Latex enhanced immunoturbidimetry kit, preparation method of latex microsphere solution and its application

Abstract

The present invention discloses a latex-enhanced immunoturbidimetric assay kit, a preparation method of a latex microsphere solution, and an application thereof, and belongs to the technical field of polymer materials. The latex-enhanced immunoturbidimetric assay kit of the present invention comprises a latex microsphere solution with a dispersion coefficient (PDI) of 0.05-0.3, has the advantages of a wide detection range, high detection reactivity and sensitivity, and has a significant coupling effect on antibodies, especially immunoglobulin G4 antibodies, thereby solving the technical problem of difficulty in simultaneously achieving a wide detection range, high detection reactivity and sensitivity in latex-enhanced immunoturbidimetric assay testing.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer materials, in particular to a latex enhanced immunoturbidimetry kit, a preparation method of a latex microsphere solution and applications thereof. Background Art

[0002] Polymer microspheres are a new type of functional material that has emerged with the development of chemical materials. Nanoscale latex microspheres, in particular, offer advantages such as large surface area, strong adsorption, and, under the action of functional groups, specific surface reactivity and adsorption specificity. Consequently, in recent years, these microspheres have shown promising application prospects in fields such as immunodiagnosis and separation chromatography, and have been widely used in some areas.

[0003] In the latex-enhanced immunoturbidimetry (ETIA) test, latex microspheres are the key component, and their own performance directly affects the quality of the test results. Currently, technical personnel in this field often obtain microspheres with excellent performance by continuously optimizing the structure or composition of latex microspheres, but the operation is often complicated, and the intervention on the results does not have an immediate effect.

[0004] On the other hand, as the number of detection items increases, people have a wider pursuit for the detection range, so that when facing some items with higher or lower content, they can also ensure the accuracy of the detection. Therefore, changing the particle size of latex is a simple and direct choice. Generally speaking, choosing latex microspheres as small as possible for labeling preparation can obtain a wider detection range, but the reactivity and sensitivity of small-particle latex usually cannot meet the detection needs. Summary of the Invention

[0005] The invention provides a latex enhanced immunoturbidimetric assay kit, a preparation method of a latex microsphere solution and application thereof. The latex enhanced immunoturbidimetric assay kit aims to solve the technical problem of difficulty in simultaneously achieving a wide detection range, high detection reactivity and high sensitivity in latex enhanced immunoturbidimetric assay testing.

[0006] To achieve the above object, the present invention provides a latex-enhanced turbidimetric immunoassay kit, which comprises a latex microsphere solution with a dispersion coefficient PDI of 0.05-0.3.

[0007] Optionally, the latex enhanced immunoturbidimetry kit comprises a latex microsphere solution with a dispersion coefficient PDI of 0.1-0.3.

[0008] Optionally, the particle size of the latex microspheres in the latex microsphere solution is 20-100 nm.

[0009] Optionally, the particle size of the latex microspheres in the latex microsphere solution is 50-70 nm.

[0010] Optionally, the latex microsphere solution is a carboxyl latex microsphere solution.

[0011] In another aspect, the present invention discloses a method for preparing a latex microsphere solution, comprising the following steps:

[0012] preparing a monodisperse latex microsphere solution;

[0013] The dispersion index (PDI) of the monodisperse latex microsphere solution is adjusted to 0.05-0.3 with salt to obtain a latex microsphere solution.

[0014] Optionally, the latex microsphere solution includes olefin monomers, acrylic monomers, surfactants and initiators.

[0015] Further optionally, the olefin monomer includes at least one of styrene, styrene propylene, methyl styrene, para-styrene or methyl methacrylate;

[0016] And / or, the acrylic monomer includes at least one of methacrylic acid, acrylic acid, itaconic acid, methyl methacrylate, and methyl acrylate;

[0017] And / or, the initiator includes at least one of ammonium persulfate, potassium persulfate, sodium persulfate, and sodium sulfite;

[0018] And / or, the surfactant includes at least one of sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium dodecylsulfonate.

[0019] Optionally, the salt includes at least one of sodium carbonate, sodium bicarbonate, ammonium carbonate, ammonium bicarbonate, potassium carbonate, and potassium bicarbonate.

[0020] On the other hand, the present invention also provides a use of a latex microsphere solution in latex enhanced immunoturbidimetry, wherein the dispersion coefficient PDI of the latex microsphere solution is 0.05-0.3.

[0021] The beneficial effects that can be achieved by the present invention are:

[0022] The present invention provides a latex-enhanced immunoturbidimetric assay kit with a dispersion index (PDI) of 0.05-0.3. The kit is applied to the latex-enhanced immunoturbidimetric assay and adopts a non-uniform latex microsphere solution to achieve high sensitivity, high reactivity and wide range detection, breaking the conventional understanding in the field that only latex microsphere solutions with good dispersion (i.e., PDI < 0.05) can bring good detection effects.

[0023] The present invention further provides a latex-enhanced immunoturbidimetry kit with a carboxyl latex microsphere solution, which can significantly improve the reactivity and sensitivity of the detection because the carboxyl latex microspheres carry a large number of highly active carboxyl groups, further ensuring that it has a strong antibody detection capability while having a wide detection range.

[0024] Compared with the traditional seed method and hydrolysis method, the preparation method of the latex microsphere solution with a dispersion coefficient PDI of 0.05-3 in the latex-enhanced immunoturbidimetric assay kit of the present invention is very simple. It is obtained by a one-step polymerization method, has a wide detection range, and has strong reactivity and sensitivity, solving the technical problem of latex-enhanced immunoturbidimetric assay that is difficult to achieve both a wide detection range and high detection reactivity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0026] Figure 1 These are SEM images of the latex microsphere solutions in Examples 1 to 5 of the present invention.

[0027] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0028] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] In the present invention, descriptions such as "first" and "second" are for descriptive purposes only and should not be understood as indicating or implying their relative importance or implicitly indicating the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions of various embodiments may be combined with each other, but this must be based on the fact that they can be implemented by a person of ordinary skill in the art. If the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0031] The invention provides a latex enhanced immunoturbidimetry kit. The latex enhanced immunoturbidimetry kit comprises a latex microsphere solution with a dispersion coefficient PDI of 0.05-0.3.

[0032] Furthermore, the dispersion coefficient PDI of the latex microsphere solution in the latex enhanced immunoturbidimetry kit of the present invention is 0.1-0.3.

[0033] In some embodiments, the dispersion index (PDI) of the latex microsphere solution can be 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.23, 0.25, 0.28, 0.29 or 0.3, or any point in the range of 0.05-0.3.

[0034] The latex-enhanced immunoturbidimetric assay kit uses a latex microsphere solution to perform detection functions. The latex-enhanced immunoturbidimetric assay kit of the present invention uses a non-uniform latex microsphere solution, which can achieve high sensitivity, high reactivity and wide range detection, breaking the conventional understanding in the field that only a latex microsphere solution with good dispersibility (i.e., PDI < 0.05) can bring good detection results.

[0035] In some embodiments, the particle size of the latex microspheres in the latex microsphere solution is 20-100 nm. Further, the particle size of the latex microspheres in the latex microsphere solution is 50-70 nm.

[0036] In some embodiments, the particle size of the latex microspheres in the latex microsphere solution can be any point value in the range of 20-100 nm, such as 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm.

[0037] The latex microsphere solution having the above-mentioned particle size of latex microspheres is conducive to achieving a wider detection range of the latex enhanced immunoturbidimetry kit.

[0038] In some embodiments, the latex microsphere solution is a carboxyl latex microsphere solution. The carboxyl latex microspheres carry a large number of highly active carboxyl groups, which can significantly improve the reactivity and sensitivity of the latex-enhanced immunoturbidimetric assay kit, further ensuring that the latex-enhanced immunoturbidimetric assay kit still has strong detection capabilities while having a wide detection range.

[0039] The present invention does not limit the preparation method of the latex microsphere solution with a dispersion coefficient PDI of 0.05-0.3 in the above-mentioned latex enhanced immunoturbidimetry kit. A preparation method commonly used by those skilled in the art can be used to obtain a monodisperse latex microsphere solution, and then the dispersion coefficient PDI of the monodisperse latex microsphere solution is adjusted to 0.05-0.3 to obtain the latex microsphere solution required by the present invention. In addition, the present invention does not limit the method for adjusting the dispersion coefficient of the latex microsphere solution.

[0040] To achieve the object of the present invention, the present invention further provides a method for preparing the latex microsphere solution in the latex enhanced immune turbidimetry kit, comprising the following steps:

[0041] preparing a monodisperse latex microsphere solution;

[0042] The dispersion index (PDI) of the monodisperse latex microsphere solution is adjusted to 0.05-0.3 with salt to obtain a latex microsphere solution.

[0043] In some embodiments, the latex microsphere solution includes an olefin monomer, an acrylic monomer, a surfactant, and an initiator. The olefin monomer selected in this embodiment is insoluble in water because olefin monomers soluble in water cannot form latex microspheres.

[0044] In some embodiments, the olefin monomer includes at least one of styrene, styrene-propylene, methyl styrene, para-styrene, or methyl methacrylate. These olefin monomers are insoluble in water and can be polymerized with an acrylic monomer, a surfactant, and an initiator to produce latex microspheres with uniform particle size and good stability.

[0045] The present invention does not limit the type of acrylic monomer. In some embodiments, the acrylic monomer includes at least one of methacrylic acid, acrylic acid, itaconic acid, methyl methacrylate, and methyl acrylate.

[0046] The present invention does not limit the types of initiators and surfactants. In some embodiments, the initiator includes at least one of ammonium persulfate, potassium persulfate, sodium persulfate, and sodium sulfite; in some embodiments, the surfactant includes at least one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and sodium dodecyl sulfonate.

[0047] In some embodiments, a monodisperse latex microsphere solution is obtained by subjecting the above raw materials to a polymerization reaction.

[0048] The present embodiment does not limit the reaction temperature and reaction time of the polymerization reaction. The reaction temperature is preferably 50-100° C., and the reaction time is preferably 4-48 hours.

[0049] In some embodiments, the temperature of the polymerization reaction can be 50°C, 53°C, 56°C, 57°C, 59°C, 60°C, 62°C, 64°C, 65°C, 67°C, 69°C, 70°C, 73°C, 76°C, 79°C, 80°C, 82°C, 85°C, 87°C, 88°C, 89°C, 90°C, 91°C, 94°C, 95°C, 97°C or 100°C.

[0050] In some embodiments, the polymerization reaction time can be 4h, 5h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 15h, 18h, 19h, 20h, 23h, 25h, 27h, 28h, 30h, 32h, 34h, 35h, 38h, 39h, 40h, 41h, 43h, 45h, 47h or 48h.

[0051] In the above reaction temperature range, the initiator can initiate the polymerization reaction of olefin monomers and acrylic monomers, so that the reaction proceeds more completely and obtains higher yield and more stable monodisperse latex microspheres.

[0052] The present invention also does not limit the amount of the above-mentioned olefin monomer, acrylic monomer, surfactant and initiator added for the polymerization reaction. Based on 100% by mass of the olefin monomer, the mass percentage of the acrylic monomer is 5%-30%, the mass percentage of the initiator is 5%-20%, and the mass percentage of the surfactant is ≤2.5%.

[0053] In some embodiments, based on 100% by weight of the olefin monomer, the weight percentage of the acrylic monomer can be 5%, 8%, 10%, 15%, 18%, 20%, 24%, 25%, 28%, or 30%; the weight percentage of the initiator can be 5%, 8%, 10%, 15%, 18%, or 20%; and the weight percentage of the surfactant can be 2.5%, 2%, 1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, etc. With the above-mentioned ratios, a monodisperse latex microsphere solution with smaller particle size, higher yield, and better stability can be obtained.

[0054] In some embodiments, the polymerization reaction can be carried out under stirring conditions, and the stirring speed is preferably 100-400 rpm, which can promote a more complete reaction.

[0055] Under the above raw materials and reaction conditions, the latex microspheres in the latex microsphere solution have a smaller particle size in the range of 20-100 nm; more preferably, the latex microsphere particle size range is 50-70 nm, which can make the latex microsphere solution have a wider detection range and good antibody coupling effect.

[0056] In some embodiments, the particle size of the latex microspheres in the latex microsphere solution is 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 55 nm, 50 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm.

[0057] It should be noted that the above-mentioned restrictions on the types of olefin monomers, acrylic monomers, surfactants and initiators can be met at the same time, or only one of them can be met. If they are met at the same time, a latex microsphere solution with higher yield, better stability, smaller particle size and good stability can be obtained. At the same time, the latex microspheres are also given a large number of highly active carboxyl groups, which makes the detection range of the latex microspheres wider and the detection reactivity and sensitivity higher.

[0058] In some embodiments, the salt for adjusting the dispersion coefficient of the monodisperse latex microsphere solution is an inorganic salt, including at least one of sodium carbonate, sodium bicarbonate, ammonium carbonate, ammonium bicarbonate, potassium carbonate, and potassium bicarbonate. The above salts can be adjusted to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0059] With the assistance of the above salts, a latex microsphere solution with a dispersion coefficient PDI of 0.05-0.3 can be obtained, thereby expanding the detection range of the latex enhanced immunoturbidimetry kit and enhancing its detection sensitivity.

[0060] The present invention also provides an application of the latex microsphere solution as described above in latex-enhanced immunoturbidimetry. The latex microsphere solution having a dispersion coefficient (PDI) of 0.05-0.3 is applied to latex-enhanced immunoturbidimetry, which can also achieve the advantages of a wide detection range, high detection reactivity and sensitivity.

[0061] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention and are not used to limit the present invention.

[0062] Example 1

[0063] In 1000g of pure water, 0.5g of sodium lauryl sulfate, 0.3g of sodium bicarbonate, 50g of styrene, 5g of acrylic acid, and 5g of potassium persulfate were added, and the mixture was stirred at 90°C and 150rpm for 24h to obtain a monodisperse latex microsphere solution through polymerization reaction, which was then adjusted with sodium carbonate to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0064] Example 2

[0065] In 1000g of pure water, 0.8g of sodium dodecylbenzenesulfonate, 0.7g of sodium carbonate, 50g of styrene, 7g of methacrylic acid, and 7g of sodium persulfate were added, and the mixture was stirred at 80°C and 150rpm for 24h to obtain a monodisperse latex microsphere solution through polymerization reaction, which was then adjusted with sodium bicarbonate to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0066] Example 3

[0067] In 1000g of pure water, 0.5g of sodium lauryl sulfate, 50g of styrene, 4g of itaconic acid, 4g of methyl methacrylate, and 8g of ammonium persulfate were added, and the mixture was stirred at 80°C and 200rpm for 24h to obtain a monodisperse latex microsphere solution by polymerization reaction, which was then adjusted with sodium bicarbonate to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0068] Example 4

[0069] In 1000g of pure water, 0.5g of sodium lauryl sulfate, 50g of styrene, 4g of acrylic acid, 4g of methyl methacrylate, 3g of sodium persulfate and 3g of sodium sulfite were added, and stirred at 50°C and 200rpm for 24h to obtain a monodisperse latex microsphere solution through polymerization reaction, which was then adjusted with sodium bicarbonate to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0070] Example 5

[0071] In 1000g of pure water, 0.8g of sodium lauryl sulfate, 50g of styrene, 0.3g of sodium bicarbonate, 8g of methacrylic acid, and 7.5g of sodium persulfate were added, and the mixture was stirred at 80°C and 200rpm for 24h to obtain a monodisperse latex microsphere solution through polymerization reaction, which was then adjusted with sodium bicarbonate to obtain a latex microsphere solution with a dispersion coefficient of 0.05-0.3.

[0072] Performance Verification

[0073] Test 1: Topography test and PDI test

[0074] The latex microspheres in the latex microsphere solutions of Examples 1 to 5 were characterized using a scanning electron microscope. Figure 1, Figure 1 A to E represent the characterization results of Examples 1 to 5, respectively. In addition, the particle size results are shown in Table 1.

[0075] Table 1 Particle size of latex microspheres in latex microsphere solutions of Examples 1-5

[0076] Test items Example 1 Example 2 Example 3 Example 4 Example 5 Particle size (nm) 55 60 70 78 67

[0077] Depend on Figure 1 It can be seen that the particle sizes of the latex microspheres in the latex microsphere solutions of Examples 1 to 5 are uniform.

[0078] Test 2: Coupling test

[0079] Test sample:

[0080] 1. By adding salt at different concentrations, the PDI of the 60 nm latex microsphere solution obtained in Example 2 was adjusted to 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5, respectively, and expressed as the corresponding dispersion coefficient.

[0081] 2. Thermo Fisher brand 60nm carboxyl microspheres, represented by D1.

[0082] Conjugation target: IgG4 latex has a higher content than other biochemical test items. Given the specificity of IgG4-related diseases, the detection reagent needs to maximize its detection range. Therefore, labeling preparation requires latex microspheres with the smallest possible particle size. However, the reactivity and sensitivity of small-particle latex generally cannot meet detection requirements. Given this, the conjugated antibody selected in this example is immunoglobulin G4 antibody. However, it should be noted that the antibodies that can be conjugated to the latex microsphere solution of the present invention are not limited to immunoglobulin G4 antibodies.

[0083] The coupling effects of the above samples and immunoglobulin G4 are shown in Table 2. It should be noted that, since the latex microsphere solution plays a role in detection in the latex-enhanced immunoturbidimetric assay kit, the coupling effects of the latex microsphere solution with a dispersion coefficient of 0.05-0.3 below represent the coupling effects of the latex-enhanced immunoturbidimetric assay kit of the present invention.

[0084] Table 2 Coupling results of test samples and immunoglobulin G4

[0085]

[0086]

[0087] As shown in Table 2, compared with the more dispersed latex microsphere solution (PDI is 0.01, 0.03), the agglomerated latex microsphere solution (PDI is 0.05-0.3) has a better coupling effect. However, excessive agglomeration (PDI is 0.4, 0.5) will also affect the coupling. Therefore, the dispersion index PDI of the latex microsphere solution is preferably 0.05-0.3.

[0088] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A latex enhanced immunoturbidimetry kit, characterized in that: The latex enhanced immunoturbidimetry kit comprises a latex microsphere solution with a dispersion coefficient PDI of 0.05-0.3, The preparation steps of the latex microsphere solution include: preparing a monodisperse latex microsphere solution; Adjusting the dispersion index (PDI) of the monodisperse latex microsphere solution to 0.05-0.3 with salt to obtain the latex microsphere solution; The salt includes at least one of sodium carbonate, sodium bicarbonate, ammonium carbonate, ammonium bicarbonate, potassium carbonate, and potassium bicarbonate.

2. The latex enhanced immune turbidimetry kit according to claim 1, wherein The latex enhanced immunoturbidimetry kit comprises a latex microsphere solution with a dispersion coefficient PDI of 0.1-0.

3.

3. The latex enhanced immune turbidimetry kit according to claim 1, wherein The particle size of the latex microspheres in the latex microsphere solution is 20-100 nm.

4. The latex enhanced immune turbidimetry kit according to claim 3, wherein The particle size of the latex microspheres in the latex microsphere solution is 50-70 nm.

5. The latex enhanced immunoturbidimetry kit according to any one of claims 1 to 4, wherein The latex microsphere solution is a carboxyl latex microsphere solution.

6. A method for preparing a latex microsphere solution, characterized in that: The following steps are involved: preparing a monodisperse latex microsphere solution; The dispersion index (PDI) of the monodisperse latex microsphere solution is adjusted to 0.05-0.3 with salt to obtain a latex microsphere solution.

7. The method for preparing the latex microsphere solution according to claim 6, wherein: The latex microsphere solution comprises olefin monomers, acrylic monomers, surfactants and initiators.

8. The method for preparing the latex microsphere solution according to claim 7, wherein: The olefin monomer includes at least one of styrene, styrene-propylene, methylstyrene, parastyrene or methyl methacrylate; And / or, the acrylic monomer includes at least one of methacrylic acid, acrylic acid, itaconic acid, methyl methacrylate, and methyl acrylate; And / or, the initiator includes at least one of ammonium persulfate, potassium persulfate, sodium persulfate, and sodium sulfite; And / or, the surfactant includes at least one of sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium dodecylsulfonate.

9. The method for preparing the latex microsphere solution according to claim 6, wherein: The salt includes at least one of sodium carbonate, sodium bicarbonate, ammonium carbonate, ammonium bicarbonate, potassium carbonate, and potassium bicarbonate.

10. Application of a latex microsphere solution in latex enhanced immunoturbidimetry, characterized in that: The dispersion coefficient PDI of the latex microsphere solution is 0.05-0.3.

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