Application of low-ionic-strength buffer substance and detection reagent

Abstract

The invention relates to application of a low-ionic-strength buffer substance and a detection reagent, in particular to application of the low-ionic-strength buffer substance in preparation of a latex microsphere immunoturbidimetry detection reagent. The requirements of an immunoturbidimetry detection reagent for high buffer capacity and stable pH can be met, the defects that strong electrolyte ions in an existing buffer system influence the dispersion stability of hydrophobic latex microspheres and interfere adsorption of substances to be detected on the surfaces of the capture type latex microspheres and the like can be overcome, a stable liquid environment is provided for immunoturbidimetry reaction, and the detection sensitivity is high. The method has the capacity of resisting carrying and cross contamination, interference is reduced, and the accuracy of a detection result is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to a reagent for in vitro detection, in particular to the use of a buffer substance in the preparation of a reagent for in vitro detection and the reagent. BACKGROUND

[0002] Antigen-antibody immune reaction is a specific reaction, and the reaction forms insoluble immune complexes, which become microparticles suspended in the reaction solution, thereby causing a change in the absorbance of the solution, and the absorbance change is proportional to the concentration of microparticles, so that the content of the antigen molecules to be detected in the sample can be calculated, and the method is widely used for in vitro quantitative detection of protein molecules or polypeptides in biological samples. The antibody of the molecule to be detected is connected with the polymer latex microspheres, and the absorbance change caused by the latex-carrying immune complex formed after the combination of the antibody and the molecule to be detected is more obvious, thereby helping to improve the detection sensitivity. This method is the latex-enhanced immunoturbidimetry widely used in the field of current clinical in vitro detection. The commonly used latex microspheres are polystyrene nanoparticles.

[0003] Latex-enhanced immunoturbidimetry is a sensitive and low-cost detection method, but it has poor anti-interference performance. Immune reaction requires a stable solution environment, so buffer solution is used as the reaction medium in the field of in vitro detection. The immunoturbidimetry reagent needs to be carried to the full-automatic biochemical analyzer for use, and multiple reagents are placed in the reagent bin after being opened. Then, the R1 reagent will be affected by carbon dioxide in the air, gradually acidified, deviated from the initial pH, and if there is a carbonate system buffer substance or volatile acid such as hydrochloric acid and acetic acid in the reagent bin, the closed environment in the reagent bin will accelerate the acidification of the R1 reagent, resulting in a continuous increase or decrease in the measured value after the reagent is opened, which makes the detection result distorted and causes misdiagnosis. In addition, the reaction cup reagent needle in the biochemical analyzer system is reusable, and in a single test, the trace reagent carried by the reagent needle or the alkaline cleaning liquid remaining in the reaction cup will cause the pH change in the reaction cup, resulting in inaccurate measurement. Therefore, it is necessary to improve the buffer capacity of the reagent to improve the anti-interference performance.

[0004] When preparing the commonly used buffer solution, strong acid or strong base is often used to adjust the pH of the solution. However, the inventors have found that when the concentration of the buffer substance is large, a large amount of strong acid or strong base is introduced, and such a solution environment is not conducive to the detection of some molecules to be detected.

[0005] For example, the level of glycated hemoglobin (HbA1c) in human blood is a reliable indicator for evaluating the long-term blood glucose control level of diabetes and a cornerstone of diabetes management. There are multiple hydrophobic amino acids (such as valine, leucine, isoleucine, phenylalanine, etc.) on the surface of the hemoglobin β subunit, and the side chains of these residues are non-polar, which tend to avoid the aqueous environment. The 1-8 helix region of the β chain (A-B helix segment) is rich in leucine and valine, forming a hydrophobic surface. The hydrophobic residues (such as phenylalanine, leucine) of the H helix region of the β chain (near the heme pocket) form a hydrophobic pocket around the heme, protecting the Fe 2+ and stabilizing the heme binding. When red blood cells are treated with a hemolytic agent, hemoglobin is released, loses the quaternary structure, and is pulled by hydrophobic force, (glycated) hemoglobin will be adsorbed to the surface of polystyrene microspheres, and due to the multiple hydroxyl groups after the glycation of valine, the hydrophilicity is enhanced, making the antigen structure outward and easy to be recognized by antibodies.

[0006] Based on this characteristic, the general turbidimetry method commonly used in the industry is to add latex microspheres in reagent R1, which non-specifically adsorb multiple (glycated) hemoglobin to form a multi-epitope large microparticle sensitized by glycated hemoglobin, and then add anti-human HbA1c antibody in reagent R2, and after mixing, insoluble microparticles are generated, and the absorbance at 660 nm is read to calculate the glycated hemoglobin content.

[0007] In the glycated hemoglobin detection kit, the surface of the polystyrene latex microspheres in reagent R1 is modified by sulfate groups, which carry strong negative charges in the reagent R1 (pH > 7) buffer. Under the action of electrostatic force, the microspheres repel each other and remain stable. After neutral salt is dissolved in water, the cations ionized from the water will neutralize the charges on the surface of the microspheres, causing the distance between the microspheres to decrease or even to aggregate.

[0008] The polystyrene molecular chain lacks polar groups, resulting in a low surface energy, making it difficult for water molecules to spread on the surface, thereby forming a hydrophobic interface. Moreover, the non-polar benzene ring structure of polystyrene makes the intermolecular interaction mainly rely on van der Waals force, lacking strong interactions such as hydrogen bonding, further strengthening the hydrophobicity. Therefore, the surface of the polystyrene microspheres can adsorb proteins rich in hydrophobic residues to capture glycated hemoglobin in the sample. When Cl-, I-, K + or Na + ions are present in the solution, the ions will disturb the order of water, destroy the original hydrogen bond network of water, reduce the arrangement resistance of water molecules, and make water more easily contact the hydrophobic surface, thereby causing the stability of the hydration layer around the hydrophobic molecules to decrease, and the driving force for hydrophobic aggregation (the need for entropy increase) to weaken. In addition, by shielding the charges, the salt ions reduce the polarity of water, which also reduces the repulsion of water molecules to hydrophobic groups, which is not conducive to the adsorption of protein molecules with hydrophobic residues.

[0009] Another example is the detection of oxidized low density lipoprotein. Oxidized low density lipoprotein (Ox-LDL) is a product formed after low density lipoprotein is oxidatively modified in vivo, and its level can accurately and effectively reflect the progression of atherosclerosis and has potential diagnostic value in patients with chronic heart failure (CHF).

[0010] In an oxidized low density lipoprotein detection kit, the main component of the R1 reagent is latex microparticles sensitized with an anti-human oxidized low density lipoprotein antibody (4E6 epitope). When the sample is mixed with the R1 reagent, the oxidized low density lipoprotein in the sample will be captured by the antibody on the microspheres. Since the 4E6 epitope is unique in Ox-LDL, no insoluble microparticles are formed at this stage. The R2 reagent is a latex microparticle sensitized with an anti-human apolipoprotein B antibody (non-4E6 epitope). After adding the R2 reagent, a double-antibody sandwich insoluble microparticle is formed. Since there is a large amount of free apolipoprotein B in the sample, it will quickly bind to R2, causing the R2 antibody to be occupied and unable to form effective turbidity. The inventors found that this effect can be achieved by reducing the salt concentration.

[0011] Therefore, for such reactions, it is necessary to solve the contradiction between the high buffer capacity and the low ionic strength, and to develop a new buffer solution system. SUMMARY

[0012] To solve the above technical problems, the present application uses a low ionic strength buffer substance to prepare a detection reagent, providing a stable liquid environment for the immune turbidimetry reaction, having the ability to resist carrying and cross-contamination, reducing interference, and ensuring the accuracy of the detection results.

[0013] The present application first provides a use of a low ionic strength buffer substance.

[0014] The use of a low ionic strength buffer substance in the preparation of a latex microsphere immune turbidimetry detection reagent, wherein the total concentration of strong electrolyte ions in the detection reagent is 0-10 mmol / L, and the strong electrolyte ions include at least one of metal ions, ammonium ions, or strong acid anions. Strong electrolyte ions are examples generated by strong electrolytes, which are generally considered to be completely ionized in aqueous solution. These substances are commonly strong acids, strong bases, or salts.

[0015] In one embodiment, the detection reagent contains at least one buffer substance and at least one adjusting substance, wherein the buffer substance is selected from at least one of Bicine, ADA, EPPS, POPSO, MES, CHES, CAPS, Hepes, Mops, TAPS, DEA, TEA, Tris, the adjusting substance is selected from at least one of TEA, DEA, lysine, arginine, histidine, aspartic acid, glutamic acid, and the adjusting substance is not the same as the buffer substance. The buffer substance functions to buffer the change of H+or OH-concentration, thereby stabilizing the solution pH; the adjusting substance is used to adjust the solution pH to the target pH, and in combination with the buffer substance, forms a buffer system to maintain the solution pH in the working interval. These buffer substances and adjusting substances are all commonly used substances in the art, wherein the suitable working pH of the buffer substances has been reported, and the adjusting range of the adjusting substances is from pH 5.5 to 10, but each substance has a certain adjusting range.

[0016] In one embodiment, the detection reagent contains capture latex microspheres for binding the to-be-detected substance in the sample, and the detection reagent provides a liquid environment for the binding of the to-be-detected substance on the capture latex microspheres. The capture latex microspheres include two types: one type is to capture the to-be-detected substance through non-specific adsorption, i.e., physical adsorption, for example, the to-be-detected substance is adsorbed on the surface of the latex microspheres through hydrophobic interaction; the other type is to specifically bind the to-be-detected substance through immune adsorption, in which case, the surface of the latex microspheres is connected with antibody or antigen molecules for binding the to-be-detected substance. The binding of the to-be-detected substance on the capture latex microspheres does not bring about a significant change in the solution absorbance, and does not produce a significant immune turbidity change.

[0017] Preferably, the above-mentioned detection reagent is reagent R1 of the latex-enhanced immune turbidimetry.

[0018] Preferably, the above-mentioned detection reagent is used for preparing a glycated hemoglobin detection kit, or for preparing an oxidized low-density lipoprotein detection kit.

[0019] The present application also provides a detection reagent.

[0020] A detection reagent for latex microsphere immune turbidimetry, which is characterized in that the detection reagent is an aqueous solution containing a buffer substance, and the total concentration of strong electrolyte ions in the detection reagent is not more than 10 mmol / L; preferably, not more than 5 mmol / L; more preferably, not more than 2.5 mmol / L.

[0021] Preferably, the buffer substance is selected from at least one of Bicine, ADA, EPPS, POPSO, MES, CHES, CAPS, Hepes, Mops, TAPS, DEA, TEA, Tris;

[0022] The detection reagent also contains a regulating substance selected from at least one of TEA, DEA, lysine, arginine, histidine, aspartic acid, glutamic acid, and the regulating substance is not the same as the buffering substance.

[0023] Preferably, the strong electrolyte ion is a substance of at least one of alkali metal ions, alkaline earth metal ions, ammonium ions, halogen ions, sulfate ions, bisulfate ions, nitrate ions, dihydrogen phosphate ions, hydrogen phosphate ions, phosphate ions. The alkali metal ions include Na + , K + , Li + , etc., the alkaline earth metal ions include Mg 2+ , Ca 2+ , etc., and the halogen ions include Cl - , Br - , I - , etc.

[0024] Preferably, the detection reagent also contains at least one of a chelating agent, a surfactant, a freeze-thaw stabilizer, a protein protective agent, and a preservative. The chelating agent can be ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate (EDTA-2Na), or other commonly used substances in the art. The protein protective agent can be bovine serum albumin (BSA), and the preservative can be PC-300, PC-950, or sodium azide, etc. according to the detection object.

[0025] Preferably, the detection reagent also contains a capture type latex microsphere.

[0026] Preferably, the concentration of the buffering substance in the detection reagent is 30-300 mM. For example, it can be 30 mM, 50 mM, 100 mM, 200 mM, or 300 mM.

[0027] Preferably, when the concentration of the buffering substance is not more than 100 mM, the regulating substance is selected from TEA or DEA. At this time, the regulating substance can not only adjust the pH of the buffer, but also further improve the buffering capacity. When the concentration of the buffering substance is relatively high, such as greater than 100 mM, the regulating substance can be selected as needed, for example, a buffering system composed of MES and lysine, or a buffering system composed of ADA and lysine.

[0028] Since the addition of some components in the reagent can introduce strong electrolyte ions, resulting in the increase of the overall ionic strength of the solution, thereby affecting the binding of the capture type latex microspheres and the measured substance or affecting the dispersion stability of the latex microspheres, or causing the combined measured substance to be quickly combined with other substances in the sample, thereby affecting the subsequent immune turbidization reaction of the latex microsphere coupled antibody and the measured substance, therefore, in addition to the buffer system, the strong electrolyte ions introduced by other components should be as little as possible. However, under certain circumstances, the addition of some functional components will inevitably introduce strong electrolyte ions, such as the use of sodium azide as a preservative for some detection reagents, which will introduce sodium ions, but as long as the concentration of the strong electrolyte ions does not exceed the upper limit specified in the application, the influence on the detection result is relatively small.

[0029] The application also provides a kit.

[0030] A kit comprising the detection reagent according to any one of the above as reagent R1, further comprising reagent R2, the key is that,

[0031] The reagent R1 comprises:

[0032] A buffer system composed of a buffer substance and an adjusting substance, wherein the concentration of the buffer substance is 30-300mM;

[0033] A preservative with a mass-volume content of 1-3‰;

[0034] Capture type latex microspheres with a mass-volume content of 0.1-0.2%;

[0035] The solvent is ultrapure water;

[0036] The reagent R2 comprises:

[0037] A buffer agent with a concentration of 50-300mM;

[0038] A protein protective agent with a mass-volume content of 0.5%-2%;

[0039] A preservative with a mass-volume content of 1-3‰;

[0040] An inorganic salt with a mass-volume content of 0.5%-2%;

[0041] Latex microsphere coupled antibodies with a content of 35-55mg / L;

[0042] The solvent is ultrapure water.

[0043] The buffer agent in the reagent R2 can be the same as the buffer substance in the reagent R1, or it can be different, and it can be a low ionic strength buffer agent, or it can not be a low ionic strength buffer agent. The latex microsphere coupled antibody in the reagent R2 can be obtained by coupling a monoclonal antibody on a polystyrene latex microsphere.

[0044] The capturing latex microspheres can capture the molecules to be detected by physical adsorption or by means of the antibody or antigen functional molecules connected to the surface of the microspheres through antigen-antibody reaction, as described above. The particle size of the capturing latex microspheres is 50-200 nm, preferably 100-150 nm.

[0045] In one embodiment, the reagent R1 further contains 0.5-1 g / L of a surfactant such as imidazole, 0.2-0.6 g / L of a chelating agent such as ethylenediaminetetraacetic acid, and 1.2-2.5% of a freeze-thaw stabilizer such as DMSO, the pH of the reagent R1 is 7.9-8.1, and the capturing latex microspheres in the reagent R1 are physically adsorbed latex microspheres. This reagent R1 can be used for the detection of glycated hemoglobin, and the added capturing latex microspheres are sulfate group modified latex microspheres. The corresponding reagent R2 buffer can use Good's buffer with a pH of 5.6-5.8.

[0046] In one embodiment, the reagent R1 further contains 0.5-5% of a protein protective agent, and the capturing latex microspheres in the reagent R1 are immunosorbed microspheres. This reagent R1 can be used for the detection of oxidized low-density lipoprotein.

[0047] After the inventors found that strong electrolyte ions had adverse effects on the reagent containing capturing latex microspheres, a buffer system composed of a low ionic strength buffer material and a regulating material was used, which not only met the requirements of high buffer capacity and stable pH of the immunoturbidimetric method detection reagent, but also avoided the shortcomings of the existing buffer system, such as the influence of strong electrolyte ions on the dispersion stability of hydrophobic latex microspheres and the interference of the measured substance with the binding of the measured substance to the surface of the capturing latex microspheres. The immunoturbidimetric method detection reagent using a low ionic strength buffer system can obtain reliable detection results with good consistency with the reference method, and its open bottle stability, freeze-thaw stability, detection precision and sensitivity are superior to those of the existing conventional reagent containing a higher concentration of strong electrolyte ions.

[0048] In particular, for the detection of glycated hemoglobin and oxidized low-density lipoprotein, the detection kit of the present application has outstanding advantages. In the detection of glycated hemoglobin, the kit of the present application can improve the dispersion stability of sulfate group modified capturing latex microspheres in solution and improve the adsorption effect of glycated hemoglobin on the surface of the latex microspheres; in the detection of oxidized low-density lipoprotein, the kit of the present application can slow down the reaction rate of the latex microsphere captured oxidized low-density lipoprotein with free apolipoprotein B in the sample to be measured, so that the latex microsphere coupled anti-human apolipoprotein B antibody (non-4E6 epitope) and the captured oxidized low-density lipoprotein can more fully immunoreact, thereby forming effective turbidity and improving the detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Correlation of the results of the immuno-turbidimetry method using the glycosylated hemoglobin kit of the present application with the results of the reference method (HPLC);

[0050] Figure 2 Absolute deviation of the results of the immuno-turbidimetry method using the glycosylated hemoglobin kit of the present application from the results of the reference method (HPLC);

[0051] Figure 3 Correlation of the results of the immuno-turbidimetry method using the oxidized low density lipoprotein kit of the present application with the results of the reference method (ELISA);

[0052] Figure 4 Absolute deviation of the results of the immuno-turbidimetry method using the oxidized low density lipoprotein kit of the present application from the results of the reference method (ELISA). DETAILED DESCRIPTION

[0053] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will be combined with the accompanying drawings for the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0054] In this document, "and / or" includes any and all combinations of one or more of the associated items.

[0055] In this document, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0056] The "buffering substance" in the present application refers to a substance capable of maintaining the pH stability of a solution. The low ionic strength buffering substance refers to a substance that is a weak electrolyte, and in a solution, only part of the molecules are ionized, and the rest still exist in the form of molecules.

[0057] The "adjusting substance" in the present application refers to a substance that can adjust the pH of a solution after being added. In the present application, the adjusting substance and the buffering substance constitute a buffer system.

[0058] The chemical names and corresponding abbreviations of the buffering substances and adjusting substances of the present application are shown in Table 1.

[0059] Table 1 Chemical names and abbreviations of low ionic strength buffering substances

[0060] Abbreviations Compounds ADA N-(2-acetamido)iminodiacetic acid Bicine 2-Bis(2-hydroxyethyl)aminoethanesulfonic acid CAPS 3-(Cyclohexylamino)-1-propanesulfonic acid CHES 2-(N-Cyclohexylamino)ethanesulfonic acid EPPS 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid MES 2-(N-Morpholino)ethanesulfonic acid Mops 3-(N-Morpholino)propanesulfonic acid POPSO Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) TAPS Tris(hydroxymethyl)methylaminopropanesulfonic acid TEA Triethanolamine DEA Diethanolamine Tris Tris(hydroxymethyl)aminomethane

[0061] In the present application, the content or the amount of a functional substance in a detection reagent is generally expressed in molar concentration or mass-volume concentration or percentage content, which are common expressions in the art. Unless otherwise specified, the percentage content is generally approximately equivalent to the mass fraction or mass-volume percentage. For example, the content of a preservative in a reagent is 1 ‰, which represents that the amount of preservative added in 100 mL of the reagent is 0.1 g.

[0062] (I) Glycated hemoglobin detection kit and performance verification

[0063] Example 1

[0064] A glycated hemoglobin detection kit includes reagent R1 containing capture type latex microspheres and R2 coated with latex microsphere-coated monoclonal antibodies. The specific reagent formula and concentration range are as shown in Table 2.

[0065] Table 2 Reagent composition of Example 1

[0066] Reagent R1 Reagent R2 Bicine / TEA buffer system: 30 mM Buffer (Good's buffer): 50 mM Imidazole: 0.5 g / L Protein protectant (BSA): 0.5% Ethylenediaminetetraacetic acid: 0.2 g / L Preservative (PC-300): 1 ‰ Preservative (PC-950): 1 ‰ Inorganic salt (NaCl): 0.5% DMSO: 1.2% Latex microspheres coated monoclonal: 35 mg / L pH: 7.9 pH: 5.6 Solvent: ultrapure water Solvent: ultrapure water Capturing latex microspheres (particle size 100-150 nm): 0.1%

[0067] Note: The capacity of the buffer system in reagent R1 is calculated based on the concentration of buffer substance Bicine.

[0068] Each chemical is an analytical pure reagent commercially available. The capture type latex microspheres are from Beijing Bolmai Biotechnology Co., Ltd. (P2116). The latex microsphere-coated monoclonal antibodies are from Chongqing Aisens Biological Engineering Co., Ltd. (DAA130), wherein the monoclonal antibodies are anti-human HbA1c antibodies.

[0069] The preparation process of the kit is as follows:

[0070] (1) Reagent R1

[0071] A. Weigh an appropriate amount of ultrapure water, add the buffer system and stir until dissolved completely.

[0072] B. After all the chemicals are dissolved, adjust the pH to the required pH. In Example 1, the final concentration of TEA is 1.6 g / L.

[0073] C. Make up to the target volume to obtain R1 buffer.

[0074] D. Add the target concentration of capture type latex microspheres to the R1 buffer (the volume change is negligible), mix well to obtain reagent R1.

[0075] (2) Reagent R2

[0076] A. Weigh an appropriate amount of ultrapure water, add the buffer and stir until dissolved completely.

[0077] B. After all the chemicals are dissolved, adjust the pH to the required pH.

[0078] C, add water to the target volume to obtain R2 buffer solution.

[0079] D, add latex microsphere-coated monoclonal antibody to the buffer solution to obtain reagent R2.

[0080] The reagents Rl and R2 are separately packaged to obtain the kit. Optionally, the kit also provides a glycated hemoglobin calibration solution.

[0081] Example 2

[0082] A glycated hemoglobin detection kit includes reagent Rl containing capture latex microspheres and reagent R2 containing latex microsphere-coated monoclonal antibody. The specific reagent formulation and concentration range are shown in Table 3. The final concentration of TEA in reagent Rl is about 7.5 g / L.

[0083] Table 3 Reagent composition of Example 2

[0084]

[0085] Note: The buffer system capacity in reagent Rl is calculated based on the concentration of buffer substance Bicine.

[0086] The preparation method of the kit is the same as that of Example 1.

[0087] Comparative Examples 1-5

[0088] A glycated hemoglobin detection kit includes reagent Rl containing capture latex microspheres and reagent R2 containing latex microsphere-coated monoclonal antibody. The difference from the example is that NaOH is used instead of TEA. The buffer capacity of Comparative Examples 1-5 is calculated based on Bicine, which is 8 mM, 17 mM, 26 mM, 37 mM, and 48 mM, respectively. At this time, the concentration of NaOH in reagent Rl of each comparative example is 2 mM, 5 mM, 7 mM, 10 mM, and 13 mM, respectively. + The concentration of NaOH in reagent Rl of each comparative example is 2 mM, 5 mM, 7 mM, 10 mM, and 13 mM, respectively.

[0089] The specific reagent formulation and concentration range are shown in Table 4. The kit preparation process is referred to Example 1.

[0090] Table 4 Reagent composition of Comparative Examples

[0091]

[0092] Note: The buffer system capacity in reagent Rl is calculated based on the concentration of buffer substance Bicine.

[0093] Example 3

[0094] The performance of the detection reagent of the application was verified using the glycosylated hemoglobin kit of Example 1, while Comparative Examples 1-5 were detected as a comparison. The test samples were from Chongqing High-tech Medical Instrument Research Institute Co., Ltd.

[0095] Principle of detection: glycosylated hemoglobin in the sample can bind with the anti-glycosylated hemoglobin antibody in the reagent to form an antigen-antibody complex, producing a certain turbidity, and the turbidity is proportional to the content of the antigen. The turbidity is measured at a certain wavelength, and the glycosylated hemoglobin can be quantitatively determined by a multi-point calibration curve.

[0096] The glycosylated hemoglobin content calculation formula is

[0097] HbA1c(%) = CS x ΔAT / ΔAS(mg / L)

[0098] In the formula, HbA1c(%) is the glycosylated hemoglobin content in the sample, ΔAT is the absorbance value of the sample tube compared with the blank tube, ΔAS is the absorbance value of the calibration tube compared with the blank tube, and CS is the content of HbA1c in the calibration solution.

[0099] (1) Immune turbidimetry detection

[0100] The Beckman AU680 full-automatic biochemical analyzer was used for detection.

[0101] ① Parameter settings are as follows.

[0102] Method: end-point method;

[0103] Reaction direction: upward;

[0104] Sample: reagent R1: reagent R2 = 4: 150: 50;

[0105] Main wavelength: 660 nm;

[0106] Sub-wavelength: 800 nm;

[0107] Reaction temperature: 37°C;

[0108] Reaction time: 10 min.

[0109] ② Sample addition mode and determination scheme are shown in Table 5.

[0110] Table 5 Detection scheme of glycosylated hemoglobin by immune turbidimetry

[0111]

[0112] (3) Calculation: using multi-point non-linear / semi-log calibration mode, with spline function as calculation mode, according to the value of the calibrator and the absorbance change value, the dose / response curve is drawn, and the content of the target detection substance in the sample can be calculated according to the absorbance change value on the dose / response curve.

[0113] (2) High performance liquid chromatography (HPLC)

[0114] Since the HPLC method is the reference method for glycated hemoglobin, the HPLC method is used as a comparison in the experiment. The detection process is as follows:

[0115] When the sample enters the chromatographic column, different hemoglobin components have slight differences in the number of surface charges (net positive charge) due to their molecular structures (especially whether the N-terminal amino acid of the β chain is glycated). The positively charged hemoglobin molecules are attracted by the negatively charged chromatographic column filler (ion exchange). HbA1c has less net positive charge than non-glycated HbA0 because glucose attached to the valine at the N-terminal of the β chain masks a positive charge group (amino group).

[0116] During the gradient elution process, the ionic strength of the mobile phase gradually increases (or the pH gradually changes), competitively eluting the hemoglobin components bound to the filler. By precisely controlling the gradient and flow rate of the buffer, different hemoglobin components (including HbA1c) will flow out of the chromatographic column in a specific order. The components (eluate) flowing out of the chromatographic column enter the detector, which calculates the total hemoglobin amount and the glycated hemoglobin amount and reports the test results.

[0117] (3) Clinical relevance

[0118] As can be seen from Table 6, Figure 1 and Figure 2 the consistency of the glycated hemoglobin clinical test results of the present application with the HPLC detection results is good (R 2 ≥ 0.975).

[0119] Table 6 Comparison of immunoturbidimetry detection results with HPLC method

[0120]

[0121]

[0122] (4) Precision

[0123] Using the detection kit of the present application, different concentrations of natural samples were detected by immunoturbidimetry, and the results are shown in Table 7. The precision is good (CV < 2%) in the linear high (H), medium (M), and low (L) sections, as well as near the clinical reference range.

[0124] Table 7 Detection precision of samples in different concentration intervals

[0125]

[0126] (5) Freeze-thaw accelerated stability

[0127] To simulate the freeze-thaw phenomenon of the kit during transportation and user storage, the performance of the kit was studied through freeze-thaw accelerated stability experiments. The experimental process was as follows: the reagent was placed in a -20°C environment for 24 h to simulate the freezing condition, and then was transferred to a 2-8°C environment for 24 h. After repeating one to two times, the experiment was carried out.

[0128] The test results of the examples are shown in Table 8, respectively. In the table, RB1 and RB2 are the reading point data of the blank sample (water), and S1-S5 are the multiple calibration point data of the glycosylated hemoglobin calibration solution. After adjusting the buffer combination, Bicine-triethanolamine was used as the buffer system, and the stability of the reagent was enhanced. The deviation was less than 10% after 15 days of acceleration at 35°C, and the reaction rate deviation was less than 10% after two freeze-thaw cycles at -20°C.

[0129] Table 8 Test results of the kit of Example 1 after freeze-thaw and heat acceleration treatment

[0130]

[0131]

[0132] (6) Test results of the control examples

[0133] Table 9 Test results of the kits of Control Examples 1-5 using sodium hydroxide to adjust pH

[0134]

[0135] When using the Bicine-sodium hydroxide buffer system, as shown in Table 9, with the increase of the buffer concentration, in order to adjust to the same pH, the introduced sodium ions increased, and the reagent reaction rate decreased significantly, and the analytical sensitivity decreased.

[0136] (7) Open bottle stability

[0137] To simulate the storage state of the reagent in the full-automatic biochemical instrument after opening the bottle when used by the user, the test was carried out after opening the bottle for different times. The kits of Example 1 and Control Example 5 were used for the test, respectively.

[0138] From the results of Table 10, it can be seen that the Bicine-sodium hydroxide system is affected by carbon dioxide in the air after the bottle is opened for 7 days, and the measured value increases significantly. From the results of Table 11, it can be seen that after adjusting the buffer combination, Bicine-triethanolamine is used as the buffering material, and triethanolamine is added based on the 30 mM Bicine buffer, and there is no large change in the measured value after the bottle is opened for 14 days. Since the Bicine concentration in this embodiment is relatively low, the addition of triethanolamine can greatly increase the buffering capacity, and the ability of the reagent to resist carbon dioxide is enhanced. If the concentration of the base buffer used is > 100 mM, amino acids can be used to adjust the pH, and diethanolamine or triethanolamine does not need to be used.

[0139] Table 10 Test results of the control example 5 kit after opening and storing

[0140]

[0141] Table 11 Test results of the example 1 kit after opening and storing

[0142] Days (d) 0 2 3 6 9 14 17 22 30 L 3.75 3.68 3.62 3.65 3.66 3.73 3.83 3.71 3.71 L 3.75 3.63 3.64 3.66 3.70 3.75 3.79 3.68 3.68 H 8.36 8.24 8.23 8.22 8.27 8.39 8.55 8.33 8.38 H 8.34 8.20 8.23 8.24 8.34 8.43 8.57 8.35 8.35

[0143] Note: L and H represent low and high concentrations of the sample to be tested, respectively.

[0144] (II) Oxidized low-density lipoprotein detection kit and performance verification

[0145] Example 4

[0146] An oxidized low-density lipoprotein detection kit includes reagent R1 containing capture latex microspheres and R2 latex microsphere-coated monoclonal antibodies. The specific reagent formula and concentration range are as follows in Table 12.

[0147] Table 12 Reagent composition of example 4

[0148]

[0149]

[0150] Note: The capacity of the buffer system in reagent R1 is calculated based on the concentration of the buffering material MES.

[0151] Each chemical is a commercially available analytical pure reagent.

[0152] The preparation process of the kit is as follows:

[0153] (1) Preparation of reagent R1

[0154] A. Prepare 30 mM labeled buffer (including buffering material and preservative), which contains 30 mM MOPS and 1 ‰ PC-300.

[0155] B, Take the appropriate amount of labeling buffer, add the appropriate amount of microspheres (Beijing Bolmai Biotechnology Co., Ltd., P0117) to the labeling buffer, and stir evenly.

[0156] C, Continue to add the appropriate amount of anti-human oxidized low-density lipoprotein antibody (4E6 epitope) monoclonal antibody (Chongqing Aisens Biological Engineering Co., Ltd., DCO351), and stir evenly.

[0157] D, Slowly add EDC activator (1%, 0.4ul / mL, prepared fresh), continue to stir during the addition process, and react for 8-16h.

[0158] E, Add blocking agent (including BSA, PC-300, etc.), and block for 1h.

[0159] F, Centrifuge, remove the supernatant, and resuspend the latex microspheres in the preservation solution; the preservation solution contains 200mM MES, lysine to adjust the target pH, 3% BSA, and 1‰ PC-300.

[0160] G, Transfer the resuspended reagent into a 37℃ oven, and age for 48h to obtain reagent R1.

[0161] (2) Preparation of reagent R2

[0162] A, Prepare 50mM labeling buffer (including buffer and preservative). The labeling buffer is 50mM phosphate buffer (PBS) with 1‰ PC-300 added.

[0163] B, Take the appropriate amount of labeling buffer, add the appropriate amount of microspheres to the labeling buffer, and stir evenly.

[0164] C, Continue to add the appropriate amount of anti-human apolipoprotein B antibody (non-4E6 epitope) monoclonal antibody (Chongqing Aisens Biological Engineering Co., Ltd., DCO475), and stir evenly.

[0165] D, Slowly add EDC activator (1%, 0.4ul / mL, prepared fresh), continue to stir during the addition process, and react for 8-16h.

[0166] E, Add blocking agent (including BSA, PC-300, etc.), and block for 1h.

[0167] F, Centrifuge, remove the supernatant, and resuspend the latex microspheres in the preservation solution; the preservation solution contains 200mM MES, lysine to adjust the target pH, 3% BSA, and 1‰ PC-300.

[0168] G, Transfer the resuspended reagent into a 37℃ oven, and age for 48h to obtain reagent R2.

[0169] (3) The reagents R1 and R2 are packaged to obtain a kit. Alternatively, the kit further comprises an oxidized low density protein calibration solution.

[0170] Control Example 6

[0171] An oxidized low density lipoprotein detection kit, which is different from Example 4 in that NaOH is used instead of lysine, and the rest of the components remain unchanged, and the buffer capacity is still 200 mM in terms of MES.

[0172] The kit preparation process is referred to Example 4.

[0173] Control Example 7

[0174] An oxidized low density lipoprotein detection kit, which is different from Example 4 in that 2 g / L NaCl is additionally added to reagent R1 to confirm the equivalent effect of sodium ions. The rest of the components remain unchanged, and the buffer capacity is still 200 mM in terms of MES.

[0175] The kit preparation process is referred to Example 4.

[0176] Example 5

[0177] The performance of the detection reagent of the present application is verified using the oxidized low density lipoprotein detection kit of Example 4. The test sample is from Chongqing Gaoke Medical Instrument Research Institute Co., Ltd.

[0178] Detection principle: oxidized low density lipoprotein in the sample can bind with anti-oxidized low density lipoprotein monoclonal antibody in reagent R2 to form an antigen-antibody complex, producing a certain turbidity, and the turbidity is proportional to the content of the antigen. The turbidity is measured at a certain wavelength, and the quantitative determination of oxidized low density lipoprotein can be carried out through a multi-point calibration curve. The content of oxidized low density lipoprotein is calculated using the following formula:

[0179] ox-LDL (%) = CS x ΔAT / ΔAS (mg / L)

[0180] In the formula, ox-LDL (%) is the content of oxidized low density lipoprotein, ΔAT is the absorbance value of the sample tube compared with the absorbance of the blank tube, ΔAS is the absorbance value of the calibration tube compared with the absorbance of the blank tube, and CS is the content of ox-LDL in the calibration solution.

[0181] (1) Immunoturbidimetry

[0182] A Beckman AU680 full-automatic biochemical analyzer is used for detection.

[0183] ① The program parameter settings are as follows.

[0184] Method: end-point method;

[0185] Reaction direction: up;

[0186] Sample: reagent R1: reagent R2 = 8:120:40;

[0187] Main wavelength: 540nm;

[0188] Sub-wavelength: 800nm;

[0189] Reaction temperature: 37℃;

[0190] Reaction time: 10min.

[0191] ②Sample adding mode and determination scheme as shown in Table 13.

[0192] Table 13 Detection scheme of immunoturbidimetry for detecting oxidized low density lipoprotein

[0193]

[0194] ③Calculation: using multi-point nonlinear / half-log calibration mode, with spline function as the calculation mode, the dose / response curve is made according to the value of the calibration sample and the absorbance change value, and the content of the target detection substance in the sample can be calculated according to the absorbance change value on the dose / response curve.

[0195] (2) Enzyme-linked immunoassay (ELISA)

[0196] Since most of the commercial oxidized low density lipoprotein detection kits are ELISA method, the ELISA method is used as a comparison in the experiment. Detection method: first, the capture antibody specific to the epitope of ox-LDL is pre-fixed on the bottom surface of the microwell plate, and the ox-LDL in the sample will specifically bind to the capture antibody fixed on the bottom of the hole, so as to be fixed on the bottom of the hole.

[0197] (3) Clinical relevance

[0198] From Table 14, Figure 3 and Figure 4 It can be seen that the clinical detection results of the oxidized low density lipoprotein of the present application are consistent with the ELISA detection results (R 2 ≥0.975).

[0199] Table 14 Comparison of detection results of immunoturbidimetry and ELISA method

[0200]

[0201]

[0202] (4) Precision

[0203] The test kit of the present application was used to detect test samples of different concentrations (Chongqing Hi-Tech Medical Laboratory Co., Ltd.) by immunoturbidimetry, and the results are shown in Table 15. The linear high (H), medium (M) and low (L) precision was good (CV < 3%).

[0204] Table 15 Detection precision of samples of different concentration intervals

[0205]

[0206] (5) Analysis sensitivity

[0207] The kits of Example 4 and Comparative Examples 6 and 7 were used for detection, respectively. The test samples were from Chongqing Hi-Tech Medical Instrument Research Institute Co., Ltd.

[0208] Table 16 Comparison of detection sensitivity of different kits

[0209]

[0210]

[0211] As shown in Table 16, by comparing Example 4 with Comparative Example 6, the NaOH concentration in the reagent of Comparative Example 6 was about 34 mM, and the use of lysine instead of sodium hydroxide reduced the sodium ions in the solution, and the reagent reaction was significantly improved; in order to maintain the same Na + concentration as in Comparative Example 6, Comparative Example 7 added 2 g / L sodium chloride to the MES-lysine buffer of Example 4, i.e. the concentration of Na + , Cl - and other strong electrolyte ions in the solution was 34 mM, and the reagent reaction of Comparative Example 7 was significantly decreased, indicating that lysine instead of sodium hydroxide can effectively reduce the ionic strength of the solution, thereby helping to improve the detection sensitivity.

Claims

1. Use of a low ionic strength buffer substance in the preparation of a latex microsphere immunoturbidimetric assay, wherein the total concentration of strong electrolyte ions in the assay is 0-10 mmol / L, and wherein the strong electrolyte ions comprise at least one of metal ions, ammonium ions or strong acid ions.

2. Use according to claim 1, characterized in that: The assay comprises at least one buffer substance selected from at least one of Bicine, ADA, EPPS, POPSO, MES, CHES, CAPS, Hepes, Mops, TAPS, DEA, TEA, Tris and at least one adjusting substance selected from TEA, DEA, lysine, arginine, histidine, aspartic acid, glutamic acid, and wherein the adjusting substance is not identical to the buffer substance.

3. Use according to claim 1, characterized in that: The assay comprises capture latex microspheres for binding of a substance to be detected in a sample, and the assay provides a liquid environment for the binding of the substance to be detected on the capture latex microspheres.

4. Use according to claim 1 or 2 or 3, characterized in that: The assay is used in the preparation of a glycated hemoglobin assay kit or in the preparation of an oxidized low density lipoprotein assay kit.

5. A latex microsphere immunoturbidimetry test reagent, characterized by: The assay is an aqueous solution comprising a buffer substance, and wherein the total concentration of strong electrolyte ions in the assay is not more than 10 mmol / L; preferably not more than 5 mmol / L; more preferably not more than 2.5 mmol / L.

6. The test reagent according to claim 5, characterized in that: The buffer substance is selected from at least one of Bicine, ADA, EPPS, POPSO, MES, CHES, CAPS, Hepes, Mops, TAPS, DEA, TEA, Tris; The assay further comprises an adjusting substance selected from at least one of TEA, DEA, lysine, arginine, histidine, aspartic acid, glutamic acid, and wherein the adjusting substance is not identical to the buffer substance.

7. The detection reagent according to claim 5 or 6, characterized in that: The strong electrolyte ions are substances comprising at least one of alkali metal ions, alkaline earth metal ions, ammonium ions, halogen ions, sulfate ions, bisulfate ions, nitrate ions, dihydrogen phosphate ions, hydrogen phosphate ions, phosphate ions.

8. The test reagent according to any one of claims 5 to 7, characterized in that: The assay further comprises at least one of a chelating agent, a surfactant, a freeze-thaw stabilizer, a protein protective agent, a preservative.

9. The test reagent according to any one of claims 5 to 7, characterized by: The concentration of the buffer substance in the assay is 30-300 mM.

10. A kit comprising the assay of any one of claims 5-9 as reagent Rl, and further comprising reagent R2, wherein: The reagent Rl comprises: a buffer system consisting of a buffer substance and an adjusting substance, wherein the concentration of the buffer substance is 30-300 mM; a preservative in a mass / volume content of 1-3 ‰; capture latex microspheres in a mass / volume content of 0.1-0.2%; a solvent being ultrapure water; The reagent R2 comprises: a buffer in a concentration of 50-300 mM; a protein protective agent in a mass fraction of 0.5-2%; a preservative in a mass / volume content of 1-3 ‰; an inorganic salt in a mass / volume content of 0.5-2%; latex microsphere coupled antibodies in a content of 35-55 mg / L; a solvent being ultrapure water.

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