Insulin homogeneous detection kit and detection method

Abstract

The present invention relates to the field of analytical detection technology, and in particular to an insulin homogeneous detection kit and a detection method. The kit includes an R1 buffer and an R2 buffer; the R1 buffer includes an insulin-specific antibody, tris(hydroxymethylaminomethane), bovine serum albumin, glucose and an oxidized coenzyme; the R2 buffer includes tris(hydroxymethylaminomethane), bovine serum albumin and a glucose dehydrogenase-insulin conjugate. The insulin homogeneous detection method and kit of the present invention have the advantages of simple and rapid operation, accurate test results, high sensitivity, good repeatability, and low cost, and have a good prospect for large-scale clinical promotion and application in the future, especially for small and medium-sized hospitals lacking expensive machines.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to an insulin homogeneous detection kit and a detection method. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar. Various different causes of diabetes cause insufficient insulin secretion by pancreatic β cells and / or insufficient insulin action in peripheral tissues, leading to elevated blood sugar. Currently, diabetes has become the world's third largest health enemy after tumors and cardiovascular and cerebrovascular diseases, and the progression of the disease continues to accelerate.

[0003] Insulin is a small globular protein with a molecular weight of 5.7×10 6 D, isoelectric point pI is 5.3, soluble in dilute acid solution, heat stable under neutral conditions, not alkali-resistant, and inactivated after oxidation or reduction. Insulin in human plasma exists mainly in two forms: one part is free, and the other part is bound to plasma globulin. Under normal physiological conditions, pancreatic β cells secrete an appropriate amount of insulin into the blood circulation every day to regulate sugar, protein and fat metabolism. The most important physiological function of insulin is to lower blood sugar concentration, promote tissue glycogen synthesis and glucose oxidation, and regulate the body's energy supply. When the glucose content in the body increases, insulin biosynthesis and release accelerate. The glucose concentration in the blood is a signal to stimulate the excitement of pancreatic β cells and promote insulin synthesis.

[0004] At present, the main methods for determining insulin are bioassay, immunoassay and instrumental analysis. Bioassay requires more animals, constant temperature conditions, cumbersome data processing and long time. Instrumental analysis mainly includes spectrophotometry, electrophoresis analysis and high performance liquid chromatography. The instruments and equipment required for instrumental analysis are expensive, the operation is cumbersome and time-consuming, and it is not suitable for clinical batch processing. Immunological assays mainly include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, etc. Radioimmunoassay is radioactive, cumbersome to operate, time-consuming, and the reagent stability period is short, which limits its clinical application. Enzyme-linked immunosorbent assay is suitable for batch processing, but the repeatability is poor, and the processing time of a single sample is long, which limits its application. Chemiluminescence immunoassay has good repeatability and is suitable for batch processing. The processing time of a single sample is moderate, but because a separate chemiluminescence device is required and the equipment price is relatively expensive, the promotion of this technology is limited.

[0005] Therefore, how to provide a method for detecting insulin with low detection cost and simple operation steps has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] First, the present invention provides an insulin homogeneous detection kit, comprising: an R1 buffer and an R2 buffer; the R1 buffer comprises an insulin-specific antibody, tris(hydroxymethylaminomethane) (Tris), bovine serum albumin (BSA), glucose and oxidized coenzyme; the R2 buffer comprises Tris, BSA and a glucose dehydrogenase-insulin conjugate.

[0007] Preferably, in the R1 buffer, the concentration of insulin-specific antibody is 0.001-200 mg / L, the concentration of Tris is 5-500 mmol / L, the concentration of BSA is 0.05-5 g / L, the concentration of glucose is 0.5-1000 mmol / L, and the concentration of oxidized coenzyme is 0.1-100 mmol / L.

[0008] More preferably, in the R1 buffer, the concentration of insulin-specific antibody is 1-5 mg / L, the concentration of Tris is 30-80 mmol / L, the concentration of BSA is 0.5-1.5 g / L, the concentration of glucose is 20-80 mmol / L, and the concentration of oxidized coenzyme is 5-15 mmol / L.

[0009] Preferably, in the R2 buffer, the concentration of Tris is 5-500 mmol / L, the concentration of BSA is 0.05-5 g / L; and the concentration of glucose dehydrogenase-insulin conjugate is 0.05-5 mg / L.

[0010] More preferably, in the R2 buffer, the concentration of Tris is 30-80 mmol / L, the concentration of BSA is 0.5-1.5 g / L; and the concentration of glucose dehydrogenase-insulin conjugate is 1-5 mg / L.

[0011] Preferably, the oxidized coenzyme is oxidized coenzyme I (NAD + ) or oxidized coenzyme II (NADP + ).

[0012] Preferably, the glucose dehydrogenase-insulin conjugate is obtained by dissolving insulin in an organic solvent, activating it, performing a cross-linking reaction with a glucose dehydrogenase solution, and purifying and dialyzing it.

[0013] More preferably, the preparation method of the glucose dehydrogenase-insulin conjugate comprises: dissolving glucose dehydrogenase in Tris buffer at room temperature, wherein the final concentration of glucose dehydrogenase is 0.01-100 mg / mL (preferably 2-6 mg / mL), and the final concentration of Tris is 5-1000 mmol / L (preferably 10-100 mmol / L, more preferably 10-20 mmol / L), to prepare a glucose dehydrogenase solution; dissolving insulin in an organic solvent so that the final concentration of insulin is 0.01-100 mg / mL (preferably 2-6 mg / mL), performing a cross-linking reaction with the glucose dehydrogenase solution after activation, and obtaining the conjugate after purification and dialysis.

[0014] Preferably, the activation reagent comprises EDC, EDC / NHS, glutaraldehyde, sodium periodate or sodium borohydride.

[0015] Preferably, the reagent used for the activation is EDC / NHS.

[0016] Preferably, the organic solvent includes at least one of methanol, ethanol, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) (preferably DMF).

[0017] In a specific implementation process, the insulin-specific antibody can be a commonly purchased commercial antibody.

[0018] Furthermore, the present invention provides a method for detecting insulin using the above-mentioned kit, comprising: adding a sample to a biochemical analyzer, then adding R1 buffer and R2 buffer in sequence, measuring the 340nm absorbance value at different time points, obtaining the sample reaction rate value, and then obtaining the insulin concentration value in the sample according to the calibration curve.

[0019] Preferably, the sample is serum, plasma or whole blood.

[0020] The detection method of the present invention is a homogeneous detection method, which is a competitive reaction. The insulin bound to the antibody and the free insulin in the reaction system do not need to be separated. The basic reaction principle is as follows:

[0021] The insulin in the sample competes with the insulin coupled to the glucose dehydrogenase for the binding site of the insulin-specific antibody. The insulin in the liquid sample competitively displaces the glucose dehydrogenase-insulin conjugate bound to the insulin-specific antibody and releases it from the binding site of the insulin-specific antibody, thereby restoring the activity of the enzyme. Therefore, the higher the insulin concentration in the sample, the more free glucose dehydrogenase-insulin conjugate there is, and thus the OD 340nmThe faster the reaction rate, the stronger the signal. Therefore, the insulin concentration in the sample can be quantified by making a calibration curve based on the relationship between the reaction rate and the concentration of the calibrator in the calibration curve.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The insulin homogeneous detection method and kit of the present invention have the advantages of simple and rapid operation, accurate detection results, high sensitivity, good repeatability, low cost, etc., and have good prospects for large-scale clinical promotion and application in the future, especially for small and medium-sized hospitals lacking expensive machines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a calibration curve comparison chart. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0027] Example 1

[0028] This embodiment provides an insulin homogeneous detection kit, comprising: R1 buffer and R2 buffer; the R1 buffer comprises insulin-specific antibody (purchased from CalBioreagents, Inc.; item number: M483; source: mouse; concentration: 2 mg / L), Tris, BSA, glucose and oxidized coenzyme I, and the solvent is water; the R2 buffer comprises Tris, BSA and glucose dehydrogenase-insulin conjugate, and the solvent is water.

[0029] The preparation method of glucose dehydrogenase-insulin conjugate is as follows:

[0030] Glucose dehydrogenase was dissolved in Tris buffer at room temperature, the final concentration of glucose dehydrogenase was 4 mg / mL), the final concentration of Tris was 10 mmol / L, and glucose dehydrogenase solution was prepared; insulin was dissolved in organic solvent DMF, so that the final concentration of insulin was 4 mg / mL, and after EDC / NHS activation, it was cross-linked with the glucose dehydrogenase solution, and after purification and dialysis, a glucose dehydrogenase-insulin conjugate with a final concentration of 1 mg / mL was obtained.

[0031] In R1 buffer, the concentration of insulin-specific antibody was 2 mg / L, the concentration of Tris was 50 mmol / L, the concentration of BSA was 1.0 g / L, the concentration of glucose was 50 mmol / L, and the concentration of oxidized coenzyme I was 10 mmol / L;

[0032] In R2 buffer, the concentration of Tris was 50 mmol / L, the concentration of BSA was 1.0 g / L; the final concentration of glucose dehydrogenase-insulin conjugate was 3 mg / L.

[0033] Example 2

[0034] This embodiment provides an insulin homogeneous detection kit, the components of which are different from those of Example 1 only in that during the preparation of the glucose dehydrogenase-insulin conjugate, glutaraldehyde is used to cross-link glucose dehydrogenase and insulin to obtain the glucose dehydrogenase-insulin conjugate.

[0035] Example 3

[0036] This embodiment provides an insulin homogeneous detection kit, the components of which are different from those of Example 1 only in that during the preparation of the glucose dehydrogenase-insulin conjugate, sodium periodate is used to cross-link glucose dehydrogenase and insulin to obtain the glucose dehydrogenase-insulin conjugate.

[0037] Comparative Example 1

[0038] This comparative example provides an insulin homogeneous detection kit, the components of which are different from those of Example 1 only in that the Tris in the R1 buffer and the R2 buffer is replaced with HEPES of the same concentration.

[0039] Comparative Example 2

[0040] This comparative example provides an insulin homogeneous detection kit, the components of which are different from those of Example 1 only in that the BSA in the R1 buffer and the R2 buffer is replaced with casein of the same concentration.

[0041] Test Example 1

[0042] This test example uses the insulin homogeneous detection kits in the above embodiment and comparative example to detect insulin in the same serum sample, and the steps are as follows:

[0043] (1) Using a Hitachi 7180 biochemical analyzer, add the sample into the sample chamber, put R1 buffer and R2 buffer into the reagent chamber, and set the instrument parameters according to Table 1.

[0044] Table 1 Instrument parameters

[0045]

[0046] (2) Prepare calibrants of known concentration, measure the absorbance at 340 nm at different time points, calculate the reaction rate values ​​of calibrants of different concentrations, and draw a calibration curve of absorbance reaction rate and calibrant concentration.

[0047] (3) Determine the reaction rate value of the sample and determine the insulin concentration in the sample based on the calibration curve.

[0048] The calibration data of different embodiments and comparative examples are shown in Table 2. Figure 1 shown.

[0049] Table 2 Calibration data

[0050]

[0051] Test Example 2

[0052] This test example detects the yield of glucose dehydrogenase-insulin conjugates obtained by different cross-linking methods in the above different embodiments, and the specific method is as follows:

[0053] The enzymatic activity of glucose dehydrogenase before cross-linking and the enzymatic activity of glucose dehydrogenase-insulin conjugate after cross-linking were tested according to the recognized glucose dehydrogenase enzymatic activity test method. The ratio of the enzymatic activity of glucose dehydrogenase-insulin conjugate after cross-linking to the enzymatic activity of glucose dehydrogenase before cross-linking was calculated as the yield. The yield calculation formula is as follows:

[0054]

[0055] For example, in Example 1, the specific enzyme activity of 4 mg / mL glucose dehydrogenase before cross-linking is 150 U / ml, the amount used during cross-linking is 1 ml, the volume of glucose dehydrogenase-insulin conjugate after cross-linking is 2.3 ml, and the specific enzyme activity is 60 U / ml, then the calculated yield is 92%.

[0056] The test results are shown in Table 3.

[0057] Table 3 Yields of glucose dehydrogenase-insulin conjugates with different cross-linking methods

[0058]

[0059] It can be seen that the yield of the product is the highest when glucose dehydrogenase-insulin conjugate is prepared using the EDC / NHS method.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An insulin homogeneous detection kit, characterized in that: include: R1 buffer and R2 buffer; the R1 buffer comprises insulin-specific antibody, tris(hydroxymethylaminomethane), bovine serum albumin, glucose and oxidized coenzyme I; the R2 buffer comprises tris(hydroxymethylaminomethane), bovine serum albumin and glucose dehydrogenase-insulin conjugate; The glucose dehydrogenase-insulin conjugate is obtained by dissolving insulin in an organic solvent, performing a cross-linking reaction with a glucose dehydrogenase solution after activation, and then purifying and dialyzing; the reagent used for the activation is EDC / NHS; In the R1 buffer, the concentration of insulin-specific antibody is 1-5 mg / L, the concentration of tris(hydroxymethyl)aminomethane is 30-80 mmol / L, the concentration of bovine serum albumin is 0.5-1.5 g / L, the concentration of glucose is 20-80 mmol / L, and the concentration of oxidized coenzyme I is 5-15 mmol / L; In the R2 buffer, the concentration of tris(hydroxymethyl)aminomethane is 30-80 mmol / L, the concentration of bovine serum albumin is 0.5-1.5 g / L, and the concentration of glucose dehydrogenase-insulin conjugate is 1-5 mg / L.

2. The kit according to claim 1, characterized in that The organic solvent includes at least one of methanol, ethanol, N,N-dimethylformamide and dimethyl sulfoxide.

3. A method for detecting insulin using the kit according to claim 1 or 2, characterized in that: include: Add the sample to the biochemical analyzer, then add R1 buffer and R2 buffer in sequence, measure the 340nm absorbance value at different time points, obtain the sample reaction rate value, and then obtain the insulin concentration value in the sample according to the calibration curve; the method is for non-disease diagnosis purposes.

4. The method according to claim 3, characterized in that The sample is serum, plasma or whole blood.

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