Immunoturbidimetric Detection Sensitizer

Abstract

The present invention provides an immunoturbidimetric detection sensitizer / detection reagent containing cross-linked cyclodextrin, which can greatly improve the detection sensitivity of the target analyte tested, and hardly increases the detection noise and does not reduce the specificity, and has great industrial application value.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, in particular to an immunoturbidimetric detection sensitizer. Background Art

[0002] Among immunological assays, immunoturbidimetry has poorer detection sensitivity than enzyme-linked immunosorbent assay and chemiluminescence assay, which greatly limited its application in clinical diagnosis in the early stage. In order to improve the sensitivity of immunoturbidimetry and expand the scope of application of the methodology, latex-enhanced immunoturbidimetry was developed on the basis of immunoturbidimetry, that is, the antibody or antigen corresponding to the substance to be tested is coated on nanometer-level latex particles, so that the volume of the antigen-antibody complex is increased, the turbidity signal changes more significantly, and thus the sensitivity of the test is improved.

[0003] In order to further improve the sensitivity of immunoturbidimetry, people have developed a variety of sensitizers that can be used in immunoturbidimetric detection reagents, such as polyethylene glycol, polyvinyl pyrrolidone, dextran, etc. However, due to the wide variety of analytes to be tested in clinical practice and their different physical and chemical properties, and the unclear principle of turbidimetric reagent sensitization, the existing sensitizers have encountered many limitations in practical applications. Some test items cannot screen for suitable sensitizers, or the use of sensitizers leads to increased detection noise or reduced detection specificity. Therefore, screening more sensitizers that can be used in immunoturbidimetric detection reagents is of great significance to expand the application scope of immunoturbidimetry. Summary of the invention

[0004] Cyclodextrin is a general term for a series of cyclic oligosaccharides produced by amylose under the action of cyclodextrin glucosyltransferase produced by Bacillus, usually containing 6 to 12 D-pyranose units. Among them, the molecules containing 6, 7, and 8 glucose units are more studied and have important practical significance, which are called α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin respectively (see Figure 6 ). Cyclodextrin molecules are barrel-shaped, with a hydrophobic cavity formed inside the barrel, which can absorb hydrophobic small molecules or groups of a certain size and shape to form a stable non-covalent complex. Cyclodextrin is used in many fields such as food, medicine, cosmetics, pesticides, etc. Its inclusion complexation principle is mainly used to improve the solubility and stability of specific ingredients. Cyclodextrin also has a small number of application cases in immunoturbidimetry. For example, CN106932594A discloses that the addition of β-cyclodextrin improves the sensitivity of detecting cystatin C; CN109613257B discloses that the addition of γ-cyclodextrin improves the sensitivity of detecting transferrin.

[0005] However, these existing immunoturbidimetric technologies all use monomeric cyclodextrin as a sensitizer. The inventors of this application unexpectedly found in some research on detection projects that adding monomeric cyclodextrin had no sensitization effect, but adding cross-linked cyclodextrin could greatly improve the sensitivity. Cross-linked cyclodextrin is a water-soluble cyclodextrin polymer formed by cross-linking multiple cyclodextrin monomers (see Figure 7 ), has an amorphous spatial network structure, and the space within the network structure can bind guest molecules.

[0006] The first aspect of the present invention provides an immunoturbidimetric detection sensitizer, which includes cross-linked cyclodextrin.

[0007] The immunoturbidimetric detection sensitizer exists as a separate reagent product and is used in conjunction with other immunoturbidimetric detection reagent products. It can be in the form of a solid dry powder or a solution. When it is in the form of a solution, it also contains other ingredients, including buffers, salts, surfactants and the like.

[0008] Optionally, the immunoturbidimetric detection sensitizer may be a composite sensitizer, which, in addition to cross-linked cyclodextrin, further comprises at least one other sensitizing component selected from polyethylene glycol, polyvinyl pyrrolidone and dextran.

[0009] The first aspect of the present invention also provides an immunoturbidimetric detection reagent, which comprises cross-linked cyclodextrin.

[0010] The cross-linked cyclodextrin contained in the immunoturbidimetric detection reagent is preferably 0.1-5%, preferably 0.1-4%, and preferably 0.1-3%.

[0011] The product form of the immunoturbidimetric detection reagent is at least one of a sample diluent, a sample processing solution, an immunoturbidimetric R1 reagent, and an immunoturbidimetric R2 reagent.

[0012] Optionally, in addition to the cross-linked cyclodextrin, the immunoturbidimetric detection reagent further comprises at least one of a buffer, a salt, a surfactant, a stabilizer and a preservative.

[0013] Optionally, in addition to cross-linked cyclodextrin, the immunoturbidimetric detection reagent further comprises at least one other sensitizing component selected from polyethylene glycol, polyvinyl pyrrolidone and dextran.

[0014] The first aspect of the present invention further provides an immunoturbidimetric detection kit, which comprises the aforementioned immunoturbidimetric detection sensitizer or the aforementioned immunoturbidimetric detection reagent.

[0015] The second aspect of the present invention provides the use of cross-linked cyclodextrin in preparing an immunoturbidimetric detection sensitizer, an immunoturbidimetric detection reagent or an immunoturbidimetric detection kit.

[0016] The third aspect of the present invention provides a method for increasing the detection sensitivity of an immunoturbidimetric detection method, comprising adding cross-linked cyclodextrin to a sample.

[0017] For the "immunoturbidimetric assay" described in the first to third aspects of the present invention, preferably, the immunoturbidimetric assay is latex-enhanced immunoturbidimetric assay.

[0018] For the "immunoturbidimetric detection" described in the first to third aspects of the present invention, preferably, the target analyte to be detected is at least one selected from hemoglobin, glycosylated hemoglobin and CCP antibody.

[0019] Technical effect: The immunoturbidimetric detection sensitizer / detection reagent containing cross-linked cyclodextrin provided by the present invention can greatly improve the detection sensitivity of the target analyte tested, and almost does not increase the detection noise or reduce the specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .The influence of different sensitizers on the detection signal of Anti-CCP project

[0021] Figure 2 .The influence of different concentrations of cross-linked cyclodextrin on the detection signal of Anti-CCP project

[0022] Figure 3 .Effects of different concentrations of cross-linked cyclodextrin on the detection signal of hemoglobin test (FIT)

[0023] Figure 4 Effect of adding different concentrations of cross-linked cyclodextrin to HbA1c R1 reagent on the detection signal

[0024] Figure 5 Effect of adding different concentrations of cross-linked cyclodextrin to HbA1c R2 reagent on the detection signal

[0025] Figure 6 . Schematic diagram of the molecular structure of monomeric cyclodextrin

[0026] Figure 7 . Schematic diagram of the molecular structure of cross-linked cyclodextrin DETAILED DESCRIPTION

[0027] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0028] Herein, the term "immunoturbidimetry" refers to a dynamic determination method for antigen-antibody binding. Its basic principle is: when the antigen and antibody react specifically in a special dilution system and the ratio is appropriate (generally, the antibody is excessive), the soluble immune complex formed is precipitated from the liquid phase under the action of the polymerization promoter (polyethylene glycol, etc.) in the dilution system, forming particles, so that the reaction solution has turbidity. When the antibody concentration is fixed, the amount of the immune complex formed increases with the increase of the amount of antigen in the test sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standard products, the content of the antigen in the test sample can be calculated. The latex-enhanced immunoturbidimetry is to coat the antibody corresponding to the substance to be tested on the latex particles, so that the volume of the antigen-antibody conjugate is increased. After the light passes through, the intensity changes of the transmitted light and the scattered light are more significant, thereby improving the sensitivity of the test.

[0029] As used herein, the term "reagent" may be understood as a substance or product, etc., regardless of its form or state, and may be either liquid or solid.

[0030] Herein, the term "cross-linked cyclodextrin" refers to a cross-linked polymer of cyclodextrin monomers, typically a network polymer obtained by cross-linking α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin monomers with cross-linking agents such as epichlorohydrin, polycarboxylic acid or citric acid derivatives, with a molecular weight of 2-300KD, and is a common industrial raw material on the market.

[0031] As used herein, the term "buffer" refers to a substance used to stabilize the pH of a solution, such as HEPES buffer, PB buffer, PBS buffer, Tris buffer or phosphate buffer.

[0032] As used herein, the term "salt" refers to salt substances used to regulate the ionic strength of a solution, such as NaCl, KCl and NH4Cl, MgCl2, MgSO4, CaCl2, Na2SO4, K2SO4, NH4SO4, NaNO3, and KNO3.

[0033] As used herein, the term "surfactant" refers to a substance that can significantly reduce the surface energy of water, such as Brij 35, Tween 20 or Triton X-100.

[0034] Herein, the term "stabilizer" refers to a substance that can maintain the physicochemical properties of the active ingredient in the reagent, such as disodium edetate, BSA, glycine, sucrose or trehalose.

[0035] As used herein, the term "preservative" refers to a substance that prevents the active ingredient in a reagent from deteriorating, such as Proclin 300 or sodium azide.

[0036] Herein, the term "sample diluent" refers to a solution used to dilute and liquefy a sample to be tested so as to facilitate the detection of a target analyte in the sample, and generally contains components such as a buffer and a surfactant.

[0037] Herein, the term "sample processing solution" refers to a solution used to dissolve and extract target analytes in a sample, and contains components such as a protein denaturant (eg, acid, base, disulfide bond reducing agent), a surfactant, and the like.

[0038] In this article, immunoturbidimetric R1 reagent refers to a buffer reagent used to dilute and dissolve samples, which can remove the interference of interfering substances in the samples; immunoturbidimetric R2 reagent is a reagent containing immunoreactive substances (antibodies or antigens, or latex coated with antibodies or antigens), which is used to react with the target analyte in the sample to produce a detectable signal (absorbance change).

[0039] Herein, the percentage symbol "%" means weight percentage unless otherwise specified.

[0040] The main experimental materials of the present invention are as follows:

[0041] 1. Epichlorohydrin cross-linked polymerized cyclodextrin (purchased from Zhiyuan Biotechnology)

[0042] 2. α-cyclodextrin (purchased from Aladdin)

[0043] 3. β-cyclodextrin (purchased from Aladdin)

[0044] 4. γ-cyclodextrin (purchased from Aladdin)

[0045] 5. 2-Hydroxypropyl-β-cyclodextrin (purchased from Aladdin)

[0046] 6. Polyethylene glycol 6000 (PEG6000, purchased from Xibao Biotechnology)

[0047] 7. Polyvinylpyrrolidone 10000 (PVP10000, purchased from Aladdin)

[0048] 8. Dextran 200000 (purchased from Fujifilm Wako)

[0049] Unless otherwise specified, other materials are commercially available conventional materials.

[0050] Example 1 Reagent Preparation

[0051] 1.1 Preparation of R2 reagents for each test item

[0052] 1) Add 100 μL of latex microspheres (PS1016A) to 400 μL of cross-linking solution (50 mM HEPES, pH 6.1) and shake on a shaker to obtain a latex microsphere solution;

[0053] 2) Add 15 μL of EDAC solution to the latex microsphere solution and activate it at 37°C with shaking for 1 hour;

[0054] 3) Add 60 μL of hemoglobin polyclonal antibody (Dako Company: Dako A0118 human hemoglobin polyclonal antibody) to the latex microsphere solution and shake cross-link at 37°C for 4 hours;

[0055] 4) Add 60 μL of TW-20 solution and shake at 37°C for 1 hour;

[0056] 5) Add the cross-linking liquid to the centrifuge tube. After centrifugation, carefully discard the supernatant with a pipette;

[0057] 6) Add 2.5 mL of cleaning solution (50 mM glycine + 0.1% NaN3, pH 8.0), vortex, centrifuge again, and carefully discard the supernatant;

[0058] 7) Add 6 mL of latex microsphere suspension (50 mM glycine + 0.1% BSA + 8% sucrose + 0.1% NaN3, pH 8.0) and perform ultrasonic dispersion treatment to obtain the R2 reagent for hemoglobin detection item.

[0059] Similarly, according to the above steps, the hemoglobin polyclonal antibody is replaced with cyclic citrullinated peptide (AG-CCP-HP) to prepare the CCP antibody detection project R2 reagent.

[0060] Take a cross-linked HbA1c monoclonal antibody (Phipeng Bio MABM-HbA1c-1004) with a final concentration of 80ug / ml and dissolve it in a buffer solution (final concentration 25mmol Tris+600mM NaCl+0.1Tween20) to prepare the R2 reagent for the glycated hemoglobin detection project.

[0061] 1.2 Preparation of R1 reagents for each test item

[0062] Hemoglobin detection project R1 reagent: 25mmol Tris+600mM NaCl+0.1Tween20.

[0063] CCP antibody detection project R1 reagent: 25mmol Tris+600mM NaCl+0.1Tween20.

[0064] Glycated hemoglobin detection item R1 reagent: Take adsorption latex microspheres (Merck: KSF010) with a final concentration of 2.5 mg / ml and dissolve them in buffer (final concentration 25 mmol Tris+600 mM NaCl+0.1 Tween20) to prepare the glycated hemoglobin detection item R1 reagent.

[0065] 1.3 Preparation of standard reagents for each project

[0066] Human hemoglobin (Sigma H7379) was added to diluent (25mM Hepes + 500mM NaCl + 10mM Arg + 10mM Glu + 0.1% EDTA·Na2 + 10% sucrose + 5% trehalose + 0.1% Tween20 + 0.05% Proclin300, pH adjusted to 7.0 with NaOH / HCl) and diluted to a gradient concentration of 0ng / mL, 62.5ng / mL, 125ng / mL, 250ng / mL, 500ng / mL and 1000U / mL.

[0067] CCP antibody gradient concentration standard is commercially available Anti-CCP calibrator (PhiPeng Bio S00704C1-S00704C5)

[0068] The glycosylated hemoglobin gradient concentration standard was a commercially available HbA1c calibrator (Phipeng Biotechnology S00110C1-S00110C5).

[0069] Example 2: On-machine test process for each test item

[0070] Instrument: Mindray BS450 fully automatic biochemical analyzer

[0071] Hemoglobin test process: Pipette 135μL of R1 into the cuvette, incubate at 37℃ for 2 minutes, then pipette 35μL of the sample on the sample plate into the cuvette, stir mechanically, and incubate at 37℃ for 7 minutes; then add 35μL of R2 to the cuvette, stir mechanically, and incubate at 37℃ for 12 minutes. The instrument records the absorbance value at a wavelength of 570nm at 7 minutes and 12 minutes from the time the sample is added to the cuvette. The difference between the absorbance at 12 minutes and 7 minutes is the instrument reading, so sometimes the instrument reading is negative.

[0072] CCP antibody detection process: Pipette 150μL of R1 into the cuvette, incubate at 37℃ for 2 minutes, then pipette 5μL of the sample on the sample plate into the cuvette, stir mechanically, and incubate at 37℃ for 7 minutes; then add 50μL of R2 to the cuvette, stir mechanically, and incubate at 37℃ for 12 minutes. The instrument records the absorbance value at a wavelength of 570nm at 7 minutes and 12 minutes from the time the sample is added to the cuvette. The difference between the absorbance at 12 minutes and 7 minutes is the instrument reading, so sometimes the instrument reading is negative.

[0073] HbA1c testing process:

[0074] Pipette 150μL of R1 into the cuvette, incubate at 37℃ for 2 minutes, then pipette 4μL of the sample on the sample plate into the cuvette, stir mechanically, and incubate at 37℃ for 8 minutes; then add 50μL of R2 into the cuvette, stir mechanically, and incubate at 37℃ for 12 minutes. The instrument records the absorbance values ​​at wavelengths of 660nm / 800nm ​​at 8 minutes and 12 minutes from the time the sample is added to the cuvette (the absorbance value is set as the absorbance value of 660nm wavelength minus the absorbance of 800nm ​​wavelength). The difference between the absorbance at 12 minutes and 8 minutes is the instrument reading, so sometimes the instrument reading is negative.

[0075] Test Example 1 Comparison of the effects of adding different sensitizers in the CCP antibody (Anti-CCP) detection project

[0076] Different sensitizers were added to the R1 reagent of the Anti-CCP detection project, and Anti-CCP standards with gradient concentrations were tested. The results are shown in Table 1.

[0077] Table 1

[0078]

[0079] From the above results, only cross-linked cyclodextrin, PEG6000 and dextran 200000 have sensitization effect (see Appendix Figure 1 ), other test sensitizers had no sensitizing effect. Monomeric α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin had no sensitizing effect, and β-cyclodextrin even reduced the detection signal. However, cross-linked cyclodextrin had an incredible sensitizing effect. This may be because the network space of cross-linked cyclodextrin brought an exponential improvement to the latex turbidimetric detection signal.

[0080] Test Example 2 Comparison of the effects of adding different concentrations of cross-linked cyclodextrin in the CCP antibody (Anti-CCP) detection project

[0081] Different concentrations of cross-linked cyclodextrin were added to the R1 reagent of the Anti-CCP detection project, and gradient concentrations of Anti-CCP standards and positive clinical samples were tested. The results are shown in Table 2.

[0082] Table 2

[0083]

[0084] From the above results, it can be seen that when 0.5% to 2% of epichlorohydrin cross-linked polymerized cyclodextrin is added to Anti-CCP R1, the detection signal increases exponentially, and the signal increase trend shows a typical dose-dependent effect with the concentration of epichlorohydrin cross-linked polymerized cyclodextrin added. However, when the concentration exceeds 1%, a higher detection background noise appears (see Appendix Figure 2). Before and after the addition of epichlorohydrin cross-linked polymerized cyclodextrin, the increasing trend of the detection signal of the calibrator and the clinical sample was inconsistent, which may be because the raw material of the calibrator is obtained by antigen immunization, and its affinity is higher than that in the natural sample. In summary, cross-linked cyclodextrin can be used as a sensitizer in the Anti-CCP project, with an optimal concentration of 0.5%-1%.

[0085] Test Example 3 Comparison of the effects of adding different concentrations of cross-linked cyclodextrin in the hemoglobin test project (FIT project)

[0086] Different concentrations of cross-linked cyclodextrin were added to the FIT project R1 reagent, and gradient concentrations of hemoglobin standards and positive clinical samples were tested. The results are shown in Table 3.

[0087] Table 3

[0088]

[0089] From the above results, it can be seen that when 0.5% to 2% epichlorohydrin cross-linked polymerized cyclodextrin is added to FIT R1, the detection signal increases exponentially, and the signal increase trend shows a typical dose-dependent effect with the concentration of epichlorohydrin cross-linked polymerized cyclodextrin added (see Appendix Figure 3 ). The increasing trend of the detection signal of hemoglobin standard and clinical samples is consistent, indicating that the addition of cross-linked cyclodextrin does not affect the detection specificity. In summary, cross-linked cyclodextrin can be used as a sensitizer in FIT projects, with an optimal concentration of 1%-2%.

[0090] Test Example 4 Comparison of the effects of adding different concentrations of cross-linked cyclodextrin in the glycated hemoglobin (HbA1c) test

[0091] Different concentrations of cross-linked cyclodextrin were added to the R1 reagent or R2 reagent of the HbA1c test project, and positive clinical samples were tested. The results are shown in Tables 4 and 5.

[0092] Table 4

[0093]

[0094] Table 5

[0095]

[0096]

[0097] From the above results, it can be seen that when 0.5% to 2% epichlorohydrin cross-linked polymerized cyclodextrin is added to HbA1c R1 reagent or R2 reagent, the detection signal has a certain upward trend, and the signal increase trend shows a typical dose-dependent effect with the concentration of epichlorohydrin cross-linked polymerized cyclodextrin added. The sensitization effect of adding to R2 reagent in HbA1c detection is better than that of adding to R1 reagent (see Figure 4 and Figure 5 ). In summary, cross-linked cyclodextrin can be used as a sensitizer in HbA1c projects, and the preferred added concentration is 2%. It can be seen that different test items are affected by the sensitization of cross-linked cyclodextrin to different degrees, and the dosage of the sensitizer needs to be adjusted accordingly.

[0098] In summary, cross-linked cyclodextrin showed significant sensitization effects in multiple latex turbidimetric projects, and its use within a certain range did not affect the reagent background and specificity. It is a new sensitizer suitable for a variety of latex turbidimetric reagents.

[0099] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0100] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. Application of epichlorohydrin cross-linked polymerized cyclodextrin in the preparation of immunoturbidimetric detection sensitizer.

2. The use according to claim 1, characterized in that: The sensitizer also includes at least one of a buffer, a salt and a surfactant.

3. The use according to claim 1, characterized in that: The sensitizer also includes at least one selected from the following: polyethylene glycol, polyvinyl pyrrolidone, dextran.

4. The use according to claim 1, characterized in that: The target analyte detected by the immunoturbidimetric detection sensitizer is at least one of hemoglobin and CCP antibody.

5. The use according to claim 1, characterized in that: The target analyte detected by the immunoturbidimetric detection sensitizer is glycated hemoglobin.

6. An immunoturbidimetric detection reagent, characterized in that: It comprises epichlorohydrin cross-linked polymerized cyclodextrin, wherein the immunoturbidimetric detection reagent is at least one of a sample diluent, a sample processing liquid, an immunoturbidimetric R1 reagent, and an immunoturbidimetric R2 reagent.

7. The immunoturbidimetric detection reagent according to claim 6, wherein Also included is at least one of a buffer, a salt, a surfactant, a stabilizer, and a preservative.

8. The immunoturbidimetric detection reagent according to claim 6, characterized in that: It also includes at least one selected from the following: polyethylene glycol, polyvinyl pyrrolidone, and dextran.

9. The immunoturbidimetric detection reagent according to claim 6, characterized in that: The immunoturbidimetric assay is a latex-enhanced immunoturbidimetric assay.

10. The immunoturbidimetric detection reagent according to claim 6, characterized in that: The target analyte detected by the immunoturbidimetric detection reagent is at least one of hemoglobin and CCP antibody.

11. The immunoturbidimetric detection reagent according to claim 6, characterized in that: The target analyte detected by the immunoturbidimetric detection reagent is glycated hemoglobin.

12. An immunoturbidimetric detection kit, characterized in that: The invention comprises the immunoturbidimetric detection reagent according to any one of claims 6 to 11.

13. The immunoturbidimetric detection kit according to claim 12, characterized in that: The immunoturbidimetric assay is a latex-enhanced immunoturbidimetric assay.

14. The immunoturbidimetric detection kit according to claim 12, characterized in that: The target analyte detected by the immunoturbidimetric detection kit is at least one of hemoglobin and CCP antibody.

15. The immunoturbidimetric detection kit according to claim 12, characterized in that: The target analyte detected by the immunoturbidimetric detection kit is glycated hemoglobin.

16. Use of epichlorohydrin cross-linked polymerized cyclodextrin in the preparation of immunoturbidimetric detection reagents or immunoturbidimetric detection kits.

17. The use according to claim 16, characterized in that The immunoturbidimetric assay is a latex-enhanced immunoturbidimetric assay.

18. The use according to claim 16, characterized in that The target analyte for detection is at least one of hemoglobin and CCP antibody.

19. The use according to claim 16, characterized in that The target analyte of the test is glycated hemoglobin.

20. A method for increasing the detection sensitivity of an immunoturbidimetric detection method, characterized in that: Epichlorohydrin was added to the sample to cross-link polymerized cyclodextrin.

21. The method according to claim 20, characterized in that The immunoturbidimetric assay is a latex-enhanced immunoturbidimetric assay.

22. The method according to claim 20, characterized in that The target analyte to be detected is at least one of hemoglobin and CCP antibody.

23. The method according to claim 20, characterized in that The target analyte of the test is glycated hemoglobin.

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