding estimated values. The results are as follows:

[0233] Table 13 Results of Linearity Assay for Kit in Example 1

[0234]

[0235] Table 14. Results of Linearity Assay for Kit in Example 3

[0236]

[0237] Table 15. Results of linearity assay for the kit in Example 4.

[0238]

[0239]

[0240] Table 16. Results of Linearity Assay for Kit in Example 5

[0241]

[0242] Table 17. Results of Linearity Assay for Kit in Example 6

[0243]

[0244]

[0245] Table 18. Results of Linearity Assay for Kit in Example 9

[0246]

[0247] Table 19. Results of Linearity Assay for Kit in Example 10

[0248]

[0249]

[0250] Table 20: Results of Linearity Assay for Kit in Example 11

[0251]

[0252] The test results show that the linear relative deviation of Example 1 is within 10%, which is significantly better than that of other examples.

[0253] 8) Accuracy

[0254] Clinical comparative experiment: The kit of this invention was compared with the prostate-specific antigen isoform assay kit (control) from Beckman Coulter (USA) Inc. 40 clinical serum samples were tested to verify the correlation between the two kits. The method was implemented according to Example 18. The detection results are shown in Table 20. The comparison results between the detection results of the kit in Example 1 and the detection results of the Beckman Coulter kit are as follows. Figure 7 As shown.

[0255] Table 18 Comparison of Clinical Detection Results of the Reagent Kit of the Present Invention (Unit: pg / mL)

[0256]

[0257]

[0258] The test results show that the clinical correlation R between Example 1 and the prostate-specific antigen isoform assay kit from Beckman Coulter (USA) Inc. is 0.9982, which is significantly better than other examples.

[0259] 9) Anti-interference

[0260] Anti-interference experiment: Using the kits in the following examples, the methods were implemented according to Example 18. Samples were tested, and blank water (as a reference), fat emulsion, bilirubin, RF, and hemoglobin were added to the samples respectively. The relative deviation was calculated as (mean - water) / water. In this invention, the combined use of the three blocking agents in Example 1 (THBR-1, THBR-2, A1101) showed the best effect and the smallest relative deviation, superior to other examples 12 (THBR-1), 13 (THBR-2), 14 (A1101), 15 (THBR-1, THBR-2), 16 (THBR-1, A1101), and 17 (THBR-2, A1101).

[0261] Table 19

[0262]

[0263]

[0264]

[0265]

[0266] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A kit for measuring p2PSA based on latex-enhanced immunoturbidimetry, characterized in that, The kit includes: latex antibody reagent; The latex antibody reagent comprises a mixture of a first latex antibody reagent and a second latex antibody reagent; the first latex antibody reagent is formed by coupling a polyclonal antibody of p2PSA with large-particle latex microspheres; the particle size range of the large-particle latex microspheres is 172~405nm; the second latex antibody reagent is formed by coupling a monoclonal antibody of p2PSA with small-particle latex microspheres; the particle size range of the small-particle latex microspheres is 71~121nm; the monoclonal antibody of p2PSA comprises 3 Monoclonal antibodies against different strains of p2PSA; the monoclonal antibodies against p2PSA include p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies; the p2PSA-1 monoclonal antibody is p2PSA-mAb-1; the p2PSA-2 monoclonal antibody is p2PSA-mAb-2; the p2PSA-3 monoclonal antibody is p2PSA-mAb-3; the mass ratio of the p2PSA-1, p2PSA-2, and p2PSA-3 monoclonal antibodies is 1:1:9 to 9:1:

1.

2. The kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay according to claim 1, characterized in that, The polyclonal antibody against p2PSA is p2PSA-pAb.

3. The kit for measuring p2PSA based on latex-enhanced immunoturbidimetry according to claim 2, characterized in that, The particle size of the large-diameter latex microspheres is 405nm, 218nm, 192nm or 172nm, preferably 405nm; And / or, the particle size of the small-diameter latex microspheres is 71nm, 89nm, 100nm, 108nm or 121nm, preferably 71nm; And / or, the volume ratio of the p2PSA polyclonal antibody to the large-particle latex microspheres is 2~5:1, preferably 2:1; the concentration of the p2PSA polyclonal antibody is 2mg / L~20mg / L; the concentration of the large-particle latex microspheres is 0.5% w / v~1% w / v; And / or, the volume ratio of the p2PSA monoclonal antibody to the small-particle latex microspheres is 2~5:1, preferably 2:1; the concentration of the p2PSA monoclonal antibody is 2mg / L~20mg / L; the concentration of the small-particle latex microspheres is 0.5% w / v~1% w / v; And / or, the mass ratio of p2PSA-1, p2PSA-2 and p2PSA-3 monoclonal antibodies is 1:1:1; And / or, the volume ratio of the first latex antibody reagent to the second latex antibody reagent is 1:3 to 3:1, preferably 1:2 to 2:1; preferably, it is 1:

1. And / or, the concentration of the p2PSA polyclonal antibody conjugate with large-particle latex microspheres in the first latex antibody reagent is 0.1~0.5 w / v, preferably 0.3 w / v; And / or, the concentration of the p2PSA monoclonal antibody conjugate with small-particle latex microspheres in the second latex antibody reagent is 0.05~0.3 w / v, preferably 0.1 w / v.

4. The kit for the determination of p2PSA based on latex-enhanced immunoturbidimetry according to any one of claims 1 to 3, characterized in that, It also includes a reaction solution; the reaction solution includes at least one of a buffer solution, an inorganic salt, a sensitizer, a surfactant, a blocking agent, and a preservative. The buffer solution includes any one of N(2-acetamido)-2-iminodiacetic acid buffer, piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer. And / or, the inorganic salt includes at least one of sodium molybdate, ammonium molybdate, sodium bisulfate, or ammonium bisulfate; And / or, the sensitizer includes at least one of Biopidure 103, Biopidure 405, and PEG6000; preferably Biopidure 103 and / or Biopidure 405; And / or, the preservative is at least one of sodium azide, gentamicin, and isothiazolinones; And / or, the surfactant is at least one of polyoxyethylene fatty alcohol ether, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, and polysorbate; And / or, the blocking agent is at least one of THBR-1, THBR-2, and A1101; preferably, it is THBR-1, THBR-2, and A1101 in a volume ratio of 0.5~1.5: 0.5~1.5: 0.5~1.5, more preferably 1:1:1; Optionally, the reaction solution comprises: buffer solution 13~18.2 g / L, inorganic salt 8.5~20 g / L, sensitizer 20~35 g / L, surfactant 0.1~0.2 v / v%, blocker 0.002~0.006 v / v%, and preservative 0.5~1.5 g / L; pH is 7.2±0.05~8.2±0.

05.

5. The kit for determining p2PSA based on latex-enhanced immunoturbidimetry according to any one of claims 1-3, characterized in that, It also includes calibrators; the calibrators contain human p2PSA antigen; Optionally, the calibrator comprises a buffer solution of 13-18.2 g / L, a protective agent of 0.5-1 g / L, and a preservative of 13-18.2 g / L; Optionally, the buffer solution includes any one of phosphate buffer, N(2-acetamido)-2-iminodiacetic acid buffer, piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer. Optionally, the protective agent is at least one of bovine serum albumin, casein, gelatin, or NaCl; Optionally, the preservative is at least one of sodium azide, gentamicin, and isothiazolinones.

6. A method for preparing a kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay as described in any one of claims 1-5, characterized in that, Includes the following steps: Large-diameter latex microspheres and small-diameter latex microspheres were activated with carbodiimide (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) respectively, and then put into use. The activated large-particle latex microspheres were conjugated with p2PSA polyclonal antibodies, blocked, centrifuged to remove the supernatant, and resuspended to obtain the first latex antibody reagent. The activated small-particle latex microspheres were coupled with the p2PSA monoclonal antibody, blocked, centrifuged to remove the supernatant, and resuspended to obtain the second latex antibody reagent. The first latex antibody reagent and the second latex antibody reagent are mixed in a certain proportion to obtain the latex antibody reagent.

7. The preparation method of the kit for determining p2PSA based on latex-enhanced immunoturbidimetric assay according to claim 6, characterized in that, Prior to coupling, the large-diameter latex microspheres were washed, centrifuged, and reconstituted. And / or, prior to coupling, the small-diameter latex microspheres are washed, centrifuged, and reconstituted; And / or, the volume ratio of the carbodiimide and N-hydroxythiosuccinimide is 3~5:6~10, preferably 1:2, the concentration of carbodiimide is 10~30 mg / mL, and the concentration of succinimide is 20~30 mg / mL. And / or, the sealing liquid used in the sealing step includes at least one of DB1130, CE510, and CE210; And / or, in the resuspension step, a preservation solution is used for resuspension, the preservation solution comprising a buffer, a stabilizer, a surfactant, and a preservative; wherein the buffer comprises any one of piperazine-N,N-di(2-ethanesulfonic acid) buffer, 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer; the stabilizer comprises at least one of gelatin or casein; the surfactant is at least one of polyoxyethylene fatty alcohol ether, fatty acid glyceride, sucrose fatty acid ester, fatty acid sorbitan, and polysorbate; and the preservative is at least one of sodium azide, gentamicin, and isothiazolinones.

8. The use of a kit for determining p2PSA based on latex-enhanced immunoturbidimetry as claimed in any one of claims 1-5 in the preparation of products for determining p2PSA.

9. A method for determining p2PSA based on latex-enhanced immunoturbidimetry, characterized in that, Using the sample to be tested, an immune reaction was performed using the kit for determining p2PSA based on latex-enhanced immunoturbidimetry according to any one of claims 1-5, and the turbidity of the resulting reaction product was detected.

10. The method for determining p2PSA based on latex-enhanced immunoturbidimetry according to claim 9, characterized in that, include: The test sample and calibrator were mixed with the reaction solution and incubated. Then, latex antibody reagent was added, and the absorbance of the test sample and calibrator was measured separately. A standard curve was plotted based on the content of the calibrator and the measured absorbance. The p2PSA content in the test sample was calculated based on the standard curve. Optionally, the volume ratio of the test sample, reaction solution, and latex antibody reagent is 10~20:90~270:30~90, preferably 15:180:60.

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