Latex reagent and preparation method, urine microalbumin detection kit and preparation method

Abstract

The application discloses a latex reagent and a preparation method thereof, a urine microalbumin detection kit and a preparation method thereof. The latex reagent is mainly obtained by coupling a latex microsphere and an antibody, a first blocking agent, a second blocking agent and a third blocking agent. The first blocking agent comprises BlockmasterTM DB1130; the second blocking agent comprises BlockmasterTM PA1080; and the third blocking agent comprises bovine serum albumin. In the latex reagent, the solid content of the latex microsphere is 0.05% to 0.1%, the concentration of the antibody is 0.05 mg / L to 0.2 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%. Different blocking agents with different molecular weights are mixed to improve the latex stability of the reagent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemistry, more particularly, to a latex reagent and a preparation method, a urine microalbumin detection kit and a preparation method. BACKGROUND

[0002] Microalbuminuria (mALB) refers to the presence of microalbumin in urine. Under normal human metabolism, the content of albumin in urine is extremely small, but when early kidney damage such as diabetes, nephropathy, hypertension nephropathy, and complications of cardiovascular diseases occurs, it will cause damage to the intrinsic cells of the kidney, resulting in changes in the structure and function of the intrinsic cells of the kidney and leading to an increase in the content of albumin in urine. The existing technology mainly uses the latex immunoturbidimetry method to detect mALB. The latex immunoturbidimetry method is a method in which an antigen or an antibody is labeled on a latex particle to prepare a latex solution, and after mixing with a corresponding antigen or antibody solution, an antigen-antibody reaction is caused to form a certain turbidity, and the change in turbidity is detected to determine the concentration of the antibody or antigen.

[0003] With the development of medical testing, the sensitivity and linear range of the latex immunoturbidimetry method are increasingly required. Currently, large-particle-size latex microspheres and small-particle-size latex microspheres are mixed on the market, and the average diameters and concentrations of the large-particle-size latex microspheres and the small-particle-size latex microspheres are optimized to simultaneously achieve the effects of improving the sensitivity and linear range of the reagent. Chinese Patent CN111766381A discloses a determination kit based on the latex immunoturbidimetry method and its application. The amounts of activators and antibodies are optimized for latex microspheres of different particle sizes. The reagent prepared by mixing two kinds of latex microspheres with different average particle sizes in a certain proportion can improve the sensitivity and linear width of the kit. However, the large-particle-size latex microspheres have a large volume, and the Brownian motion and surface charge have a relatively weak effect on the suspension stability of the microspheres, which is often insufficient to ensure the stable storage of the microsphere components within the effective period. SUMMARY

[0004] The present application aims to overcome the above-mentioned defects in the prior art and provide a latex reagent and a preparation method, a urine microalbumin detection kit and a preparation method, which have high sensitivity, wide linear range and high stability.

[0005] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0006] A latex reagent, which is mainly obtained by coupling latex microspheres with an antibody, a first blocking agent, a second blocking agent and a third blocking agent;

[0007] The first blocking agent includes Blockmaster TM DB1130;

[0008] The second blocking agent comprises Blockmaster TM PA1080;

[0009] The third blocking agent comprises bovine serum albumin;

[0010] In the latex reagent, the solid content of the latex microspheres is 0.05% to 0.1%, the concentration of the antibody is 0.05 mg / L to 0.2 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%.

[0011] The application further provides a preparation method of the latex reagent.

[0012] The latex microspheres are mixed with an activation buffer, and then an activation agent is added to activate, to obtain an activation reaction solution;

[0013] The antibody is diluted with a coupling buffer, and then added to the activation reaction solution to couple, to obtain a coupling reaction solution;

[0014] The first blocking agent is added to the coupling reaction solution to perform primary blocking, to obtain a reaction solution after primary blocking;

[0015] The second blocking agent is added to the reaction solution after primary blocking to perform secondary blocking, to obtain a reaction solution after secondary blocking;

[0016] The third blocking agent is added to the reaction solution after secondary blocking to perform tertiary blocking, to obtain a reaction solution after tertiary blocking;

[0017] The reaction solution after tertiary blocking is subjected to solid-liquid separation, and then resuspended to obtain the latex reagent.

[0018] The application further provides a urine microalbumin detection kit, comprising an R2 reagent.

[0019] The R2 reagent comprises the following components at the following concentrations:

[0020] 25 mM to 50 mM of an R2 buffer, 50% to 90% of a first latex reagent, and 10% to 50% of a second latex reagent;

[0021] The first latex reagent is mainly obtained by coupling first latex microspheres with a first antibody, a first blocking agent, a second blocking agent, and a third blocking agent;

[0022] The second latex reagent is mainly obtained by coupling second latex microspheres with a second antibody, a first blocking agent, a second blocking agent, and a third blocking agent;

[0023] The solid content of the first latex microspheres in the first latex reagent is 0.05% to 0.1%, the concentration of the first antibody is 0.05 mg / L to 0.2 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%;

[0024] The solid content of the second latex microspheres in the second latex reagent is 0.05% to 0.1%, the concentration of the first antibody is 0.05 mg / L to 0.1 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%;

[0025] The first blocking agent comprises BlockmasterTM DB1130;

[0026] The second blocking agent comprises BlockmasterTM PA1080;

[0027] The third blocking agent comprises bovine serum albumin;

[0028] The first antibody and the second antibody are both urine microalbumin antibodies;

[0029] The particle size of the first latex microspheres is 60 nm to 90 nm;

[0030] The particle size of the second latex microspheres is 350 nm to 400 nm.

[0031] The application further provides a preparation method of the urine microalbumin detection kit.

[0032] The first latex microspheres and the second latex microspheres are mixed with an activation buffer and then activated by an activation agent to obtain a first reaction solution and a second reaction solution, respectively;

[0033] The first antibody is diluted by a coupling buffer and then added to the first reaction solution for coupling, and the second antibody is diluted by a coupling buffer and then added to the second reaction solution for coupling to obtain a first coupling reaction solution and a second coupling reaction solution, respectively;

[0034] The first coupling reaction solution and the second coupling reaction solution are added with a first blocking agent for primary blocking to obtain a first reaction solution after primary blocking and a second reaction solution after primary blocking, respectively;

[0035] The first reaction liquid after the secondary blocking and the second reaction liquid after the secondary blocking are respectively added with a third blocking agent for tertiary blocking, to obtain the first reaction liquid after the tertiary blocking and the second reaction liquid after the tertiary blocking respectively;

[0036] The first reaction liquid after the secondary blocking and the second reaction liquid after the secondary blocking are respectively added with a third blocking agent for tertiary blocking, to obtain the first reaction liquid after the tertiary blocking and the second reaction liquid after the tertiary blocking respectively;

[0037] The first reaction liquid after the tertiary blocking and the second reaction liquid after the tertiary blocking are respectively subjected to solid-liquid separation, to obtain the first latex reagent and the second latex reagent respectively;

[0038] The first latex reagent and the second latex reagent are respectively added into R2 buffer solution for mixing, to obtain the R2 reagent.

[0039] The embodiment of the present application has the following beneficial effects:

[0040] The first blocking agent, the second blocking agent and the third blocking agent are combined for use in the embodiment of the present application, the first blocking agent with a small molecular weight is added first to block the groups on the microspheres that are not connected with the antibodies, especially the groups that are shielded by the antibodies, and then the second blocking agent and the third blocking agent with a large molecular weight are added in sequence, the macromolecular chains in the second blocking agent introduce hydrophilic groups and hydrophobic groups, so as to be adsorbed to the surface area and the surface groups of the latex microspheres, thereby preventing the latex microspheres from adsorbing components that do not participate in the immune reaction, inhibiting the non-specific agglutination of the latex, and blocking the groups on the latex microspheres that are not connected with the antibodies again, and the third blocking agent can block other sites of the antibodies except for the epitopes that react with the antigens. Therefore, the different molecular weight blocking agents are mixed for use in the embodiment of the present application, so that all the sites exposed on the surface of the latex microspheres can be blocked more effectively, thereby achieving the purpose of improving the latex stability of the reagent. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0042] The application discloses a latex reagent, which is mainly obtained by coupling of latex microspheres, an antibody, a first blocking agent, a second blocking agent and a third blocking agent; the first blocking agent comprises BlockmasterTM DB1130; the second blocking agent comprises BlockmasterTM PA1080; and the third blocking agent comprises bovine serum albumin; in the latex reagent, the solid content of the latex microspheres is 0.05% to 0.1%, the concentration of the antibody is 0.05 mg / L to 0.2 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%.

[0043] Specifically, the blocking effect of the latex in the latex immunoturbidimetry directly influences the stability of the reagent, and the blocking of the latex microspheres refers to blocking of blank areas on the surface of the latex microspheres which are not occupied by the antibody or the antigen by using the blocking reagent through hydrophobic bonds, electrostatic bonds, Van der Waals forces and the like. In the existing blocking process in the latex immunoturbidimetry, only bovine serum albumin is used for blocking. However, the bovine serum albumin alone cannot obviously improve the background signal caused by non-specific adsorption, cannot guarantee the storage stability of the latex microspheres, and after the antibody is coupled to groups on the latex microspheres, some groups on the microspheres are shielded. In the application, the first blocking agent, the second blocking agent and the third blocking agent are combined and used, the first blocking agent with a small molecular weight is first added to block the groups on the microspheres which are not connected to the antibody, especially the groups shielded by the antibody, and then the second blocking agent and the third blocking agent with large molecular weights are sequentially added, wherein the macromolecular chains in the second blocking agent introduce hydrophilic groups and hydrophobic groups, so that the second blocking agent can be adsorbed to the surface areas and surface groups of the latex microspheres, thereby preventing the latex microspheres from adsorbing components not participating in the immune reaction, inhibiting the non-specific agglutination of the latex, and blocking the groups on the latex microspheres which are not connected to the antibody again, and the third blocking agent can sufficiently block other sites of the antibody except for the epitopes reacting with the antigen. Therefore, the different molecular weight blocking agents are mixed and used in the application, so that the exposed sites on the surface of the latex microspheres can be more effectively blocked, and the purpose of improving the latex stability of the reagent is achieved.

[0044] The application further discloses a preparation method of the latex reagent.

[0045] 1) mixing the latex microspheres and an activation buffer, and then adding an activation agent to activate to obtain an activation reaction solution.

[0046] In a specific embodiment, the latex microspheres are carboxyl polystyrene latex microspheres.

[0047] Specifically, the surface modification group of the polystyrene latex microspheres is a carboxyl group, the latex microspheres and the activation buffer are mixed, and then the activation agent is added to activate, that is, the carboxyl group is activated, which is beneficial to subsequent coupling.

[0048] In an embodiment, the temperature of the activation is 30-37°C, and the time of the activation is 10-20 minutes.

[0049] In an embodiment, the mass ratio of the latex microspheres to the activation buffer is 1:(5-20).

[0050] In an embodiment, the mass ratio of the latex microspheres to the activation agent is (20-50):1.

[0051] In an embodiment, the activation agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0052] In an embodiment, the activation buffer comprises a 2-morpholinoethanesulfonic acid buffer, and the pH value is 6.5-7.0.

[0053] 2) The antibody is diluted in a coupling buffer and added to the activation reaction solution to obtain a coupling reaction solution.

[0054] In an embodiment, the temperature of the coupling is 30-37°C, and the time of the coupling is 0.5-2 hours to obtain the coupling reaction solution.

[0055] In an embodiment, the coupling buffer comprises a 2-morpholinoethanesulfonic acid buffer, and the pH value is 7.5-8.0.

[0056] 3) A first blocking agent is added to the coupling reaction solution to perform a first blocking to obtain a reaction solution after the first blocking.

[0057] In an embodiment, the time of the first blocking is 1-2 hours, and the temperature of the first blocking is 30-37°C.

[0058] 4) A second blocking agent is added to the reaction solution after the first blocking to perform a second blocking to obtain a reaction solution after the second blocking.

[0059] In an embodiment, the time of the second blocking is 2-24 hours, and the temperature of the second blocking is 2-8°C.

[0060] 5) A third blocking agent is added to the reaction solution after the second blocking to perform a third blocking to obtain a reaction solution after the third blocking.

[0061] In an embodiment, the time of the third blocking is 1-2 hours, and the temperature of the third blocking is 30-37°C.

[0062] 6) The reaction solution after the third blocking is subjected to solid-liquid separation, and resuspended to obtain a latex reagent.

[0063] The application further discloses a urine microalbumin detection kit, comprising the R2 reagent.

[0064] The reagent R2 includes the following components at the following concentrations:

[0065] 25mM-50mM of R2 buffer, 50%-90% of the first latex reagent, and 10%-50% of the second latex reagent.

[0066] Further, the first latex reagent is mainly obtained by coupling the first latex microspheres with the first antibody, the first blocking agent, the second blocking agent, and the third blocking agent.

[0067] Further, the second latex reagent is mainly obtained by coupling the second latex microspheres with the second antibody, the first blocking agent, the second blocking agent, and the third blocking agent.

[0068] Further, in the first latex reagent, the solid content of the first latex microspheres is 0.05%-0.1%, the concentration of the first antibody is 0.05mg / L-0.2mg / L, the content of the first blocking agent is 0.02%-1%, the content of the second blocking agent is 0.05%-5%, and the content of the third blocking agent is 0.1%-5%.

[0069] Further, in the second latex reagent, the solid content of the second latex microspheres is 0.05%-0.1%, the concentration of the first antibody is 0.05mg / L-0.1mg / L, the content of the first blocking agent is 0.02%-1%, the content of the second blocking agent is 0.05%-5%, and the content of the third blocking agent is 0.1%-5%.

[0070] Further, the first blocking agent includes Blockmaster TM DB1130.

[0071] Further, the second blocking agent includes Blockmaster TM PA1080.

[0072] Further, the third blocking agent includes bovine serum albumin.

[0073] Further, the first antibody and the second antibody are both urine microalbumin antibodies.

[0074] Further, the particle size of the first latex microspheres is 60nm-90nm.

[0075] In a specific embodiment, the particle size of the first latex microspheres includes but is not limited to 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, etc.

[0076] Further, the particle size of the second latex microspheres is 350nm-400nm.

[0077] In a specific embodiment, the particle size of the first latex microspheres includes, but is not limited to, 350 nm, 360 nm, 370 nm, 375 nm, 380 nm, 390 nm, 400 nm, etc.

[0078] Specifically, the embodiments of the present application use the first latex microspheres with small particle size and the second latex microspheres with large particle size at the same time. The second latex microspheres with large particle size can increase the volume and turbidity of the antigen-antibody complex when the concentration of the antigen in the sample is low, thereby enhancing the sensitivity of the latex reagent. The protein load of the first latex microspheres with small particle size can be coupled to more antibodies, which can avoid the hook effect when the concentration of the antigen in the sample is high, thereby improving the linearity range of the latex reagent. Therefore, the embodiments of the present application select two kinds of latex microspheres with different particle sizes to fully exert the advantages of the latex microspheres with different particle sizes, so that the reagent kit prepared finally can meet the requirements of high sensitivity and wide detection linearity range. In addition, the embodiments of the present application use different molecular weight blocking agents to mix, which can more effectively block all exposed sites on the surface of the latex microspheres, thereby achieving the purpose of improving the latex stability of the reagent.

[0079] In a specific embodiment, the urine microalbumin test kit further includes R1 reagent;

[0080] The R1 reagent includes the following components at the following concentrations:

[0081] 20 mM to 100 mM of R1 buffer, 1 g / L to 20 g / L of first stabilizer, and 0.05% to 0.1% of first preservative;

[0082] The R2 reagent further includes the following components at the following concentrations:

[0083] 5 g / L to 10 g / L of second stabilizer, 1% to 5% of second protective agent, 0.02% to 0.5% of second surfactant, and 0.05% to 0.1% of second preservative.

[0084] In a specific embodiment, the R1 buffer and the R2 buffer each include one or more than two of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 2-morpholinoethanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate, and tris-hydroxymethyl aminomethane buffer.

[0085] Specifically, the buffer is a mixed solution composed of weak acid and its salt, weak base and its salt, which can offset and reduce the influence of the added strong acid or strong base on the pH value of the solution to a certain extent, thereby keeping the pH value of the solution relatively stable.

[0086] In a specific embodiment, the first stabilizer and the second stabilizer each include one or more than two of sodium chloride, calcium chloride, glycine, alanine, arginine, and histidine.

[0087] Specifically, the stabilizer can increase the surface layer charge, protective layer and hydration degree of the latex microspheres, so as to avoid the early gel precipitation of the latex microspheres in the storage process.

[0088] In an embodiment, the first preservative and the second preservative each include one or more than two of sodium azide, Proclin-950, Proclin-300, krovin 100, krovin 300, krovin 500 and krovin 750.

[0089] Specifically, the preservative can inhibit the growth of microorganisms and ensure the quality of the reagent.

[0090] In an embodiment, the second protective agent includes one or more than two of trehalose, sucrose, dextran, lactose and glucose.

[0091] Specifically, the protective agent can maintain the structure of the antibody and improve the stability of the reagent.

[0092] In an embodiment, the second surfactant includes one or more than two of polysorbate 20, disodium ethylenediaminetetraacetate, ethylene glycol, glycerol and dithiothreitol.

[0093] Specifically, the surfactant can be adsorbed on the surface of the latex microspheres, reduce the surface tension of the latex microspheres, make the latex microspheres not easy to aggregate, and improve the dispersibility and stability of the latex microspheres in the solution.

[0094] The application also discloses a preparation method of the urine microalbumin detection reagent kit.

[0095] 1) Preparation of the R1 reagent.

[0096] The R1 reagent is prepared by sequentially adding the R1 reagent, the first stabilizer and the first preservative, uniformly mixing, adjusting the pH and constant volume.

[0097] 2) Preparation of the R2 reagent

[0098] 2.1) The first latex microspheres and the second latex microspheres are mixed with the activation buffer, and then activated by adding the activation agent, so as to obtain the first reaction liquid and the second reaction liquid, respectively.

[0099] 2.2) The first antibody is diluted by the coupling buffer and added to the first reaction liquid for coupling, and the second antibody is diluted by the coupling buffer and added to the second reaction liquid for coupling, so as to obtain the first coupling reaction liquid and the second coupling reaction liquid, respectively.

[0100] Specifically, the polystyrene latex microspheres are modified with carboxyl groups, and the microalbumin antibody has amino groups. The antibody is diluted with a coupling buffer and then added to the activated reaction solution for coupling, i.e., an amide bond is formed between the carboxyl groups and the amino groups to obtain the coupled reaction solution.

[0101] 2.3) First blocking agent is added to the first coupling reaction solution and the second coupling reaction solution respectively to perform first blocking, to obtain the first reaction solution after first blocking and the second reaction solution after first blocking respectively;

[0102] 2.4) Second blocking agent is added to the first reaction solution after first blocking and the second reaction solution after first blocking respectively to perform second blocking, to obtain the first reaction solution after second blocking and the second reaction solution after second blocking respectively;

[0103] 2.5) Third blocking agent is added to the first reaction solution after second blocking and the second reaction solution after second blocking respectively to perform third blocking, to obtain the first reaction solution after third blocking and the second reaction solution after third blocking respectively;

[0104] 2.6) Solid-liquid separation is performed on the first reaction solution after third blocking and the second reaction solution after third blocking respectively, to obtain the first latex reagent and the second latex reagent respectively;

[0105] 2.7) The first latex reagent and the second latex reagent are added to R2 buffer solution respectively for mixing, to obtain R2 reagent.

[0106] In a specific embodiment, the mass ratio of the first latex microspheres to the activation buffer is 1:(5-20); the mass ratio of the second latex microspheres to the activation buffer is 1:(5-20); and the mass ratio of the first latex reagent to the second latex reagent is 1:(1-9).

[0107] The following is a specific embodiment.

[0108] Example 1

[0109] 1) Preparation of R1 reagent:

[0110] 1.1) 50 mM MOPSO, 20 g / L NaCl and 0.1% sodium azide are stirred for 15 min until completely dissolved to form a mixed solution.

[0111] 1.2) After the mixed solution is clear and transparent without precipitation, the pH is adjusted to 7.2, and the final volume is adjusted to obtain the R1 reagent.

[0112] 2) Preparation of R2 reagent:

[0113] 2.1) Preparation of raw material solution.

[0114] Activation buffer: 50 mmol / L 2-morpholinoethanesulfonic acid buffer, pH 6.5.

[0115] Coupling buffer: 50 mmol / L 2-morpholinoethanesulfonic acid buffer, pH 7.5.

[0116] Activator: 10 mg / ml 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0117] First blocking agent: 0.1% Blockmaster TM DB1130.

[0118] Second blocking agent: 1% Blockmaster TM PA1080.

[0119] Third blocking agent: 5% bovine serum albumin.

[0120] R2 buffer: 50 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid, pH 7.5.

[0121] Second stabilizer: 10 g / L sodium chloride.

[0122] Second protective agent: 5% trehalose.

[0123] Second surfactant: 0.05% polysorbate 20.

[0124] Second preservative: 0.1% Proclin-300.

[0125] First latex microspheres: carboxyl polystyrene latex microspheres with a particle size of 60 nm.

[0126] Second latex microspheres: carboxyl polystyrene latex microspheres with a particle size of 350 nm.

[0127] First antibody: urine microalbumin antibody.

[0128] Second antibody: urine microalbumin antibody.

[0129] 2.2) Preparation of the first latex reagent

[0130] 2.2.1) 1 mL of the first latex microspheres was mixed with the activation buffer at a mass ratio of 1:8, then mixed with the activator at a mass ratio of 20:1, and placed in a constant temperature shaker for activation. The activation time was 20 min, and the activation temperature was 37°C. A first reaction solution was obtained.

[0131] 2.2.2) The first antibody is diluted to a concentration of 0.1 mg / mL with a coupling buffer and added to the first reaction solution, which is placed in a constant-temperature shaker for coupling, with a coupling time of 2 h and a coupling temperature of 37℃, to obtain a first coupling reaction solution;

[0132] 2.2.3) 1 mL of the first blocking agent is added to the first coupling reaction solution, which is placed in a constant-temperature shaker for primary blocking, with a primary blocking time of 2 h and a primary blocking temperature of 37℃, to obtain a first reaction solution after primary blocking;

[0133] 2.2.4) 1 mL of the second blocking agent is added to the first reaction solution after primary blocking, which is placed on a rotating disc in a refrigerator for secondary blocking, with a secondary blocking time of 15 h and a secondary blocking temperature of 4℃, to obtain a first reaction solution after secondary blocking;

[0134] 2.2.5) 2 mL of the third blocking agent is added to the first reaction solution after secondary blocking, which is placed in a constant-temperature shaker for tertiary blocking, with a tertiary blocking time of 2 h and a tertiary blocking temperature of 37℃, to obtain a first reaction solution after tertiary blocking;

[0135] 2.2.6) The first reaction solution after tertiary blocking is centrifuged and the supernatant is removed, to obtain a first latex reagent.

[0136] 2.3) Preparation of a second latex reagent

[0137] 2.3.1) 0.5 mL of the second latex microspheres is mixed with an activation buffer at a mass ratio of 1:6, and then 0.4 mL of the activator is added, which is placed in a constant-temperature shaker for activation, with an activation time of 20 min and an activation temperature of 37℃, to obtain a second reaction solution.

[0138] The other steps for preparing the second latex reagent are the same as steps 2.2.2) to 2.2.6) for preparing the first latex reagent, and the preparation method of steps 2.2.2) to 2.2.6) is repeated to obtain the second latex reagent.

[0139] 2.4) The second latex reagent and the first latex reagent are mixed at a mass ratio of 1:6, and then R2 buffer, the second stabilizer, the second protective agent, the second surfactant and the second preservative are sequentially added and mixed uniformly, and then the pH is adjusted to 7.5 to obtain an R2 reagent.

[0140] Example 2

[0141] Example 2 differs from Example 1 only in that the particle size of the first latex microspheres is 90 nm and the particle size of the second latex microspheres is 400 nm, and the other components and preparation methods are the same as those of Example 1.

[0142] Example 3

[0143] Example 3 differs from Example 1 only in that the R2 buffer is 50 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer and the Blockmaster TM DB1130 is present at a mass concentration of 0.5%, and the other components and the method of preparation are the same as in Example 1.

[0144] Example 4

[0145] Example 4 differs from Example 1 only in that the R2 buffer is 35 mM MES buffer and the Blockmaster TM PA1080 is present at a mass concentration of 2%, and the other components and the method of preparation are the same as in Example 1.

[0146] Comparative Example 1

[0147] Comparative Example 1 differs from Example 1 only in that only latex microspheres having a particle size of 60 nm are used, and the other components and the method of preparation are the same as in Example 1.

[0148] Comparative Example 2

[0149] Comparative Example 2 differs from Example 1 only in that only latex microspheres having a particle size of 350 nm are used, and the other components and the method of preparation are the same as in Example 1.

[0150] Comparative Example 3

[0151] Comparative Example 3 differs from Example 1 only in that the blocking agent is only bovine serum albumin, and the other components and the method of preparation are the same as in Example 1.

[0152] Comparative Example 4

[0153] Comparative Example 4 differs from Example 1 only in that the blocking agent is only Blockmaster TM PA1080, and the other components and the method of preparation are the same as in Example 1.

[0154] Comparative Example 5

[0155] Comparative Example 5 differs from Example 1 only in that the blocking agent is only Blockmaster TM DB1130, and the other components and the method of preparation are the same as in Example 1.

[0156] I. Evaluation of the first embodiment (linear range evaluation):

[0157] After the reagents prepared in Examples 1-2 and Comparative Examples 1-2 are calibrated using mALB calibrators, the linear range of the reagents is determined using the Korma CP880 instrument, and the evaluation method is as follows:

[0158] The sample with the concentration of 0.5 mg / L-600 mg / L was determined, each concentration value was repeated three times, the average value, relative deviation and linear correlation coefficient R were calculated; R was greater than 0.9900, and the relative deviation of each point was within 10%, then the use requirement was met.

[0159] Table 1 linear range data of examples 1-2 and comparative examples 1-2

[0160]

[0161]

[0162] As can be seen from the data in Table 1, the high value of the linearity of examples 1-2 reached 600 mg / L, and the linear correlation coefficient R and the relative deviation of each concentration met the requirements; the high value of the linearity of comparative example 1 was 352.67 mg / L, the relative deviation of the low section exceeded 10%, and the correlation coefficient R was less than 0.99, i.e. the linear correlation was poor; the high value of the linearity of comparative example 2 was only 118.54 mg / L, which indicated that the combination use of the first latex microspheres with small particle size and the second latex microspheres with large particle size made the detection reagent have a wider linear range.

[0163] As can be seen from the data in Table 1, the linear range of comparative example 1 was wider than that of comparative example 2, which indicated that the latex microspheres with small particle size could improve the linear range of the detection reagent.

[0164] II. Evaluation of the second embodiment (sensitivity evaluation)

[0165] After the reagents prepared by examples 1-2 and comparative examples 1-5 were calibrated by selecting mALB calibrators, the sensitivity of the detection reagent was detected by using a Komam CP880 instrument, and the evaluation method was as follows:

[0166] The samples with the concentration of 0 mg / L and 0.5 mg / L were determined respectively, each concentration value was repeated five times, and the average value was calculated.

[0167] Table 2 sensitivity data of examples 1-2 and comparative examples 1-5

[0168]

[0169] As can be seen from the data in Table 2, the sensitivity of examples 1-2 was higher than that of comparative examples 3-5, which indicated that the combination use of the first blocking agent, the second blocking agent and the third blocking agent made the detection reagent have higher sensitivity.

[0170] It can also be seen from the data in Table 2 that the sensitivity of Examples 1-2 is higher than that of Comparative Examples 1-2, which indicates that the combination use of the first latex microspheres with small particle size and the second latex microspheres with large particle size makes the finally prepared reagent meet the requirement of high sensitivity.

[0171] It can also be seen from the data in Table 2 that the sensitivity of Comparative Example 2 is higher than that of Comparative Example 1, which indicates that the latex microspheres with large particle size can improve the sensitivity of the detection reagent.

[0172] II. Evaluation of Embodiment Three (Precision Evaluation)

[0173] After the reagents prepared by Examples 1-2 and Comparative Examples 3-5 are calibrated by selecting mALB calibrators, the precision of the detection reagent is detected by using the Komam CP880 instrument, and the evaluation method is as follows:

[0174] The samples with concentrations of 30 mg / L and 400 mg / L are respectively measured, and each concentration value is repeated for 10 times to calculate the average value.

[0175] Table 3 Precision data of Examples 1-2 and Comparative Examples 3-5 (concentration of 30 mg / L)

[0176] Comparative Example 5 Comparative Example 4 Comparative Example 3 Example 1 Example 2 Test 1 28.93 23.62 25.28 31.05 32.52 Test 2 24.89 31.83 29.52 29.84 30.84 Test 3 27.56 28.79 23.27 28.89 31.09 Test 4 32.47 29.82 34.52 31.27 32.32 Test 5 35.59 25.39 29.84 33.09 31.33 Test 6 30.24 38.92 30.05 32.63 29.99 Test 7 31.52 36.83 33.27 31.84 30.45 Test 8 29.83 30.52 24.32 30.56 30.43 Test 9 37.02 33.29 33.67 31.83 30.02 Test 10 31.52 31.83 32.59 31.29 31.58 Mean 30.96 31.08 29.63 31.23 31.06 Standard Deviation 3.58 4.66 4.08 1.25 0.89 Coefficient of Variation 11.57％ 15.00％ 13.78％ 4.00％ 2.86％

[0177] Table 4 Precision data of Examples 1-2 and Comparative Examples 3-5 (concentration of 400 mg / L)

[0178]

[0179]

[0180] It can be seen from the data in Tables 3 and 4 that the coefficient of variation of Examples 1-2 is smaller than that of Comparative Examples 3-5, whether for low concentration samples or high concentration samples, so the precision effect of the detection reagent of Comparative Examples 3-5 using only a single blocking agent is not good, and Examples 1-2 using three blocking agents have better performance in precision, which indicates that the combination use of the first blocking agent, the second blocking agent and the third blocking agent makes the detection reagent have higher precision.

[0181] II. Evaluation of Embodiment Four (Stability Evaluation)

[0182] The reagents prepared by Comparative Examples 3-5 and Examples 1-2 are respectively placed in a 37°C constant temperature box and a 2°C-8°C refrigerator, and after calibration by selecting mALB calibrators, the stability of the detection reagent is detected by using the Komam CP880 instrument, and the evaluation method is as follows:

[0183] The sample determination concentration is 20 mg / L and 350 mg / L respectively, and the sample is taken out for testing at 1st week, 2nd week, 3rd week, 4th week, 2nd month, 4th month, 6th month, 8th month, 10th month, 11th month and 12th month under the condition of 2℃-8℃; the sample is taken out for testing at 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 10th day, 12th day, 14th day and 15th day under the condition of 37℃.

[0184] Table 5 Stability test data of Example 1-2 and Comparative Example 3-5 under the condition of 2℃-8℃ (concentration is 20 mg / L)

[0185]

[0186]

[0187] Table 6 Stability test data of Example 1-2 and Comparative Example 3-5 under the condition of 2-8℃ (concentration is 350 mg / L)

[0188]

[0189] Table 7 Stability test data of Example 1-2 and Comparative Example 3-5 under the condition of 37℃ (concentration is 20 mg / L)

[0190]

[0191]

[0192] Table 8 Stability test data of Example 1-2 and Comparative Example 3-5 under the condition of 37℃ (concentration is 350 mg / L)

[0193]

[0194] The results show that Example 1-2 has good stability under the conditions of 2℃-8℃ and 37℃ respectively, while Comparative Example 3-5 using single sealing agent has poor stability, which shows that the combination of the first sealing agent, the second sealing agent and the third sealing agent makes the detection reagent have higher stability.

[0195] Through the verification of the above-mentioned embodiments, the kit prepared finally can meet the requirements of high sensitivity and wide detection linear range by using the first latex microspheres with small particle size and the second latex microspheres with large particle size, and fully exerting the advantages of latex microspheres with different particle sizes. Moreover, the combination blocking of the first blocking agent, the second blocking agent and the third blocking agent with different molecular weights can significantly reduce the background signal without affecting the low value distinguishability and detection sensitivity, so as to improve the latex stability of the reagent and ensure the accuracy of the detection result.

[0196] The above-mentioned embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as the limitation of the patent application scope. It should be pointed out that, for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A latex reagent, characterized by, The latex reagent is mainly obtained by coupling latex microspheres with an antibody, a first blocking agent, a second blocking agent and a third blocking agent; The antibody is a urine microalbumin antibody; The first sealant is Blockmaster TM DB1130; The second blocking agent is Blockmaster TM PA1080; The third blocking agent is bovine serum albumin; In the latex reagent, the solid content of the latex microspheres is 0.05%-0.1%, the concentration of the antibody is 0.05 mg / L-0.2 mg / L, the content of the first blocking agent is 0.02%-1%, the content of the second blocking agent is 0.05%-5%, and the content of the third blocking agent is 0.1%-5%.

2. A method for preparing the latex reagent according to claim 1, characterized by, The process comprises the following steps: The latex microspheres are mixed with an activation buffer, and then an activation agent is added for activation to obtain an activation reaction solution; The antibody is diluted with a coupling buffer and then added to the activation reaction solution for coupling to obtain a coupling reaction solution; A first blocking agent is added to the coupling reaction solution for primary blocking to obtain a reaction solution after primary blocking; A second blocking agent is added to the reaction solution after primary blocking for secondary blocking to obtain a reaction solution after secondary blocking; A third blocking agent is added to the reaction solution after secondary blocking for tertiary blocking to obtain a reaction solution after tertiary blocking; The reaction solution after tertiary blocking is subjected to solid-liquid separation, and then resuspended to obtain the latex reagent.

3. The method of claim 2, wherein the latex reagent is prepared by adding the compound of formula (I) to the latex. The activation is performed at a temperature of 30-37°C for 10-20 min; The coupling is performed at a temperature of 30-37°C for 0.5-2 h; The primary blocking is performed at a temperature of 30-37°C for 1-2 h; The secondary blocking is performed at a temperature of 2-8°C for 2-24 h; The tertiary blocking is performed at a temperature of 30-37°C for 1-2 h.

4. The method of claim 2, wherein the latex reagent is prepared by adding the compound of formula (I) to the latex. The mass ratio of the latex microspheres to the activation buffer is 1:(5-20); The mass ratio of the latex microspheres to the activation agent is (20-50):1; The latex microspheres comprise carboxyl polystyrene latex microspheres; The activation agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; The activation buffer comprises a 2-morpholinoethanesulfonic acid buffer with a pH value of 6.5-7.0; The coupling buffer comprises a 2-morpholinoethanesulfonic acid buffer with a pH value of 7.5-8.

0.

5. A urinary microalbumin test kit, characterized by, The R2 reagent comprises the following components at the following concentrations: 25-50 mM of an R2 buffer, 50%-90% of a first latex reagent and 10%-50% of a second latex reagent; The first latex reagent is mainly obtained by coupling first latex microspheres with a first antibody, a first blocking agent, a second blocking agent and a third blocking agent; The second latex reagent is mainly obtained by coupling second latex microspheres with a second antibody, a first blocking agent, a second blocking agent and a third blocking agent; ​ The solid content of the first latex microspheres in the first latex reagent is 0.05% to 0.1%, the concentration of the first antibody is 0.05 mg / L to 0.2 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%; The solid content of the second latex microspheres in the second latex reagent is 0.05% to 0.1%, the concentration of the first antibody is 0.05 mg / L to 0.1 mg / L, the content of the first blocking agent is 0.02% to 1%, the content of the second blocking agent is 0.05% to 5%, and the content of the third blocking agent is 0.1% to 5%; The first sealant is Blockmaster TM DB1130; The second blocking agent is Blockmaster TM PA1080; The third blocking agent is bovine serum albumin; The first antibody and the second antibody are both urine microalbumin antibodies; The particle size of the first latex microspheres is 60 nm to 90 nm; The particle size of the second latex microspheres is 350 nm to 400 nm.

6. The urinary microalbumin test kit according to claim 5, characterized by, The R1 reagent also includes the following concentrations of components: 20 mM to 100 mM of R1 buffer, 1 g / L to 20 g / L of first stabilizer, and 0.05% to 0.1% of first preservative; The R2 reagent also includes the following concentrations of components: 5 g / L to 10 g / L of second stabilizer, 1% to 5% of second protective agent, 0.02% to 0.5% of second surfactant, and 0.05% to 0.1% of second preservative.

7. The urinary microalbumin test kit according to claim 6, characterized by, The R1 buffer and the R2 buffer each include one or more of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 2-morpholinoethanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate, and tris-hydroxymethyl aminomethane buffer.

8. The urinary microalbumin test kit according to claim 6, characterized by, The first stabilizer and the second stabilizer each include one or more of sodium chloride, calcium chloride, glycine, alanine, arginine, and histidine; The first preservative and the second preservative each include one or more of sodium azide, Proclin-950, Proclin-300, krovin 100, krovin 300, krovin 500, and krovin 750; The second protective agent includes one or more of trehalose, sucrose, dextran, lactose, and glucose; The second surfactant includes one or more of polysorbate 20, disodium ethylenediaminetetraacetate, ethylene glycol, glycerol, and dithiothreitol.

9. A method for preparing a urine microalbumin test kit according to any one of claims 5 to 8, characterized by, The following steps are included: The first latex microspheres and the second latex microspheres are each mixed with an activation buffer and then activated with an activation agent to obtain a first reaction solution and a second reaction solution, respectively; The first antibody is diluted with a coupling buffer and then added to the first reaction solution for coupling, and the second antibody is diluted with a coupling buffer and then added to the second reaction solution for coupling, to obtain a first coupling reaction solution and a second coupling reaction solution, respectively; The first antibody is diluted with a coupling buffer and then added to the first reaction solution for coupling, and the second antibody is diluted with a coupling buffer and then added to the second reaction solution for coupling, to obtain a first coupling reaction solution and a second coupling reaction solution, respectively; A first blocking agent is added to the first coupling reaction solution and the second coupling reaction solution respectively to perform a first blocking, to obtain a first reaction solution after a first blocking and a second reaction solution after a first blocking respectively; A second blocking agent is added to the first reaction solution after a first blocking and the second reaction solution after a first blocking respectively to perform a second blocking, to obtain a first reaction solution after a second blocking and a second reaction solution after a second blocking respectively; A third blocking agent is added to the first reaction solution after a second blocking and the second reaction solution after a second blocking respectively to perform a third blocking, to obtain a first reaction solution after a third blocking and a second reaction solution after a third blocking respectively; Solid-liquid separation is performed on the first reaction solution after a third blocking and the second reaction solution after a third blocking respectively, to obtain the first latex reagent and the second latex reagent respectively; The first latex reagent and the second latex reagent are mixed in R2 buffer solution respectively to obtain the R2 reagent.

10. The method for preparing the urine microalbumin detection kit according to claim 9, characterized in that, The mass ratio of the first latex microspheres to the activation buffer solution is 1: (5-20); The mass ratio of the second latex microspheres to the activation buffer solution is 1: (5-20); The mass ratio of the second latex reagent to the first latex reagent is 1: (1-9).

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