阅读说明：本技术 干性免疫比浊试剂及其制备方法和应用 (Dry immunoturbidimetric reagent and preparation method and application thereof ) 是由 张屹 刘真 于 2019-10-30 设计创作，主要内容包括：本文提供了一种制备干性免疫比浊试剂的方法,其包括1)将偶联有抗体的微球与水溶性成膜剂在溶液中混合；2)让步骤1)得到的混合液形成膜状物并进行干燥处理。通过该方法制备的干性免疫比浊试剂能够在常温稳定保存,复溶速度快,尤其适合于预保存在微流控试剂卡中。(A process for preparing a dry immunoturbidimetric reagent is provided, which comprises 1) mixing microspheres coupled with an antibody with a water-soluble film-forming agent in a solution; 2) forming the mixed solution obtained in the step 1) into a film-shaped substance and drying the film-shaped substance. The dry immunoturbidimetric reagent prepared by the method can be stably stored at normal temperature, has high redissolution speed, and is particularly suitable for being pre-stored in a microfluidic reagent card.)

1. A method for preparing a dry immunoturbidimetric reagent, comprising:

1) mixing the microspheres coupled with the antibody and a water-soluble film-forming agent in a solution;

2) forming the mixed solution obtained in the step 1) into a film-shaped substance and drying the film-shaped substance.

2. The method of claim 1, wherein the microspheres are latex microspheres.

3. The method of claim 1, wherein the water soluble film forming agent is selected from the group consisting of PVP, PVA, PEG, PEO, and combinations thereof.

4. The method of claim 3, wherein the PVP has a K value of no greater than 90; the alcoholysis degree of the PVA is not more than 89%, and the polymerization degree is not more than 2300.

5. The method of claim 3, wherein the PVP has a K value in the range of 15 to 90; the alcoholysis degree of the PVA ranges from 65% to 89%, and the polymerization degree ranges from 500 to 2300.

6. The method of claim 3, wherein the film-forming agent is PVA1788, PVA0580, PVPK30 or PVPK 15.

7. The method of claim 1, wherein the concentration of the antibody-conjugated microspheres in the mixture is not greater than 2.5% (wt).

8. The method of claim 1, wherein the concentration of the antibody-coupled microspheres in the mixed liquor is 0.05% to 0.5% (wt) and the concentration of the film-forming agent in the mixed liquor is 0.5% to 5% (wt).

9. The method of claim 1, wherein the drying process in step 2) is performed in a mold.

10. The method of claim 1, wherein the drying treatment in step 2) comprises heat drying at 37 ℃ for 2 to 3 hours.

11. The method of claim 1, wherein the film formed by the drying process in step 2) has a thickness of 5 μm to 530 μm.

12. The method of claim 1, wherein the film formed by the drying process in step 2) has a thickness of 50 μm to 300 μm.

13. The method of claim 1, wherein the membrane in step 2) is obtained by spray dry forming or ultra-thin film forming.

14. The method of claim 1, wherein the antibody is an anti-CRP, anti-D-Dimer, or anti-MALB antibody.

15. A dry immunoturbidimetric reagent prepared by the process of any one of claims 1 to 14.

16. An immunoassay kit comprising the dry immunoturbidimetric reagent of claim 15.

17. A microfluidic reagent card comprising the dry immunoturbidimetric reagent of claim 15.

18. An immunoturbidimetric assay comprising contacting a whole blood, serum or plasma sample with a dry immunoturbidimetric reagent according to claim 15.

Technical Field

The present invention relates to an immunoturbidimetric reagent for immunoturbidimetric assay, in particular a dry immunoturbidimetric reagent. The invention also relates to a preparation method and application of the dry immunoturbidimetric reagent.

Background

Immunoturbidimetry is an antigen-antibody binding dynamic assay that has wide application in detecting some disease diagnostic markers in blood. The basic principle is as follows: when the antigen and the antibody react in the solution, the formed soluble complex forms particles under the action of the polymerization promoter, so that the reaction solution generates turbidity. When the antibody concentration is fixed, the amount of the immunocomplex formed increases with the increase in the amount of the antigen in the sample, and the turbidity of the reaction solution also increases. Compared with the traditional biochemical reaction, the immunoturbidimetry detects the change of absorbance, but the reaction principle is completely different, and the performance of the reagent is greatly different. Immunoturbidimetric reagents to increase sensitivity, antibodies are typically coupled to microspheres such as latex. Changes in reagent conditions can cause non-specific aggregation of antibody-sensitized microspheres, leading to bias results.

Immunoturbidimetric reagents are usually stored in a liquid state and no commercial dry immunoturbidimetric reagents are available. Liquid reagents require storage at 2-8 ℃ and separate components (e.g., it is common to store reagent 1 containing a buffer and reagent 2 containing an antibody separately). In practical use, for example, in single-person testing, it is necessary to hermetically store liquid reagents in a plurality of reagent storage structures, and separate reaction chambers and mixing chambers are also necessary. The traditional drying methods such as freeze-drying, air-drying, vacuum low-temperature drying and the like mainly take water extraction as a main means. Antibody-coupled microbeads that have been freed of moisture are particularly susceptible to nonspecific agglutination, which undermines the application of turbidimetry by detecting agglutination. In most cases, the microspheres coupled with the antibody are solidified by freeze-drying and the like and then dissolved to aggregate, and cannot be used for detection continuously.

Researchers have long sought methods for preparing dry immunoturbidimetric reagents. In Japanese patent laid-open No. 11-258241, a method for preparing a latex reagent for detecting HCV by freeze-drying is described, which comprises adding a dispersion stabilizer and an antioxidant to increase the stability of the latex particles after freeze-drying; but the latex particles still need to be stored at 0-8 ℃ after freeze-drying; chinese patent application publication No. CN107255727A describes a method for preparing an avian transferrin immunoturbidimetric reagent by low temperature lyophilization in the presence of excipients and preservatives, which comprises freezing the reagent containing the antibody at-80 ℃ overnight and lyophilizing in a freeze vacuum dryer for 20 hours. Chinese patent application publication No. CN107255727A describes a method for preparing a latex turbidimetric lyophilized reagent by lyophilization in the presence of bovine serum albumin, vitamin C and glycine, which comprises repeated freeze-thawing and sealing in an inert gas atmosphere. Obviously, these preparation methods are complicated, e.g. they all require lyophilization and take more than 10 hours. For the latter method, the lyophilized reagent prepared thereof needs to be separately reconstituted for 15 minutes before detection. These all result in high manufacturing costs, long time consumption and inefficient detection. In addition, no dry reagent products are available on the market from these patent applicants.

Disclosure of Invention

In one aspect, provided herein is a method of preparing a dry immunoturbidimetric reagent comprising:

1) mixing the microspheres coupled with the antibody and a water-soluble film-forming agent in a solution;

2) forming the mixed solution obtained in the step 1) into a film-shaped substance and drying the film-shaped substance.

In some embodiments, the microspheres are latex microspheres.

In some embodiments, the water soluble film forming agent is selected from PVP, PVA, PEG, PEO, and combinations thereof.

In some embodiments, the PVP has a K value of no greater than 90; the alcoholysis degree of the PVA is not more than 89%, and the polymerization degree is not more than 2300.

In some embodiments, the PVP has a K value in the range of 15 to 90; the alcoholysis degree of the PVA ranges from 65% to 89%, and the polymerization degree ranges from 500 to 2300.

In some embodiments, the film forming agent is PVA1788, PVA0580, PVPK30, or PVPK 15.

In some embodiments, the concentration of the antibody-conjugated microspheres in the mixture is no greater than 2.5% (wt).

In some embodiments, the concentration of the antibody-coupled microspheres in the mixture is 0.05% to 0.5% (wt) and the concentration of the film-forming agent in the mixture is 0.5% to 5% (wt).

In some embodiments, the drying treatment in step 2) is performed in a mold.

In some embodiments, the drying treatment in step 2) comprises heat drying at 37 ℃ for 2 to 3 hours.

In some embodiments, the film formed after the drying treatment in step 2) has a thickness of 5 μm to 530 μm.

In some embodiments, the film formed after the drying treatment in step 2) has a thickness of 50 μm to 300 μm.

In some embodiments, the film in step 2) is obtained by spray dry forming or ultra-thin film forming.

In some embodiments, the antibody is an anti-CRP, anti-D-Dimer, or anti-MALB antibody.

In another aspect, provided herein is a dry immunoturbidimetric reagent prepared by the above method.

In another aspect, provided herein is an immunoassay kit comprising the dry immunoturbidimetric reagent of claim.

In another aspect, provided herein is a microfluidic reagent card comprising the dry immunoturbidimetric reagent.

In another aspect, provided herein is an immunoturbidimetric assay comprising contacting a whole blood, serum, or plasma sample with the dry immunoturbidimetric reagent.

The dry immunoturbidimetric reagent provided by the invention has the advantages of simple preparation process, low cost, stable performance, capability of being stored at normal temperature for a long time, high detection efficiency and higher academic significance and commercial value.

Drawings

FIG. 1 shows the detection performance of the CRP immunoturbidimetric reagent prepared in example 4 after filming and redissolving.

FIG. 2 shows a comparison of the detection performance of the D-Dimer immunoturbidimetric assay prepared in example 6 before and after membrane formation.

FIG. 3 is a comparison of the detection performance of immunoturbidimetric reagents prepared with different film-forming agents.

FIG. 4 shows the room temperature stability of the MLAB dry immunoturbidimetric assay prepared in example 8.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, "microsphere" refers to a sphere having a particle size on the micrometer or nanometer scale. Preferably, the microsphere has a particle size of between a few nanometers and several hundred nanometers, for example, about 50, 100, 150, 200, 250, 300, or 350 nm. The material of the microsphere can be latex, such as polystyrene, polycaprolactone, polymethyl methacrylate, polylactic acid-glycolic acid copolymer, and the like. In some cases, colloidal gold microspheres may also be used.

As used herein, "antibody-conjugated microspheres" or "antibody-conjugated microspheres" refer to microspheres having antibody molecules attached to the surface. The antibody-coupled microspheres can be prepared by allowing microspheres having active groups (e.g., carboxyl or amino groups) on their surfaces to react directly with antibody molecules. Or in some cases, the antibody molecule may be indirectly attached to the microsphere surface via a conjugate (small peptide or small compound molecule).

As used herein, "water-soluble film former" refers to a hydrophilic organic polymer having film-forming properties. Common film-forming polymers include, for example, but are not limited to, one or a combination of several of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), cellulose derivatives (e.g., carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose), polyethylene glycol (PEG), polyethylene oxide (PEO), polyacrylamide, sodium alginate, carboxymethyl chitosan, and the like. The water-soluble film-forming agent should be chemically stable and substantially non-reactive with the antibody molecule or other test sample with which it may come into contact. In addition to ease of film formation, it is desirable that these water-soluble film forming agents be capable of dissolving rapidly (within 2 minutes) in aqueous solutions. Particularly, when the dry turbidimetric reagent is used in automatic detection equipment, the dry turbidimetric reagent is quickly dissolved, so that the detection efficiency of an instrument is improved.

As used in reference to PVP, the term "K value" refers to a characteristic value associated with the molecular weight of PVP. Generally, the greater the molecular weight, the greater the K value. The K value may be obtained, for example, by viscometry measurements, or may be obtained from the manufacturer of the particular PVP product.

As used herein, "film" or "film" refers to a thin film formed by a water-soluble film forming agent, the thickness of which is generally much less than the length and width. Preferably, after drying, the thickness of these films is from 5 to 530 μm. More preferably, the thickness of these films is 50 to 300 μm, such as 80 μm, 100 μm, 120 μm, 150 μm, 170 μm, 200 μm, 230 μm, 250 μm, or 280 μm. The formation of a "film" or "membrane" facilitates rapid drying. In some embodiments of the invention, this rapid drying prevents aggregation of the microspheres after reconstitution of the membrane. On the other hand, the membrane is beneficial to being divided and quantified. For example, in the case where the film thickness is known, the volume of the selected film can be accurately determined by calculating the area. The film-forming agent in a solution state may be formed into a film by various suitable methods, for example, by a blowing method, a casting method, an electrospinning method, and the like. In order to enable rapid drying, in some preferred embodiments of the invention, the film is formed using spray dry forming or ultra-thin film forming. On the contrary, the traditional casting method has thick film forming and long film forming time, is easy to cause local micro-aggregation and influences the stability of the final product.

As used herein, "drying" refers to the process by which a liquid suspension of microspheres gradually loses moisture in the presence of a film-forming agent to form a solid. Typically, more than 80% of the water is removed during the drying process. Alternatively, preferably, 90%, 95%, 99% or more or even 100% of the moisture is removed.

As used herein, "dry immunoturbidimetric reagent" refers to an immunoturbidimetric reagent in a substantially solid state having a moisture content of less than 20%, 15%, 10%, 5%, 1%, or substantially free of moisture. In some embodiments of the present invention, "immunoturbidimetric reagent" refers to a liquid reagent containing microspheres coupled to an antibody but not containing a film-forming agent, "dry immunoturbidimetric reagent" or the like refers to an immunoturbidimetric reagent to which a film-forming agent is added and which is dried after film formation, "redissolved immunoturbidimetric reagent" or the like refers to a reagent obtained by dissolving a dry immunoturbidimetric reagent in water (or an aqueous solution). In some embodiments of the invention, the amount of water (or aqueous solution) used for reconstitution is substantially such that the concentration of microspheres in the resulting liquid reagent for reconstitution is the same as the concentration in the immunoturbidimetric reagent without the film-forming agent prior to film formation.

The present invention will be described in detail with reference to specific examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific techniques or conditions not specified in the examples can be performed according to the techniques, conditions or product specifications described in the literature in the field. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase. Unless otherwise indicated, concentrations in percent are weight percent (wt%).