Matrix metalloproteinase 3 latex enhanced immunoturbidimetry kit as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of in-vitro diagnostic reagents, and relates to a matrix metalloproteinase 3 latex enhanced immunoturbidimetry kit and a preparation method and application thereof.The matrix metalloproteinase 3 latex enhanced immunoturbidimetry kit comprises a reagent R1 and a reagent R2, and the reagent R1 comprises N-hydroxysuccinimide, a rheumatoid factor blocking agent and a protein denaturing agent; the reagent R2 comprises a first buffer solution, a second buffer solution, latex microspheres, a matrix metalloproteinase 3 antibody, an activating agent, a protective agent, a sealing agent and a stabilizing agent; wherein the rheumatoid factor blocking agent is selected from at least two of anti-human IgM (Immunoglobulin M), anti-human IgG (Immunoglobulin G), anti-human IgA (Immunoglobulin A), anti-human IgE (Immunoglobulin E), anti-human IgD (Immunoglobulin D), an anti-heterotropism antibody and an anti-rheumatoid factor antibody. According to the application, the interference of rheumatoid factors can be effectively eliminated, the specificity and the accuracy of a detection result are improved, and the kit is simple in reagent composition, relatively high in sensitivity and relatively wide in universality.

Description

Technical Field

[0001] The present application relates to the technical field of in vitro diagnostic reagents, and in particular to a matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit and a preparation method and application thereof. Background Art

[0002] Matrix metalloproteinase-3 (MMP-3) can degrade the extracellular matrix and has a destructive effect on articular cartilage. It is believed to be related to a variety of rheumatic immune diseases and has good sensitivity and specificity. Foreign studies have shown that the level of MMP-3 in the peripheral serum of patients with early rheumatoid arthritis is significantly higher than that of normal people. The inflammatory state of RA (Rheumatoid Arthritis) patients can induce the expression of MMP-3 molecules. The serum MMP-3 content is positively correlated with ESA and inflammatory factor CRP (C-reaction protein). Therefore, the serum MMP-3 level is a marker of persistent joint inflammation. The degree of increase in serum MMP-3 reflects the joint damage that will occur in the next 6 to 12 months, and can also be used as an indicator for evaluating the effect of concomitant treatment.

[0003] Latex particle-enhanced turbidimetric immunoassay (PETIA) is a relatively stable and accurate homogeneous immunoturbidimetric detection method for body fluid proteins that has emerged in recent years. It can dynamically measure the binding of antigens and antibodies. This method has many advantages over other commonly used body fluid protein detection methods: it eliminates the tedious operation steps of repeated incubation and plate washing in the enzyme-linked immunosorbent assay (ELISA), and the results can be obtained in a few minutes. It has good stability and repeatability, and can more truly reflect the content of the substance being tested; it has higher sensitivity than colloidal gold chromatography (commonly known as "gold label") and can be used for quantitative detection; compared with radioimmunoassay (RIA), it has better stability and can avoid radioactive contamination.

[0004] One limitation of routine latex-enhanced immunoturbidimetry is that it is susceptible to interference caused by heterophilic antibodies that may be present in the test sample, especially interference caused by rheumatoid factor. Rheumatoid factor (RF) is an antibody against the antigenic determinant cluster of the Fc fragment of human or animal IgG molecules. It is an autoantibody with denatured IgG as the target antigen. RA patients have B cell clones that produce RF, which can synthesize RF in large quantities under the direct action of denatured IgG or Epstein-Barr virus. RF is mainly IgM autoantibody, but there are also IgG, IgA, IgD and IgE. IgM and IgG rheumatoid factors in human serum can bind to the non-specific binding of the Fc segment of antibodies (mainly IgG) in the detection system, resulting in false positives. The binding of RF to the Fc segment of IgG is random, and there is no binding pattern to follow. Moreover, both high and low concentrations of RF will cause interference in immunoassays. The antibodies used in commercial immunoassays are mostly animal-derived IgG antibodies, whose Fc segments have a high homology with the Fc segments of human IgG and can be recognized and bound by RF. Therefore, in test samples where the test substance is negative and RF is positive, the immunoassay reagents may be captured by RF to generate signals, leading to false positive results.

[0005] In the current development of immunoassay reagents, samples are pretreated before detection and analysis to eliminate or minimize RF in the samples, so as to eliminate or reduce the interference of RF on the detection system. The methods include: (1) Sample pretreatment: adding animal serum or IgG or blocking reagents to the samples before analysis; adsorption with solid particles coated with IgG; using blocking test tubes to collect test samples; using diluent containing 2-mercaptoethanol to dilute the samples to degrade IgM rheumatoid factor; using polyethylene glycol 6000 to treat the samples to precipitate IgM rheumatoid factor, etc. However, this will complicate the detection operation and may reduce the content or activity of the substance to be tested while removing interfering proteins. (2) Use blocking agents that reduce interference, mostly IgG, and there are many commercial blocking agents on the market. However, due to the diversity of rheumatoid factors or heterophilic antibodies, and the different antibodies used in various products, there is currently no product that can effectively eliminate all interference. It is usually necessary to use different products for combination and screening, which not only increases the workload and reagent costs, but also introduces new factors that interfere with the detection results uncontrollably due to the use of multiple unknown blocking agents. (3) Using antibody engineering to truncate the YI part (RF binding site) in the immunoassay and only retain the Fab segment of the antibody as the detection reagent, or using a living single-chain biotin is a more ideal method to solve RF interference. However, in the detection system, whether it is the coating and solidification of the capture antibody or the labeling of the signal antibody, it is mostly on the Fc segment of the antibody. Such modifications may increase the difficulty of antibody coating and labeling, and there are still certain difficulties in engineering. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit and a preparation method and application thereof, so as to solve the technical problem that the prior art cannot effectively eliminate or reduce the interference of rheumatoid factors.

[0007] In order to solve the above technical problems, the present application provides a matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit, which adopts the following technical solution:

[0008] The invention comprises reagent R1 and reagent R2, wherein the reagent R1 comprises N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant; the reagent R2 comprises a first buffer, a second buffer, latex microspheres, a matrix metalloproteinase 3 antibody, an activator, a protective agent, a blocking agent and a stabilizer; wherein the rheumatoid factor blocker is selected from at least two of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody.

[0009] Furthermore, the reagent R1 includes 1-40 g / L of N-hydroxysuccinimide, 0.001%-0.01% (W / V) of a rheumatoid factor blocker, and 0.001%-0.2% (W / V) of a protein denaturant.

[0010] Furthermore, the reagent R1 also includes 25-200 mmol / L phosphate buffer, 0.9-120 g / L sodium chloride, 5.0-15.0 g / L polyethylene glycol 6000, 0.1-5 g / L biological preservative Proclin 300 and 1-5 g / L Tween 20.

[0011] Furthermore, the protein denaturant is one of sodium dodecyl sulfate, urea and guanidine hydrochloride; the content of the protein denaturant in the reagent R1 is 0.005% to 0.05% (W / V).

[0012] Furthermore, the pH value of the reagent R1 is 5.0-8.0.

[0013] Furthermore, the reagent R2 includes 5 to 50 mmol / L of a first buffer, 5 to 50 mmol / L of a second buffer, 0.5 to 2 g / L of latex microspheres, 0.03 to 0.15 g / L of matrix metalloproteinase 3 antibody, 0.08 to 0.8 g / L of an activator, 0.1 to 0.8 g / L of a protective agent, 0.01% to 10% (W / V) of a blocking agent, and 15 to 40 g / L of a stabilizer.

[0014] Furthermore, the reagent R2 includes 50mmol / L of a first buffer, 50mmol / L of a second buffer, 2g / L of latex microspheres, 0.08g / L of matrix metalloproteinase 3 antibody, 0.3g / L of an activator, 0.6g / L of a protective agent, 1% (W / V) of a blocking agent, and 30g / L of a stabilizer.

[0015] Furthermore, the first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer, and Hepes buffer;

[0016] The second buffer is selected from one of 2(N-morpholine)ethanesulfonic acid buffer, glycine buffer, Hepes buffer, and Tris-HCl buffer;

[0017] The particle size of the latex microspheres is 100nm to 200nm, and the solid content is 10%;

[0018] The matrix metalloproteinase 3 antibody is an animal anti-human matrix metalloproteinase 3 monoclonal antibody or polyclonal antibody, and the animal species include rabbits, goats, sheep, chickens and mice;

[0019] The activator is 1-ethyl (3-dimethylaminopropyl) carbodiimide;

[0020] The protective agent is N-hydroxysuccinimide;

[0021] The stabilizer is selected from one of trehalose, sucrose and glucose;

[0022] The blocking agent is selected from one of bovine serum albumin and casein.

[0023] In order to solve the above technical problems, the present application also provides a method for preparing a matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit, which adopts the following technical solution:

[0024] Using N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant, a reagent R1 is prepared;

[0025] Prepare reagent R2, and subpackage the reagent R1 and reagent R2 to obtain a test kit;

[0026] Wherein, the reagent R2 is prepared by the following preparation steps:

[0027] Take a container, add the second buffer solution and latex microspheres, mix them well, then add an activator and a protective agent respectively, mix them well, and obtain activated latex microspheres;

[0028] Adding matrix metalloproteinase 3 antibody to the activated latex microspheres in the container, mixing, and obtaining labeled latex microspheres-matrix metalloproteinase 3 antibody;

[0029] Adding a blocking agent to the labeled latex microspheres-MMP-3 antibody in the container, fully dissolving and blocking, to obtain blocked latex microspheres;

[0030] Add the first buffer solution and the stabilizer to the blocked latex microspheres in the container and mix thoroughly to obtain reagent R2.

[0031] In order to solve the above technical problems, the present application also provides an application of the matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit as described above, wherein the kit is used to detect the content of matrix metalloproteinase 3 in human serum or plasma.

[0032] Compared with the prior art, this application has the following beneficial effects:

[0033] The matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit provided by the present application comprises N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant in reagent R1, which can effectively eliminate the interference of rheumatoid factors and improve the specificity and accuracy of the detection results. In the present application, an activator and a protective agent are first added to the latex microspheres to activate the matrix metalloproteinase 3, and then an antibody marker is added, and the matrix metalloproteinase 3 antibody is directionally immobilized on the latex microspheres to finally form reagent R2. The preparation process is time-consuming and has low raw material loss, thereby improving production efficiency. The matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit of the present application has a simple reagent composition, high sensitivity and relatively wide versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 It is a flow chart of an embodiment of a method for preparing a matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit according to the present application;

[0036] Figure 2 This is a schematic diagram of the linear results in Example 4 of the present application. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0038] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments. The experimental methods used in the following embodiments are all common methods unless otherwise specified; the reagents used in the following embodiments are all commercially available unless otherwise specified.

[0039] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0040] The matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit provided in the present application includes reagent R1 and reagent R2, and can be used to inhibit the interference of rheumatoid factors.

[0041] Reagent R1 is a solution for promoting the reaction between antigen and antibody, and includes N-hydroxysuccinimide (NHS), a rheumatoid factor blocker and a protein denaturant. The rheumatoid factor blocker is selected from at least two of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody.

[0042] The reagent R2 includes a first buffer, a second buffer, latex microspheres, matrix metalloproteinase 3 antibody, an activator, a protective agent, a blocking agent and a stabilizer, wherein the first buffer is mainly used as a buffer system of the reagent R2, and the second buffer is used for activating the latex microspheres.

[0043] The reagent R1 of the present application uses a combination of NHS, a rheumatoid factor blocker and a protein denaturant. This is based on the fact that rheumatoid factors or heterophilic antibodies are mostly multi-specific antibodies. Compared with the specific binding reaction of general antibodies and antigens, their binding ability to detection reagent antibodies of different species is relatively weak. By utilizing this difference in affinity, the appropriate addition of an appropriate amount of protein denaturant can dissociate the interaction between proteins, and the appropriate addition of an appropriate amount of blocker can block the interference of rheumatoid factors. Under the premise of not affecting the specific reaction of the detection antibody with the antigen to be detected, the non-specific binding reaction of the rheumatoid factor to the detection antibody can be weakened, thereby effectively eliminating the interference of the rheumatoid factor on the test results and improving the specificity and accuracy of the test results.

[0044] Specifically, NHS is added to reagent R1. After the addition of NHS, the ability of rheumatoid factor in the sample to bind to antibodies such as anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibodies and anti-rheumatoid factor antibodies is reduced, while the ability of the labeled antibody to bind to the antigen to be tested is not affected. In addition, the antibody in reagent R1 can bind to the attenuated rheumatoid factor, thereby completely eliminating the interference of the rheumatoid factor on the test results.

[0045] The kit of the present application includes reagent R1 and reagent R2, and the reagent composition is simple and has wide versatility.

[0046] In some embodiments, reagent R1 includes 1-40 g / L N-hydroxysuccinimide, 0.001%-0.01% (W / V) rheumatoid factor blocker, and 0.001%-0.2% (W / V) protein denaturant.

[0047] The contents of NHS, rheumatoid factor blocker and protein denaturant in reagent R1 are appropriate, which can more effectively eliminate the interference of rheumatoid factor on the test results.

[0048] In some embodiments, reagent R1 also includes 25-200 mmol / L phosphate buffer, 0.9-120 g / L sodium chloride, 5.0-15.0 g / L polyethylene glycol 6000, 0.1-5 g / L biological preservative Proclin 300 and 1-5 g / L Tween 20.

[0049] In some embodiments, the protein denaturant is one of sodium dodecyl sulfate, urea and guanidine hydrochloride; the content of the protein denaturant in reagent R1 is 0.005% to 0.05% (W / V).

[0050] As a specific example, reagent R1 includes 5mmol / L~200mmol / L phosphate buffer, 0.9g / L~120g / L sodium chloride, 5.0g / L~15.0g / L polyethylene glycol 6000, 0.1g / L~5g / L biological preservative Proclin300, 5~10g / L NHS, 0.001%~0.01% rheumatoid factor blocker, 0.005%~0.05% (W / V) urea and 1g / L~5g / L Tween 20.

[0051] In some embodiments, the pH value of reagent R1 is 5.0-8.0. Appropriate pH value is conducive to the protein denaturant to dissociate the interaction between proteins. Reagent R1 has good reaction sensitivity within this pH range and is relatively stable.

[0052] In some embodiments, reagent R2 includes 5-50 mmol / L of a first buffer, 5-50 mmol / L of a second buffer, 0.5-2 g / L of latex microspheres, 0.03-0.15 g / L of matrix metalloproteinase 3 antibody, 0.08-0.8 g / L of an activator, 0.1-0.8 g / L of a protective agent, 0.01%-10% (W / V) of a blocking agent, and 15-40 g / L of a stabilizer.

[0053] Among them, the activator and the protective agent act as coupling agents to activate the latex microspheres.

[0054] In some embodiments, reagent R2 includes 50 mmol / L of a first buffer, 50 mmol / L of a second buffer, 2 g / L of latex microspheres, 0.08 g / L of a matrix metalloproteinase 3 antibody, 0.3 g / L of an activator, 0.6 g / L of a protective agent, 1% (W / V) of a blocking agent, and 30 g / L of a stabilizer.

[0055] In some embodiments, the first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer, and Hepes buffer.

[0056] The second buffer is selected from one of 2(N-morpholine)ethanesulfonic acid buffer, glycine buffer, Hepes buffer and Tris-HCl buffer.

[0057] The particle size of the latex microspheres is 100nm-200nm, and the solid content is 10%.

[0058] The matrix metalloproteinase 3 antibody is an animal anti-human matrix metalloproteinase 3 monoclonal antibody or polyclonal antibody, and the animal species include rabbit, goat, sheep, chicken and mouse;

[0059] The activator is 1-ethyl (3-dimethylaminopropyl) carbodiimide (EDC); the protective agent is N-hydroxysuccinimide (NHS);

[0060] The stabilizer is selected from one of trehalose, sucrose and glucose;

[0061] The blocking agent is selected from one of bovine serum albumin and casein.

[0062] Among them, the presence of NHS improves the efficiency of EDC-mediated coupling and further improves the activation efficiency of latex microspheres. The role of the blocking agent is to bind to the free -COOH site in the latex microspheres to prevent the free carboxyl site from causing adverse effects on the test results. The main function of the stabilizer is to protect the activity of the antibody and prevent the aggregation and sinking of the latex microspheres. At the same time, it can also increase the stability of the components in the kit and extend the shelf life of the product and the use time after opening the bottle.

[0063] Based on the above-mentioned matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit, the present application also provides a method for preparing a matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit, comprising the following steps:

[0064] Using N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant, a reagent R1 is prepared;

[0065] Reagent R2 was prepared, and reagent R1 and reagent R2 were packaged separately to obtain a test kit.

[0066] In this embodiment, reagent R1 is prepared according to conventional methods, and the preparation method of two liters of reagent R2 is as follows: Figure 1 As shown, the following steps are included:

[0067] Step S101, taking a container, adding a second buffer solution and latex microspheres, mixing them evenly, and then adding an activator and a protective agent respectively, mixing them evenly, to obtain activated latex microspheres.

[0068] This step is to activate the latex microspheres. For example, take a suitable container, then add 500 ml of the second buffer solution, add the latex microspheres, and stir for 12 to 15 minutes in a 37°C shaker; then add NHS and EDC respectively and stir for 30 to 35 minutes to obtain activated latex microspheres.

[0069] The stirring speed is 180-200 rpm.

[0070] Step S102, adding matrix metalloproteinase 3 antibody to the activated latex microspheres in the container, mixing, and obtaining labeled latex microspheres-matrix metalloproteinase 3 antibody.

[0071] This step is to label the antibody. For example, the matrix metalloproteinase 3 antibody is added to the activated latex microspheres in the container while stirring, and the mixture is stirred in a shaker at 37° C. for 60 to 120 minutes to obtain labeled latex microspheres-matrix metalloproteinase 3 antibody.

[0072] The stirring speed is 180-200 rpm.

[0073] Step S103, adding a blocking agent to the labeled latex microspheres-MMP-3 antibody in the container, fully dissolving and blocking, and obtaining blocked latex microspheres.

[0074] This step is to block the latex microspheres. For example, a blocking agent is added to the labeled latex microspheres-MMP-3 antibody in a container, and the mixture is fully dissolved and mixed for 80 to 120 minutes to block the latex microspheres to obtain blocked latex microspheres.

[0075] Step S104, adding the first buffer solution and the stabilizer to the blocked latex microspheres in the container, and mixing them thoroughly to obtain the reagent R2.

[0076] For example, 500 ml of the first buffer solution and the stabilizer are added to the closed latex microspheres in the container and mixed thoroughly to obtain the reagent R2.

[0077] In the preparation process of reagent R2 in this embodiment, the amount of materials added is controlled and the preparation process is simplified. The preparation process is short in time and the loss of raw materials is low, which improves production efficiency and is conducive to mass production.

[0078] The present application also provides an application of the matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit as described above, wherein the kit is used to detect the content of matrix metalloproteinase 3 in human serum or plasma.

[0079] In some embodiments, the detection concentration range of matrix metalloproteinase 3 in the kit is 10 ng / mL to 1600 ng / mL.

[0080] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0081] It should be understood that the raw materials used in this application: buffer, stabilizer, blocking agent, latex microspheres, matrix metalloproteinase 3 antibody are all purchased from chemical and biological raw material companies. The particle size of the latex microspheres is 100nm to 200nm, the solid content is 10%, and they are used directly.

[0082] Example 1

[0083] This example is the preparation of a latex-enhanced immunoturbidimetric kit for matrix metalloproteinase 3.

[0084] Wherein, reagent R1 is prepared according to conventional methods, and reagent R2 is prepared by the following steps:

[0085] (1) Activation: Take a suitable container, add 500 ml of 50 mM Hepes buffer, add 10 ml of latex microspheres (180 nm, solid content 10%), and stir for 15 min in a shaker at 37°C; then add 4 ml of 30 mg / ml NHS and 2 ml of 20 mg / ml EDC, respectively, and stir for 30 min to obtain activated latex microspheres;

[0086] (2) Labeling: adding 40 mg of MMP-3 antibody to the activated latex microspheres in the container while stirring, and stirring in a shaker at 37° C. for 120 min to obtain labeled latex microspheres-MMP-3 antibody;

[0087] (3) Blocking: Add 8 mL of 10% bovine serum albumin to the labeled latex microspheres-MMP-3 antibody in the container, fully dissolve and mix for 120 min, and block to obtain blocked latex microspheres;

[0088] (5) Add 500 ml of 50 mM glycine buffer and 100 g of trehalose to the closed latex microspheres in the container and mix thoroughly to obtain reagent R2.

[0089] Example 2

[0090] This example is to compare the inhibitory effect of protein denaturants on RF interference of matrix metalloproteinase 3 detection reagents.

[0091] 1. Preparation of MMP-3 samples with different rheumatoid factor contents:

[0092] 0, 20, 100, 400 IU / mL of rheumatoid factor were added to the samples containing 50 ng / mL of pure matrix metalloproteinase 3 antibody, respectively. There were two sources of rheumatoid factor purchased from two different manufacturers (RF-I and RF-II).

[0093] 2. Matrix metalloproteinase 3 assay kit assay reagents R1-A: 100 mmol / L phosphate buffer, 9 g / L sodium chloride, 10.0 g / L polyethylene glycol 6000, 0.5 g / L biological preservative Proclin 300, 2 g / L Tween 20, 10 g / L N-hydroxysuccinimide (NHS), 0.005% rheumatoid factor blocker.

[0094] The components of the matrix metalloproteinase 3 assay kit assay reagents R1-B, C, D, E and F are the same as those of reagent R1-A, except for the different urea concentrations, which are 0.005%, 0.01%, 0.02%, 0.05% and 0.7%, respectively. Among them, reagent R1 is prepared according to conventional methods.

[0095] The above-prepared six kinds of matrix metalloproteinase 3 assay kit assay reagents R1 (reagents R1-A, R1-B, R1-C, R1-D, R1-E and R1-F, each reagent has a different urea concentration) are matched with the reagent R2 in Example 1, and the purchased matrix metalloproteinase 3 assay kit for measuring serum samples, and the same interference sample is tested. The test results are shown in Table 1.

[0096] Table 1 Test results of the inhibitory effect of protein denaturants on reagents subjected to RF interference

[0097]

[0098] From the above results, it can be seen that low-value RF (20 IU / mL) has basically no interference with the matrix metalloproteinase 3 detection reagent, and both high-value RFs will interfere with the test results. When the urea concentration of reagent R1 is in the range of 0.005% to 0.05%, the test results of the matrix metalloproteinase 3 content are basically not affected by the added rheumatoid factor, and the deviation from the target value is within 10%. It shows that adding a certain concentration of urea can effectively eliminate the interference of rheumatoid factor on the test results, and will not affect the detection content of the target matrix metalloproteinase 3.

[0099] At the same time, the above six sets of prepared reagents and purchased reagents were used to detect the content of matrix metalloproteinase 3 in 10 RF-positive serum specimens, and the rheumatoid factor detection reagent was used to detect the RF content in the samples. It was further confirmed that the addition of a certain concentration of urea can effectively inhibit the interference of RF in serum on the detection of matrix metalloproteinase 3 content, and accurately detect the content of matrix metalloproteinase 3 in serum. The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] From the above data, it can be seen that when different concentrations of urea are added to reagent R1, the results of the determination of reagents R1-B, R1-C, R1-D, and R1-E are more stable than those of R1-A without urea, and the correlation coefficient is higher than that of the determination results of the purchased reagents; while the measurement results of reagent R1-F with high concentration of urea are more unstable and have a larger range of variation compared with the purchased reagents, and the correlation coefficient is lower than that of the determination results of the purchased reagents. From the experimental results, it can be seen that when the urea concentration is between 0.005% and 0.05%, it can effectively inhibit the interference of RF in serum on the detection of matrix metalloproteinase 3 content, and accurately detect the content of matrix metalloproteinase 3 in serum.

[0103] Example 3

[0104] This example is to compare the inhibitory effect of the blocking agent on the RF interference of the matrix metalloproteinase 3 detection reagent.

[0105] 1. Preparation of MMP-3 samples with different rheumatoid factor contents:

[0106] 0, 20, 100, 400 IU / mL of rheumatoid factor were added to the samples containing 50 ng / mL of pure matrix metalloproteinase 3 antibody, respectively. There were two sources of rheumatoid factor purchased from two different manufacturers (RF-I and RF-II).

[0107] 2. Matrix metalloproteinase 3 assay kit assay reagents R1-G: 100 mmol / L phosphate buffer, 9 g / L sodium chloride, 10.0 g / L polyethylene glycol 6000, 0.5 g / L biological preservative Proclin 300, 2 g / L Tween 20, 10 g / L N-hydroxysuccinimide (NHS), and 0.01% urea.

[0108] The components of the matrix metalloproteinase 3 assay kit assay reagents R1-H, I, J, K, L, M and N are the same as those of reagent R1-G, except for the different types of blocking agents, which are 0.005% anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody, respectively. Among them, reagent R1 is prepared according to conventional methods.

[0109] The above-prepared six kinds of matrix metalloproteinase 3 assay kit assay reagents R1 (reagents R1-G, R1-H, R1-I, R1-J, R1-K, R1-L, R1-M, R1-N, each reagent has a different type of blocking agent) are matched with the reagent R2 in Example 1, and the purchased matrix metalloproteinase 3 assay kit for measuring serum samples, and the same interference sample is tested. The test results are shown in Table 3.

[0110] Table 3

[0111]

[0112]

[0113] The results are shown in Table 3. The low value RF (20 IU / mL) has basically no interference with the matrix metalloproteinase 3 detection reagent, and the two high value RFs will interfere with the test results. When 0.005% of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody are added to the reagent R1, the matrix metalloproteinase 3 content detection result is closer to the target value compared with the R1-G reagent, indicating that the addition of a certain concentration of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody can effectively eliminate the interference of rheumatoid factor on the test results, and will not affect the detection content of the target matrix metalloproteinase 3, among which the addition of anti-rheumatoid factor antibody has a better effect.

[0114] At the same time, the above six sets of prepared reagents and purchased reagents were used to detect the content of matrix metalloproteinase 3 in 10 RF-positive serum specimens, and the rheumatoid factor detection reagent was used to detect the RF content in the samples. It was further confirmed that the reagent R1 with the addition of a certain concentration of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody can effectively inhibit the interference of RF in serum on the detection of matrix metalloproteinase 3 content, and accurately detect the content of matrix metalloproteinase 3 in serum. The results are shown in Table 4.

[0115] Table 4

[0116]

[0117]

[0118] From the above data, it can be seen that when 0.005% anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody are added to reagent R1 respectively, compared with R1-G without adding, the results of the reagent test are more stable, and the correlation coefficient is higher than the test results of purchased reagents.

[0119] Example 4

[0120] The reagents in the kit used in this embodiment are reagents R1-N of Example 2 and reagent R2 of Example 1.

[0121] (1) Accuracy verification implementation: Take one copy of the high-value quality control material and one copy of the low-value quality control material with traceability, test them separately with the reagents, test each 10 times, and compare them with the quality control material target value. The results are shown in Table 5 below.

[0122] Table 5 Accuracy verification results

[0123]

[0124]

[0125] It can be seen from the deviation values ​​in the above table that the kit of the present application has a high accuracy.

[0126] (2) Implementation of precision verification: Take one copy of the high-value quality control material and one copy of the low-value quality control material with traceability, test them separately with the reagents, test each 10 times, and calculate the mean, standard deviation and coefficient of variation of the test results. The results are shown in Table 6.

[0127] Table 6 Precision verification results

[0128]

[0129]

[0130] It can be seen from the coefficient of variation in the table that the kit of the present application has a high precision.

[0131] (3) Implementation of sensitivity verification: Take 3 portions of traceable quality control materials and dilute them to near the lower limit of the detection range for measurement. Repeat the measurement 5 times and calculate the average value and compare it with the target value of the quality control material, as shown in Table 7.

[0132] Table 7 Sensitivity verification results

[0133] frequency Low value quality control product 1 (ng / mL) Low value quality control product 2 (ng / mL) Low value quality control product 3 (ng / mL) 1 10.43 12.64 15.32 2 10.41 12.96 14.32 3 10.54 12.10 13.98 4 9.39 12.68 16.24 5 9.40 12.21 16.21 average value 10.034 12.518 15.214 Target value 10 12 15 deviation 0.34％ 4.32％ 1.43％

[0134] It can be seen from the data in the above table that the kit provided by the present application has a high sensitivity.

[0135] (4) Implementation of linear verification: Take a high-value standard with traceability, dilute it with purified water, measure each point 3 times, take the average value, draw a regression line with the result and the theoretical concentration, and calculate the square of the regression coefficient r. The results are shown in Table 8 and Figure 2 .

[0136] Table 8 Linearity verification results

[0137]

[0138]

[0139] From Table 8 and Figure 2 It can be seen that within the range of 10 ng / mL-1600 ng / mL, r=0.9976, indicating that the kit of the present application has good linearity and a wide detection range.

[0140] In summary, the matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit provided by the present application includes reagent R1 and reagent R2, and the reagent composition is simple, has high sensitivity, and has relatively wide versatility; reagent R1 can effectively eliminate the interference of rheumatoid factors and improve the specificity and accuracy of the detection results; the preparation process of reagent R2 is short in time and has low raw material loss, which improves production efficiency and is conducive to mass production; in addition, the detection range of the kit is 10ng / mL-1600ng / mL, and the sensitivity can reach 10ng / mL, and the high concentration level of matrix metalloproteinase 3 of 1600ng / mL can also be accurately determined, which improves the detection range and sensitivity and can be used to diagnose various rheumatic immune diseases.

[0141] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit, characterized in that: The invention comprises reagent R1 and reagent R2, wherein the reagent R1 comprises N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant; the reagent R2 comprises a first buffer, a second buffer, latex microspheres, a matrix metalloproteinase 3 antibody, an activator, a protective agent, a blocking agent and a stabilizer; wherein the rheumatoid factor blocker is selected from at least two of anti-human IgM, anti-human IgG, anti-human IgA, anti-human IgE, anti-human IgD, anti-heterophilic antibody and anti-rheumatoid factor antibody.

2. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to claim 1, characterized in that: The reagent R1 comprises 1-40 g / L of N-hydroxysuccinimide, 0.001%-0.01% (W / V) of a rheumatoid factor blocker and 0.001%-0.2% (W / V) of a protein denaturant.

3. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to claim 2, characterized in that: The reagent R1 also includes 25-200 mmol / L phosphate buffer, 0.9-120 g / L sodium chloride, 5.0-15.0 g / L polyethylene glycol 6000, 0.1-5 g / L biological preservative Proclin 300 and 1-5 g / L Tween 20.

4. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to claim 1, characterized in that: The protein denaturant is one of sodium dodecyl sulfate, urea and guanidine hydrochloride; the content of the protein denaturant in the reagent R1 is 0.005% to 0.05% (W / V).

5. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to any one of claims 1 to 4, characterized in that: The pH value of the reagent R1 is 5.0-8.

0.

6. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to claim 1, characterized in that: The reagent R2 includes 5-50 mmol / L of a first buffer, 5-50 mmol / L of a second buffer, 0.5-2 g / L of latex microspheres, 0.03-0.15 g / L of a matrix metalloproteinase 3 antibody, 0.08-0.8 g / L of an activator, 0.1-0.8 g / L of a protective agent, 0.01%-10% (W / V) of a blocking agent, and 15-40 g / L of a stabilizer.

7. The matrix metalloproteinase 3 latex-enhanced immunoturbidimetric kit according to claim 6, characterized in that: The reagent R2 includes 50mmol / L of a first buffer, 50mmol / L of a second buffer, 2g / L of latex microspheres, 0.08g / L of a matrix metalloproteinase 3 antibody, 0.3g / L of an activator, 0.6g / L of a protective agent, 1% (W / V) of a blocking agent, and 30g / L of a stabilizer.

8. The matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to claim 1, characterized in that: The first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer, and Hepes buffer; The second buffer is selected from one of 2(N-morpholine)ethanesulfonic acid buffer, glycine buffer, Hepes buffer, and Tris-HCl buffer; The particle size of the latex microspheres is 100nm to 200nm, and the solid content is 10%; The matrix metalloproteinase 3 antibody is an animal anti-human matrix metalloproteinase 3 monoclonal antibody or polyclonal antibody, and the animal species include rabbits, goats, sheep, chickens and mice; The activator is 1-ethyl (3-dimethylaminopropyl) carbodiimide; The protective agent is N-hydroxysuccinimide; The stabilizer is selected from one of trehalose, sucrose and glucose; The blocking agent is selected from one of bovine serum albumin and casein.

9. A method for preparing a latex-enhanced immunoturbidimetric kit for matrix metalloproteinase 3, characterized in that: The following steps are involved: Using N-hydroxysuccinimide, a rheumatoid factor blocker and a protein denaturant, a reagent R1 is prepared; Prepare reagent R2, and subpackage the reagent R1 and reagent R2 to obtain a test kit; Wherein, the reagent R2 is prepared by the following preparation steps: Take a container, add the second buffer solution and latex microspheres, mix them well, then add an activator and a protective agent respectively, mix them well, and obtain activated latex microspheres; Adding matrix metalloproteinase 3 antibody to the activated latex microspheres in the container, mixing, and obtaining labeled latex microspheres-matrix metalloproteinase 3 antibody; Adding a blocking agent to the labeled latex microspheres-MMP-3 antibody in the container, fully dissolving and blocking, to obtain blocked latex microspheres; Add the first buffer solution and the stabilizer to the blocked latex microspheres in the container and mix thoroughly to obtain reagent R2.

10. Use of the matrix metalloproteinase 3 latex enhanced immunoturbidimetric kit according to any one of claims 1 to 8, characterized in that: The kit is used for detecting the content of matrix metalloproteinase 3 in human serum or plasma.

Images