Hypersensitive C-reactive protein determination kit as well as preparation method and application thereof

Abstract

The embodiment of the invention belongs to the technical field of immunodetection, and relates to a hypersensitive C-reactive protein determination kit, which comprises a reagent R1 and a reagent R2, the reagent R1 comprises lauryl sodium sulfate, a phosphate buffer solution, a first stabilizer, a biological preservative and a nonionic surfactant; and the reagent R2 comprises the following components: a latex microsphere-amino acid-C reactive protein antibody complex, a first buffer solution and a second stabilizer. The invention also provides a preparation method and application of the hypersensitive C-reactive protein determination kit. The detection sensitivity can be improved, the detection range is wide, and the detection accuracy can be improved.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a high-sensitivity C-reactive protein assay kit and a preparation method and application thereof. Background Art

[0002] C-reactive protein (CRP) is synthesized by liver epithelial cells stimulated by inflammatory lymphokines interleukin-6, interleukin-1, and tumor necrosis factor. It is named because it can react with the capsular C polysaccharide of Streptococcus pneumoniae to precipitate. High-sensitivity C-reactive protein (hs-CRP) is detected using a highly sensitive method, that is, the detection method has the ability to detect CRP ≤0.5mg / L.

[0003] Low levels of CRP (0.1-10 mg / L) are closely related to the occurrence of cardiovascular disease and are biomarkers of cardiovascular inflammatory lesions. This level of C-reactive protein is also called high-sensitivity C-reactive protein (hs-CRP). An individual's basal CRP level is closely related to the future onset of cardiovascular disease. CRP may be a more effective independent predictor of cardiovascular disease than low-density lipoprotein cholesterol, and can increase the prognostic value of blood lipid tests, metabolic syndrome, and Framingham risk scores. In addition, studies have shown that routine testing of hs-CRP in early newborns, especially those with a history of abnormal delivery, and observation of dynamic changes are of great significance for the early diagnosis of neonatal diseases and determination of therapeutic effects.

[0004] At present, when CRP is measured by conventional clinical methods, the linear range of the test is generally 3 to 200 mg / L. Because the detection method lacks sufficient sensitivity, it is impossible to detect lower CRP levels in the blood. In the early days, the enzyme-linked immunosorbent assay was mainly used to detect hs-CRP. In recent years, latex-enhanced immunoturbidimetry, immunoturbidimetry, immunoluminescence and other technologies have been used to greatly improve the sensitivity of the test, and the detection limit has been extended to 0.005 to 0.10 mg / L.

[0005] Conventional CRP and hs-CRP are essentially the same substance in the same chemical sense. Conventional CRP is mainly used to monitor inflammation in children and adults, with a reference value of 5 μg / ml. hs-CRP is mainly used to monitor inflammation in neonates and predict the risk of cardiovascular events, with a reference value of 1 μg / ml. Since the CRP measured clinically using latex turbidimetry is limited to a concentration of approximately 0.5 μg / ml, this method cannot be applied to local inflammation or small lesions. Therefore, it is necessary to further improve the sensitivity of CRP assay reagents to detect trace levels of CRP.

[0006] In addition, the current clinically commonly used hypersensitive C-reactive protein detection kits all use plasma or serum samples for detection, which requires venous blood collection and a large amount of blood collection. The sample needs to be centrifuged before use, and whole blood cannot be directly used as a test sample. The detection speed is slow, so it cannot meet the needs of hospital emergency and outpatient rapid diagnosis. Patent document CN101881777B discloses a hypersensitive C-reactive protein detection method and kit that does not require pretreatment of whole blood samples. The method directly uses fingertip whole blood or anticoagulated venous whole blood as the sample to be tested, and uses rabbit anti-human C-reactive protein antibody sensitized styrene latex as the antibody, and uses timed scattering turbidimetry to detect C-reactive protein concentration. Patent document CN102866256B discloses a hypersensitive C-reactive protein detection method and detection reagent, which can detect body fluids, especially trace biochemical substances in blood, on a specific protein analyzer, with a detection range of 0.4mg / L-350mg / L, which expands the detection range of hypersensitive C-reactive protein. However, the detection sensitivity of high-sensitivity C-reactive protein in the technologies disclosed in the above documents does not reach the requirement of being far below 0.5 μg / ml, and does not reach a relatively sensitive indicator for distinguishing low-level inflammatory states. Summary of the invention

[0007] The purpose of the embodiments of the present application is to provide a high-sensitivity C-reactive protein assay kit and a preparation method and application thereof, so as to solve the technical problems of low specificity and low sensitivity of high-sensitivity C-reactive protein detection in the prior art.

[0008] In order to solve the above technical problems, the present application provides a high-sensitivity C-reactive protein assay kit, which adopts the following technical solution:

[0009] comprising reagent R1 and reagent R2;

[0010] The components of the reagent R1 include sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant;

[0011] The components of the reagent R2 include a latex microsphere-amino acid-C-reactive protein antibody complex, a first buffer and a second stabilizer.

[0012] Furthermore, the components of the latex microsphere-amino acid-C-reactive protein antibody complex include a second buffer, a first latex microsphere, a second latex microsphere, a coupling agent, a C-reactive protein antibody, amino acids and a blocking agent.

[0013] Further, the coupling agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide;

[0014] The concentration of the first buffer is 5-100 mmol / L;

[0015] The concentration of the second buffer is 5-50mmol / L;

[0016] The concentration of the first latex microspheres is 0.5-2 g / L;

[0017] The concentration of the second latex microspheres is 0.5-2 g / L;

[0018] The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.08-0.8 g / L;

[0019] The concentration of the N-hydroxysuccinimide is 0.1-0.8 g / L;

[0020] The concentration of the C-reactive protein antibody is 0.03-0.15 g / L;

[0021] The concentration of the amino acid is 5-1200mmol / L;

[0022] The concentration of the blocking agent is 1.5-5.5 g / L;

[0023] The concentration of the second stabilizer is 15-40 g / L.

[0024] Further, the concentration of the first buffer is 50mmol / L;

[0025] The concentration of the second buffer is 50mmol / L;

[0026] The concentration of the first latex microspheres is 2 g / L;

[0027] The concentration of the second latex microspheres is 1 g / L;

[0028] The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.3 g / L;

[0029] The concentration of the N-hydroxysuccinimide is 0.6 g / L;

[0030] The concentration of the C-reactive protein antibody is 0.08 g / L;

[0031] The concentration of the amino acid is 300mmol / L;

[0032] The concentration of the blocking agent is 3 g / L;

[0033] The concentration of the second stabilizer is 30 g / L.

[0034] Further, the first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; and / or

[0035] The second buffer is selected from one of 2-(N-morpholine)ethanesulfonic acid buffer, Tris-HCl buffer, glycine buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; and / or

[0036] The second stabilizer is selected from one of trehalose, sucrose and glucose; and / or

[0037] The amino acid is selected from the group consisting of basic amino acids, sulfur-containing amino acids, oxygen-containing amino acids, aliphatic amino acids, acidic amino acids, acidic amino acid amides, and salts and hydrates thereof; and / or

[0038] The blocking agent is selected from one of bovine serum albumin and casein; and / or

[0039] The C-reactive protein antibody is a monoclonal antibody or a polyclonal antibody, wherein the animal species of the monoclonal antibody or the polyclonal antibody is selected from at least one of rabbit, goat, sheep, chicken and mouse; and / or

[0040] The particle size of the first latex microspheres is 100 nm and the solid content is 10%; and / or

[0041] The particle size of the second latex microspheres is 180 nm, and the solid content is 10%.

[0042] Further, the basic amino acid is selected from one of histidine, lysine and arginine;

[0043] The sulfur-containing amino acid is selected from one of cysteine ​​and methionine;

[0044] The oxygen-containing amino acid is selected from one of serine and threonine;

[0045] The aliphatic amino acid is selected from one of glycine, valine and leucine;

[0046] The acidic amino acid is selected from aspartic acid or glutamic acid;

[0047] The acidic amino acid amide is glutamine.

[0048] Furthermore, the first stabilizer includes sodium chloride and polyethylene glycol 6000; the biological preservative is Proclin 300; and the nonionic surfactant is Tween 20.

[0049] Further, the concentration of the sodium dodecyl sulfate is 0.005%-0.05% w / v;

[0050] The concentration of the phosphate buffer is 25-200 mmol / L;

[0051] The concentration of the sodium chloride is 0.9-120 g / L;

[0052] The concentration of polyethylene glycol 6000 is 5.0-15.0 g / L;

[0053] The concentration of Proclin300 is 0.1-5 g / L;

[0054] The concentration of Tween 20 is 1-5 g / L.

[0055] In order to solve the above technical problems, the present application also provides a method for preparing a high-sensitivity C-reactive protein assay kit, which adopts the following technical solution:

[0056] Prepare reagent R1 and reagent R2 respectively;

[0057] Packaging the reagent R1 and the reagent R2 to obtain a kit;

[0058] Wherein, the reagent R1 is prepared by the following preparation steps:

[0059] Mixing sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant to obtain the reagent R1;

[0060] The reagent R2 is prepared by the following preparation steps:

[0061] preparing latex microsphere-amino acid-C-reactive protein antibody complex;

[0062] The first buffer and the second stabilizer are added to the latex microsphere-amino acid-C-reactive protein antibody complex and mixed to obtain reagent R2.

[0063] Furthermore, the latex microsphere-amino acid-C-reactive protein antibody complex is prepared by the following preparation steps:

[0064] Take container A and container B, and add the second buffer solution respectively;

[0065] Add the first latex microspheres into container A, add the second latex microspheres into container B, and mix them respectively;

[0066] After mixing, the coupling agent is added into the A container and the B container respectively and mixed to obtain the first activated latex microsphere and the second activated latex microsphere;

[0067] Adding amino acids to the first activated latex microsphere and the second activated latex microsphere respectively, and mixing them to obtain first latex microsphere-amino acids and second latex microsphere-amino acids respectively;

[0068] Add coupling agents to the first latex microsphere-amino acid and the second latex microsphere-amino acid respectively, mix well, and obtain the corresponding first activated latex microsphere-amino acid and second activated latex microsphere-amino acid;

[0069] Adding the C-reactive protein antibody to the first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid respectively, and mixing them evenly to obtain the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody;

[0070] Blocking agents are added to the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody, respectively, to obtain first blocked latex microspheres and second blocked latex microspheres, wherein the first blocked latex microspheres and the second blocked latex microspheres constitute the latex microsphere-amino acid-C-reactive protein antibody complex.

[0071] In order to solve the above technical problems, the embodiments of the present application also provide a use of the above-mentioned high-sensitivity C-reactive protein assay kit or the kit prepared by the above-mentioned preparation method in detecting the C-reactive protein content in whole blood.

[0072] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0073] The present application provides a high-sensitivity C-reactive protein assay kit, in which the latex microsphere-amino acid-C-reactive protein antibody complex can directionally solidify the antibody on the latex microsphere, effectively reducing the antibody steric hindrance and fully exposing the antibody active site. The kit has the advantages of simple operation, fast detection speed, improved detection sensitivity and wide detection range. Secondly, the reagent R1 contains a certain amount of surfactant sodium dodecyl sulfate, which can effectively reduce the interference of the reagent in detecting whole blood and increase the accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] 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.

[0075] Figure 1 It is a preparation flow chart of reagent R2 in the high-sensitivity C-reactive protein assay kit of the present application;

[0076] Figure 2 It is a data graph of the reactivity impact results of the embodiment of the present application;

[0077] Figure 3 It is a linear verification data diagram of an embodiment of the present application;

[0078] Figure 4 It is a linear graph of the dilution of whole blood sample in the embodiment of the present application. DETAILED DESCRIPTION

[0079] 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.

[0080] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations 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 may be combined with other embodiments.

[0081] 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.

[0082] The present application provides a high-sensitivity C-reactive protein assay kit, comprising a reagent R1 and a reagent R2.

[0083] The components of reagent R1 include sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant; the components of reagent R2 include a latex microsphere-amino acid-C-reactive protein antibody complex, a first buffer and a second stabilizer.

[0084] Among them, the latex microsphere-amino acid-C-reactive protein antibody can directionally bind to the antibody Fc fragment, effectively reducing the antibody steric hindrance, allowing the antibody active site to be fully exposed and improving sensitivity; sodium dodecyl sulfate (SDS) is an anionic surfactant, which effectively reduces the interference of reagents in detecting whole blood and increases the accuracy of detection.

[0085] It should be noted that the reagents in the high-sensitivity C-reactive protein assay kit of the present application can directly use anticoagulated venous whole blood as the sample to be tested, without the need for centrifugal separation of plasma or serum, which greatly improves the detection speed and can meet the needs of rapid diagnosis in hospital emergency and outpatient departments.

[0086] In some embodiments, the components of the latex microsphere-amino acid-C-reactive protein antibody complex include a second buffer, a first latex microsphere, a second latex microsphere, a coupling agent, a C-reactive protein antibody, amino acids, and a blocking agent.

[0087] In this embodiment, amino acids are introduced as spacer molecules on the surfaces of the first latex microsphere and the second latex microsphere respectively by chemical modification, and then activated by a coupling agent, and a C-reactive protein antibody is added to be labeled. The antibody will be directionally immobilized on the latex microsphere, and finally blocked by a blocking agent to ultimately form a latex microsphere-amino acid-C-reactive protein antibody complex, which can directionally bind to the antibody Fc fragment, effectively reduce the antibody steric hindrance, and fully expose the antibody active site.

[0088] It should be understood that the particle sizes of the first latex microspheres and the second latex microspheres are different. By controlling the latex microspheres of different particle sizes to bind to C-reactive protein antibodies, a wide range of detection is achieved, with the highest detection concentration of 350 mg / L. It has the characteristics of flexibility and universality, and can be used for the detection of CRP in serum, plasma and whole blood; by adjusting the ratio of CRP latex microspheres and antibodies, the needs of immune transmission and scattering detection can be met, providing reliable raw materials for the production of test kits.

[0089] In this embodiment, the coupling agents include 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which are used to activate the latex microspheres introduced with amino acids, which is helpful for subsequent antibody labeling.

[0090] In some embodiments, the concentration of the first buffer is 5-100 mmol / L; the concentration of the second buffer is 5-50 mmol / L; the concentration of the first latex microspheres is 0.5-2 g / L; the concentration of the second latex microspheres is 0.5-2 g / L; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.08-0.8 g / L; the concentration of N-hydroxysuccinimide is 0.1-0.8 g / L; the concentration of C-reactive protein antibody is 0.03-0.15 g / L; the concentration of amino acids is 5-1200 mmol / L; the concentration of the blocking agent is 1.5-5.5 g / L; and the concentration of the second stabilizer is 15-40 g / L.

[0091] The concentration ranges of the components of the R2 reagent in this embodiment can effectively promote antigen-antibody reactions and improve detection sensitivity.

[0092] Preferably, the concentration of the first buffer is 50mmol / L; the concentration of the second buffer is 50mmol / L; the concentration of the first latex microspheres is 2g / L; the concentration of the second latex microspheres is 1g / L; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.3g / L; the concentration of N-hydroxysuccinimide is 0.6g / L; the concentration of C-reactive protein antibody is 0.08g / L; the concentration of amino acids is 300mmol / L; the concentration of the blocking agent is 3g / L; and the concentration of the second stabilizer is 30g / L.

[0093] The above concentration range can ensure that the antigen-antibody reaction proceeds with maximum efficiency.

[0094] In some embodiments, the first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer (Hepes buffer); and / or

[0095] The second buffer is selected from one of 2-(N-morpholine)ethanesulfonic acid buffer (MES buffer), Tris-HCl buffer, glycine buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; and / or

[0096] The second stabilizer is selected from one of trehalose, sucrose and glucose; and / or

[0097] The amino acid is selected from the group consisting of basic amino acids, sulfur-containing amino acids, oxygen-containing amino acids, aliphatic amino acids, acidic amino acids, acidic amino acid amides, and salts and hydrates thereof; and / or

[0098] The blocking agent is selected from one of bovine serum albumin and casein; and / or

[0099] The C-reactive protein antibody is a monoclonal antibody or a polyclonal antibody, wherein the animal species of the monoclonal antibody or the polyclonal antibody is selected from at least one of rabbit, goat, sheep, chicken and mouse; and / or

[0100] The particle size of the first latex microsphere is 100 nm and the solid content is 10%; and / or

[0101] The particle size of the second latex microspheres is 180 nm, and the solid content is 10%.

[0102] In some embodiments, the basic amino acid is selected from one of histidine (His), lysine (Lys) and arginine (Arg); the sulfur-containing amino acid is selected from one of cysteine ​​(Cys) and methionine (Met); the oxygen-containing amino acid is selected from one of serine (Ser) and threonine (Thr); the aliphatic amino acid is selected from one of glycine (Gly), valine (Val) and leucine (Leu); the acidic amino acid is selected from aspartic acid (Asp) or glutamic acid (Glu); and the acidic amino acid amide is glutamine (Gln).

[0103] In the R1 reagent of some embodiments, the first stabilizer includes sodium chloride and polyethylene glycol 6000; the biological preservative is Proclin 300; and the nonionic surfactant is Tween 20.

[0104] In some embodiments, the concentration of sodium dodecyl sulfate is 0.005%-0.05% (w / v); the concentration of phosphate buffer is 25-200 mmol / L; the concentration of sodium chloride is 0.9-120 g / L; the concentration of polyethylene glycol 6000 is 5.0-15.0 g / L; the concentration of Proclin 300 is 0.1-5 g / L; and the concentration of Tween 20 is 1-5 g / L.

[0105] The sodium dodecyl sulfate (SDS) component in the R1 reagent is an anionic surfactant. If the concentration is too high, it will inhibit the agglutination reaction of the antigen and antibody. The concentration range used in this embodiment can achieve the purpose of rapid hemolysis without inhibiting the antigen-antibody reaction. By adding a certain amount of surfactant SDS to the R1 reagent, the interference of the reagent in detecting whole blood is effectively reduced, and the accuracy of the detection is increased.

[0106] The ultra-sensitive C-reactive protein assay kit of the present application can directly use anticoagulated venous whole blood as the sample to be tested, introduce spacer molecule amino acids combined with C-reactive protein antibodies on the surface of chemically modified latex microspheres, and use transmission turbidimetry to detect the C-reactive protein concentration. The kit is easy to operate, has a fast detection speed, a wide detection range, and an extremely high detection sensitivity, thereby realizing the diagnosis and prediction of various aspects such as infectious diseases and cardiovascular and cerebrovascular diseases.

[0107] Based on the above-mentioned high-sensitivity C-reactive protein assay kit, the present application also provides a method for preparing a high-sensitivity C-reactive protein assay kit, comprising the following steps:

[0108] Prepare reagent R1 and reagent R2 respectively;

[0109] Packaging reagent R1 and reagent R2 to obtain a test kit;

[0110] Wherein, reagent R1 is prepared by the following preparation steps:

[0111] Mixing sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant to obtain a reagent R1;

[0112] Reagent R2 is prepared by the following preparation steps:

[0113] preparing latex microsphere-amino acid-C-reactive protein antibody complex;

[0114] The first buffer and the second stabilizer are added to the latex microsphere-amino acid-C-reactive protein antibody complex and mixed to obtain reagent R2.

[0115] Reagent R1 was prepared according to conventional methods, and the latex microsphere-amino acid-C-reactive protein antibody complex in reagent R2 was prepared through activation, condensation, reactivation, labeling and blocking processes. Figure 1 , specifically including the following steps:

[0116] Step S101, taking container A and container B, and adding a second buffer solution respectively.

[0117] Step S102, adding the first latex microspheres into container A, adding the second latex microspheres into container B, and mixing them respectively.

[0118] Step S103, after mixing, the coupling agent is added into container A and container B respectively and mixed to obtain first activated latex microspheres and second activated latex microspheres.

[0119] Step S104, adding amino acids to the first activated latex microspheres and the second activated latex microspheres respectively, and mixing them to obtain the first latex microspheres-amino acids and the second latex microspheres-amino acids respectively.

[0120] Step S105, adding coupling agents to the first latex microsphere-amino acid and the second latex microsphere-amino acid respectively, mixing them evenly, to obtain the corresponding first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid.

[0121] Step S106, adding the C-reactive protein antibody to the first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid respectively, mixing them evenly, to obtain the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody.

[0122] Step S107, adding blocking agents to the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody respectively to obtain the first blocked latex microsphere and the second blocked latex microsphere, the first blocked latex microsphere and the second blocked latex microsphere constitute a latex microsphere-amino acid-C-reactive protein antibody complex.

[0123] Among them, step S101 to step S103 are the activation process; step S104 is the condensation process; step S105 is the reactivation process; step S106 is the labeling process; and step S107 is the sealing process.

[0124] In this embodiment, after preparing the latex microsphere-amino acid-C-reactive protein antibody complex, the method further comprises:

[0125] The first buffer solution and the second stabilizer are added to the first blocked latex microspheres and the second blocked latex microspheres respectively, and the mixture is fully mixed. Then, the substances in the A container and the B container are mixed evenly to obtain the reagent R2.

[0126] During the preparation of reagent R2, 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, thereby improving production efficiency.

[0127] For example, the preparation method of two liters of reagent R2 comprises the following steps:

[0128] 1) Activation: Take two containers A and B, then add 500 ml of the second buffer solution respectively, add the first latex microspheres to the A container, add the second latex microspheres to the B container, stir for 12-15 minutes in a shaker at 37° C., and mix well; then add NHS and EDC respectively and stir for 30-35 minutes to mix well, to obtain the first activated latex microspheres and the second activated latex microspheres respectively;

[0129] 2) Condensation: adding amino acids to the activated latex microspheres in container A and container B respectively while stirring, stirring in a shaker at 37° C. for 60-65 min, and mixing well to obtain first latex microsphere-amino acid and second latex microsphere-amino acid, respectively;

[0130] 3) Reactivation: Add NHS and EDC to container A and container B respectively, and stir for 30-35 minutes to mix well, to obtain the first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid respectively;

[0131] 4) Labeling: adding C-reactive protein antibody to the first activated latex microsphere-amino acid in container A while stirring, adding C-reactive protein antibody to the second activated latex microsphere-amino acid in container B while stirring, stirring in a shaker at 37° C. for 60-120 min to obtain the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody;

[0132] 5) Blocking: Add blocking agents to the latex microspheres-amino acid-C-reactive protein antibodies in the two containers, fully dissolve and mix for 80-120 minutes, and block to obtain first blocked latex microspheres and second blocked latex microspheres, respectively;

[0133] 6) Add 500 ml of the first buffer solution and the second stabilizer to the closed latex microspheres in the two containers respectively, mix thoroughly, and then mix the substances in container A and container B evenly to obtain reagent R2.

[0134] The stirring speed is 180-200 rpm.

[0135] It should be understood that the raw material buffer, stabilizer, blocking agent, latex microspheres, amino acids, and C-reactive protein antibodies used in this application are all purchased from chemical and biological raw material companies. Among them, the particle size of the first latex microspheres is 100nm and the solid content is 10%; the particle size of the second latex microspheres 2 is 180nm and the solid content is 10%, and they are used directly.

[0136] The present application also provides a use of the above-mentioned high-sensitivity C-reactive protein assay kit or the kit prepared by the above-mentioned preparation method in detecting the content of C-reactive protein in whole blood.

[0137] 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.

[0138] Example 1

[0139] The reagent kit of this embodiment contains SDS of different concentrations in reagent R1.

[0140] Reagent R1 includes R1-A, R1-B, R1-C, R1-D, R1-E, R1-F, R1-G and R1-H.

[0141] The components and corresponding concentrations of reagent R1-A are as follows:

[0142] 100mmol / L phosphate buffer, 9g / L sodium chloride, 7.0g / L polyethylene glycol 6000, 0.5g / L biological preservative Proclin300, and 2g / L Tween 20.

[0143] The components of reagents R1-B, C, D, E, F, G and H are the same as those of reagent R1-A, except for the different SDS concentrations, which are 0.005% w / v, 0.01% w / v, 0.02% w / v, 0.04% w / v, 0.06% w / v, 0.08% w / v and 0.1% w / v, respectively.

[0144] Reagent R1 was prepared according to conventional methods.

[0145] The components and corresponding concentrations of reagent R2 are as follows:

[0146] 50mmol / L glycine buffer, 50mmol / L 2-(N-morpholine)ethanesulfonic acid buffer, 2g / L first latex microspheres, 1g / L second latex microspheres, 0.3g / L EDC, 0.6g / L NHS, 0.08g / L C-reactive protein antibody, 300mmol / L arginine, 3g / L bovine serum albumin, 30g / L trehalose.

[0147] The preparation method of two liters of reagent R2 includes the following steps, wherein the stirring speed is 200 rpm:

[0148] 1) Activation: Take two containers A and B, then add 500 ml of the second buffer solution respectively, add the first latex microspheres to container A, add the second latex microspheres to container B, and stir for 15 minutes in a shaker at 37°C; then add NHS and EDC respectively and stir for 30 minutes to obtain the first activated latex microspheres and the second activated latex microspheres respectively;

[0149] 2) Condensation: adding amino acids to the activated latex microspheres in container A and container B respectively while stirring, and stirring in a shaker at 37° C. for 60 min to obtain a first latex microsphere-amino acid and a second latex microsphere-amino acid, respectively;

[0150] 3) Reactivation: Add NHS and EDC to container A and container B respectively, and stir for 30 minutes to mix well, to obtain the first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid respectively;

[0151] 4) Labeling: adding C-reactive protein antibody to the first activated latex microsphere-amino acid in container A while stirring, adding C-reactive protein antibody to the second activated latex microsphere-amino acid in container B while stirring, stirring in a shaker at 37° C. for 120 min to obtain the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody;

[0152] 5) Blocking: Add blocking agents to the latex microspheres-amino acid-C-reactive protein antibodies in the two containers, respectively, fully dissolve and mix for 120 minutes, and block to obtain first blocked latex microspheres and second blocked latex microspheres;

[0153] 6) Add 500 ml of the first buffer solution and the second stabilizer to the closed latex microspheres in the two containers respectively, mix thoroughly, and then mix the substances in container A and container B evenly to obtain reagent R2.

[0154] The eight whole blood high-sensitivity C-reactive protein assay kits prepared above (among which, reagents R1-A, R1-B, R1-C, R1-D, R1-E, R1-F, R1-G and R1-H, each with a different SDS concentration) and the purchased C-reactive protein assay kit for serum samples were tested on whole blood samples and serum samples from the same source, and the test results are shown in Table 1.

[0155] Table 1

[0156]

[0157]

[0158] From the data in the table above, it can be seen that when different concentrations of SDS are added to reagent R2, the results of reagents R1-B, R1-C, R1-D, R1-E, and R1-F are more stable than those of R1-A without SDS, while the measurement results of R1-G and R1-H are more unstable and have a larger range of variation than R1-A, and the correlation coefficient is lower than that of the purchased reagents. From the experimental results, it can be seen that when the SDS concentration is between 0.005% and 0.05%, the measurement results are more stable, and the consistency of the whole blood sample and serum sample is good.

[0159] Example 2

[0160] This example shows the effect of different amino acids on the reaction of reagents.

[0161] Specifically, latex microsphere-amino acid-C-reactive protein antibody complex was prepared using 10 kinds of amino acids, and the reactivity of the obtained reagent was analyzed.

[0162] The components and concentrations of reagent R1 are as follows:

[0163] 100mmol / L phosphate buffer, 9g / L sodium chloride, 7.0g / L polyethylene glycol 6000, 0.5g / L biological preservative Proclin300, 2g / L Tween 20 and 0.02% SDS.

[0164] Wherein, reagent R1 is prepared according to conventional methods.

[0165] Reagent R2 includes R2-A, R2-B, R2-C, R2-D, R2-E, R2-F, R2-G and R2-H.

[0166] The components and concentrations of reagent R2-A are as follows:

[0167] 50mmol / L glycine buffer, 50mmol / L 2-(N-morpholine)ethanesulfonic acid buffer, 2g / L first latex microspheres, 1g / L second latex microspheres, 0.3g / L EDC, 0.6g / L NHS, 0.08g / L C-reactive protein antibody, 3g / L bovine serum albumin and 30g / L trehalose.

[0168] The components of reagents R2-B, C, D, E, F, G and H differ from those of reagent R2-A in that the types and concentrations of amino acids are different. The corresponding amino acids are 300mmol / L arginine (Arg), 300mmol / L histidine (His), 300mmol / L cysteine ​​(Cys), 300mmol / L serine (Ser), 300mmol / L glycine (Gly), 300mmol / L aspartic acid (Asp), 300mmol / L isoleucine (Leu), and 300mmol / L glutamine (Gln).

[0169] The preparation of reagent R2 is consistent with the preparation process of reagent R2 in Example 1, and will not be repeated here.

[0170] The eight whole blood high-sensitivity C-reactive protein assay kits prepared above (among which, reagents R2-A, R2-B, R2-C, R2-D, R2-E, R2-F, R2-G and R2-H, each with a different amino acid type) and the purchased serum sample C-reactive protein assay kit were analyzed to obtain the reactivity of the reagents. The results are as follows Figure 2 shown.

[0171] When different types of amino acids were added to reagent R2, compared with R2-A without amino acids and purchased reagents, reagents R2-B, R2-C, R2-D, R2-E, R2-F, and R2-H had higher reaction sensitivities, while R1-B, R2-C, and R2-H had higher sensitivities at low values ​​of 5 mg / L, but R2-F and R2-H had poor overall linearity. R2-B and R2-C were better in terms of comprehensive reaction sensitivity and linearity. From the experimental results, it can be seen that adding different types of amino acids to reagent R2 can improve the reactivity of the reagent, and different types of amino acids have different effects on the reactivity of the reagent. For example, 300mmol / L histidine and 300mmol / L glutamine both showed higher sensitivity at low values, but 300mmol / L glutamine had poor overall linearity.

[0172] Example 3

[0173] This example analyzes and verifies the performance of a high-sensitivity C-reactive protein assay kit.

[0174] The reagent R1 in the kit is the reagent R1 in Example 2, and the reagent R2 is the reagent R2-B in Example 2.

[0175] (1) Accuracy verification

[0176] 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 reagents, test each 10 times, and compare them with the target value of the quality control material. The results are shown in Table 2 below.

[0177] Table 2

[0178]

[0179]

[0180] It can be seen from the data in the above table that the C-reactive protein detection reagent in the kit provided by the present application has a high accuracy.

[0181] (2) Precision verification

[0182] 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 reagents, test each 10 times, calculate the mean value, standard deviation and coefficient of variation of the test results, and the results are shown in Table 3 below.

[0183] Table 3

[0184]

[0185]

[0186] It can be seen from the coefficient of variation in the table that the kit of the present application has high precision in detecting C-reactive protein.

[0187] (3) Sensitivity verification

[0188] Take 3 traceable quality control materials and dilute them to near the lower limit of the detection range for determination. Repeat the determination 5 times and calculate the average value to compare with the target value of the quality control material. The results are shown in Table 4.

[0189] Table 4

[0190]

[0191] It can be seen from the data in the above table that the C-reactive protein detection reagent in the kit provided by the present application has a high sensitivity.

[0192] (4) Linear verification

[0193] 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 theoretical concentration, and calculate the square of the regression coefficient r. The results are shown in Table 5 and Figure 3 .

[0194] Table 5

[0195] Linear Theoretical concentration Measured value 1 Measured value 2 Measured value 3 average value deviation Linear 1 0.00 0.05 0.00 0.03 0.03 -1.3 Linear 2 35.00 30.46 30.91 30.78 30.72 -4.8％ Linear 3 70.00 70.28 75.71 87.90 77.96 -2.5％ Linear 4 105.00 108.96 111.41 102.97 107.78 -2.1％ Linear 5 140.00 151.80 145.43 144.17 147.13 -1.8％ Linear 6 175.00 184.04 199.61 162.15 181.93 -1.6％ Linear 7 210.00 175.36 193.94 216.29 195.20 -1.6％ Linear 8 245.00 267.64 260.69 274.07 267.47 -1.4％ Linear 9 280.00 275.51 275.27 277.00 275.93 -1.4％ Linear 10 315.00 311.97 323.53 313.17 316.22 -1.3％ Linear 11 350.00 356.24 354.09 358.99 356.44 -1.3％ formula intercept 1.2823 Slope 1.0092 r-value 0.9969

[0196] From the above table and Figure 3 It can be seen that within the range of 0.04 mg / L-350 mg / L, the regression coefficient r=0.9969, indicating that the test kit of the present application has good linearity and a wide detection range for detecting C-reactive protein.

[0197] (5) Human whole blood samples were diluted at different times and the C-reactive protein concentration was tested. The linearity graph of whole blood sample dilution is shown in Figure 4 ,Depend on Figure 4 It can be seen that after a series of dilutions of the sample, the measured CRP value has a small deviation from the CRP theoretical value, which indicates that the kit of the present application has high sensitivity, high accuracy and wide detection range for detecting C-reactive protein.

[0198] 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 high-sensitivity C-reactive protein assay kit, characterized in that: include: Reagent R1 and reagent R2; The components of the reagent R1 include sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant; The components of the reagent R2 include a latex microsphere-amino acid-C-reactive protein antibody complex, a first buffer and a second stabilizer.

2. The high-sensitivity C-reactive protein assay kit according to claim 1, characterized in that: The components of the latex microsphere-amino acid-C-reactive protein antibody complex include a second buffer, a first latex microsphere, a second latex microsphere, a coupling agent, a C-reactive protein antibody, amino acids and a blocking agent.

3. The high-sensitivity C-reactive protein assay kit according to claim 2, characterized in that: The coupling agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The concentration of the first buffer is 5-100 mmol / L; The concentration of the second buffer is 5-50mmol / L; The concentration of the first latex microspheres is 0.5-2 g / L; The concentration of the second latex microspheres is 0.5-2 g / L; The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.08-0.8 g / L; The concentration of the N-hydroxysuccinimide is 0.1-0.8 g / L; The concentration of the C-reactive protein antibody is 0.03-0.15 g / L; The concentration of the amino acid is 5-1200mmol / L; The concentration of the blocking agent is 1.5-5.5 g / L; The concentration of the second stabilizer is 15-40 g / L.

4. The high-sensitivity C-reactive protein assay kit according to claim 3, characterized in that: The concentration of the first buffer is 50mmol / L; The concentration of the second buffer is 50mmol / L; The concentration of the first latex microspheres is 2 g / L; The concentration of the second latex microspheres is 1 g / L; The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.3 g / L; The concentration of the N-hydroxysuccinimide is 0.6 g / L; The concentration of the C-reactive protein antibody is 0.08 g / L; The concentration of the amino acid is 300mmol / L; The concentration of the blocking agent is 3 g / L; The concentration of the second stabilizer is 30 g / L.

5. The high-sensitivity C-reactive protein assay kit according to claim 2, characterized in that: The first buffer is selected from one of Tris-HCl buffer, glycine buffer, PBS buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; and / or The second buffer is selected from one of 2-(N-morpholine)ethanesulfonic acid buffer, Tris-HCl buffer, glycine buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; and / or The second stabilizer is selected from one of trehalose, sucrose and glucose; and / or The amino acid is selected from the group consisting of basic amino acids, sulfur-containing amino acids, oxygen-containing amino acids, aliphatic amino acids, acidic amino acids, acidic amino acid amides, and salts and hydrates thereof; and / or The blocking agent is selected from one of bovine serum albumin and casein; and / or The C-reactive protein antibody is a monoclonal antibody or a polyclonal antibody, wherein the animal species of the monoclonal antibody or the polyclonal antibody is selected from at least one of rabbit, goat, sheep, chicken and mouse; and / or The particle size of the first latex microspheres is 100 nm and the solid content is 10%; and / or The particle size of the second latex microspheres is 180 nm, and the solid content is 10%.

6. The high-sensitivity C-reactive protein assay kit according to claim 5, characterized in that: The basic amino acid is selected from one of histidine, lysine and arginine; The sulfur-containing amino acid is selected from one of cysteine ​​and methionine; The oxygen-containing amino acid is selected from one of serine and threonine; The aliphatic amino acid is selected from one of glycine, valine and leucine; The acidic amino acid is selected from aspartic acid or glutamic acid; The acidic amino acid amide is glutamine.

7. The high-sensitivity C-reactive protein assay kit according to claim 1, characterized in that: The first stabilizer includes sodium chloride and polyethylene glycol 6000; the biological preservative is Proclin 300; and the nonionic surfactant is Tween 20.

8. The high-sensitivity C-reactive protein assay kit according to claim 7, characterized in that: The concentration of the sodium lauryl sulfate is 0.005%-0.05% w / v; The concentration of the phosphate buffer is 25-200 mmol / L; The concentration of the sodium chloride is 0.9-120 g / L; The concentration of polyethylene glycol 6000 is 5.0-15.0 g / L; The concentration of Proclin300 is 0.1-5 g / L; The concentration of Tween 20 is 1-5 g / L.

9. A method for preparing a high-sensitivity C-reactive protein assay kit, characterized in that: The following steps are involved: Prepare reagent R1 and reagent R2 respectively; Packaging the reagent R1 and the reagent R2 to obtain a kit; Wherein, the reagent R1 is prepared by the following preparation steps: Mixing sodium dodecyl sulfate, phosphate buffer, a first stabilizer, a biological preservative and a nonionic surfactant to obtain the reagent R1; The reagent R2 is prepared by the following preparation steps: preparing latex microsphere-amino acid-C-reactive protein antibody complex; The first buffer and the second stabilizer are added to the latex microsphere-amino acid-C-reactive protein antibody complex and mixed to obtain reagent R2.

10. The method for preparing the high-sensitivity C-reactive protein assay kit according to claim 9, characterized in that: The latex microsphere-amino acid-C-reactive protein antibody complex is prepared by the following preparation steps: Take container A and container B, and add the second buffer solution respectively; Add the first latex microspheres into container A, add the second latex microspheres into container B, and mix them respectively; After mixing, the coupling agent is added into the A container and the B container respectively and mixed to obtain the first activated latex microsphere and the second activated latex microsphere; Adding amino acids to the first activated latex microsphere and the second activated latex microsphere respectively, and mixing them to obtain first latex microsphere-amino acids and second latex microsphere-amino acids respectively; Add coupling agents to the first latex microsphere-amino acid and the second latex microsphere-amino acid respectively, mix well, and obtain the corresponding first activated latex microsphere-amino acid and second activated latex microsphere-amino acid; Adding the C-reactive protein antibody to the first activated latex microsphere-amino acid and the second activated latex microsphere-amino acid respectively, and mixing them evenly to obtain the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody; Blocking agents are added to the first labeled latex microsphere-amino acid-C-reactive protein antibody and the second labeled latex microsphere-amino acid-C-reactive protein antibody, respectively, to obtain first blocked latex microspheres and second blocked latex microspheres, wherein the first blocked latex microspheres and the second blocked latex microspheres constitute the latex microsphere-amino acid-C-reactive protein antibody complex.

11. Use of the high-sensitivity C-reactive protein assay kit according to any one of claims 1 to 8 or the kit prepared by the preparation method according to claim 9 or 10 in detecting the content of C-reactive protein in whole blood.

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