Magnetic particle chemiluminescence detection kit for insulin and preparation method thereof

Abstract

The invention relates to the technical field of kits, in particular to a magnetic particle chemiluminiscence detection kit for insulin and a preparation method of the magnetic particle chemiluminiscence detection kit for insulin, specifically, a reagent M, a reagent R2 and a reagent R1 are used for forming the kit, and the reagent M is a magnetic particle working solution containing streptavidin magnetic beads; the reagent R2 is an enzyme-labeled working solution containing an insulin antibody labeled by alkaline phosphatase; the reagent R1 is a working solution containing a biotin-labeled insulin monoclonal antibody. Through verification, according to the chemiluminescence kit for insulin provided by the invention, the test sensitivity reaches 2.00 mu IU / mL, the test repeatability for high and low value samples is less than 3%, and the linear correlation with Rogowski reaches 0.98 within the concentration range of 2.00-450 mu IU / mL. The invention also provides a preparation method of the insulin chemiluminescence kit, the production cycle is greatly shortened, the batch difference is easy to control, and the production work of enterprises is facilitated.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, specifically to a magnetic microparticle chemiluminescence detection kit for insulin and its preparation method. Background Technology

[0002] Insulin (INS) is a hormone secreted by the beta cells of the pancreas, composed of 51 amino acids. Insulin regulates glucose uptake and utilization, and also participates in the regulation of protein synthesis and triglyceride storage. An increase in circulating glucose stimulates insulin secretion. In turn, insulin stimulates tissue uptake of glucose while inhibiting glycogenolysis in the liver. Diabetes mellitus is caused by an absolute or relative deficiency of insulin secretion, leading to impaired glucose utilization by tissues and resulting in elevated blood glucose levels. Diabetes can lead to serious complications such as kidney failure, heart disease, nerve damage, blindness, and gangrene. Severe hyperglycemic episodes can also cause ketoacidosis and coma. Early diabetes is characterized only by a decreased insulin response to glucose stimulation; measuring insulin levels at basal levels or after glucose treatment helps diagnose early diabetes and assess the pancreas's ability to secrete insulin.

[0003] Currently, INS detection kits on the market are divided into radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay. RIA is highly contaminated and has low sensitivity; ELISA is greatly affected by individual factors, has low automation, and long detection time, which is not conducive to increasingly demanding testing requirements and fully automated detection. Chemiluminescent immunoassay for INS detection is currently dominated by imported products from Roche, which are expensive and have long procurement cycles. Domestic research and development of INS detection kits, such as the Chinese patent application CN106596524A, uses carboxylated magnetic nanobeads, which have poor dispersibility and are prone to aggregation, leading to high luminescence intensity and non-specific antibody adsorption, resulting in reduced specific capture ability. The Chinese patent application CN110146692A amplifies the detection signal and improves detection sensitivity by introducing a streptavidin-biotin system, but this also results in a long manufacturing cycle and is prone to batch-to-batch variations. Summary of the Invention

[0004] The purpose of this invention is to provide a magnetic microparticle chemiluminescence detection kit for insulin and its preparation method, aiming to solve the technical problems of insufficient detection sensitivity, long production cycle, and batch-to-batch variation in existing insulin detection kits.

[0005] To achieve the above objectives, the present invention provides a magnetic microparticle chemiluminescence detection kit for insulin, comprising three parts: reagent M, reagent R2, and reagent R1. Reagent M is a magnetic microparticle working solution containing streptavidin magnetic beads; reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody; and reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibody.

[0006] The reagent M comprises 0.01–0.1 mol / L PBS buffer, 0.1 g / L–10 g / L protein stabilizer, 5–25 g / L carbohydrate molecules, 0.5 g / L–5 g / L nonionic surfactant, 0.5 g / L–1 g / L preservative, and 0.1 mg / mL–0.5 mg / mL inhibitor and streptavidin magnetic beads.

[0007] The reagent R2 consists of MES buffer (0.05–0.1 mol / L), zinc chloride, magnesium chloride, protein stabilizer (0.1 g / L–10 g / L), nonionic surfactant (0.5 g / L–5 g / L), preservative (0.5 g / L–1 g / L), and alkaline phosphatase-labeled insulin antibody (0.5 μg / mL–2 μg / mL).

[0008] The reagent R1 comprises PBS buffer, protein stabilizer 0.1 g / L to 10 g / L, carbohydrate molecules 5 to 25 g / L, nonionic surfactant 0.5 g / L to 5 g / L, preservative 0.5 g / L to 1 g / L, and biotin-labeled insulin monoclonal antibody 0.5 μg / mL to 2 μg / mL.

[0009] The PBS buffer has a pH value in the range of 7.0±8.0 and contains 0.1 to 0.2 mol / L sodium chloride; the MES buffer has a pH value in the range of 6.0±7.0 and contains 0.1 to 0.2 mol / L sodium chloride.

[0010] This invention also proposes a method for preparing a magnetic microparticle chemiluminescence detection kit for insulin, comprising the following steps:

[0011] Step 1: Prepare reagent M;

[0012] Step 2: Prepare reagent R2;

[0013] Step 3: Prepare reagent R1.

[0014] Optionally, the execution process of step 1 includes the following steps:

[0015] Step 1.1: Take out the magnetic bead stock solution at a ratio of 0.1 mg / mL to 0.5 mg / mL for streptavidin magnetic beads;

[0016] Step 1.2: Perform a liquid exchange pretreatment operation on the extracted magnetic bead stock solution. Take 3 times the volume of the magnetic bead stock solution of washing buffer B according to the calculation, add it to the magnetic microparticles after the supernatant is discarded in the magnetic separator, and place it on a mixer to mix for 30 min to 1 h.

[0017] Step 1.3: Take 3 times the volume of the original magnetic bead solution of the washing buffer B according to the calculation, add it to the magnetic microparticles after pretreatment and discarding the supernatant, wash 3 times, place it on the magnetic separator and wait for the liquid to become clear and transparent before discarding the supernatant.

[0018] Step 1.4: Add the magnetic microparticle working solution Buffer A to the magnetic microparticles after discarding the supernatant, adjust the volume to the target concentration, and add 10-100 μg / mL of the blocking agent specifically interfering with and eliminating protein to obtain reagent M.

[0019] Optionally, the magnetic microparticle working solution Buffer A in step 1 is a PBS buffer with pH 7.40 ± 0.05, with the addition of 0.15 mol NaCl, casein 5 g / L, bovine serum albumin 1 g / L, mannitol 20 g / L, 4-aminopyrine 0.1 g / L, biotin 0.01 g / L, Tween-20 1 g / L, TX-100 0.5 g / L, and PC-300 0.5 g / L.

[0020] Washing buffer B is a PBS buffer with pH 7.40 ± 0.05, with the following added: 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 10 g / L sucrose, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.2 g / L PC-300.

[0021] All buffers were prepared and then filtered through a 0.22 μm nylon filter membrane.

[0022] Optionally, the execution process of step 2 includes the following steps:

[0023] Step 2.1: Dissolve and dilute the heteroterminated bifunctional cross-linking agent with dimethyl sulfoxide, and react it with the antigen derivative diluted with buffer 1 at a molar ratio of 1:10 to 1:40 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours.

[0024] Step 2.2: Dissolve and dilute the thiol-modified crosslinking agent with buffer 2, and react it with alkaline phosphatase diluted with buffer 2 at a molar ratio of 1:10 to 1:30 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours.

[0025] Step 2.3: The activated antigen derivative and ALP modified with thiol groups are reacted at a mass ratio of 1:0.5 to 1:2 in the dark at a temperature of 20 to 37°C for 1 to 3 hours to obtain alkaline phosphatase-labeled insulin antibody.

[0026] Step 2.4: Purify the alkaline phosphatase-labeled insulin antibody obtained above using buffer 1;

[0027] Step 2.5 Prepare enzyme-labeled working solution using MES buffer (0.05–0.1 mol / L), zinc chloride, magnesium chloride, protein stabilizer (0.1 g / L–10 g / L), nonionic surfactant (0.5 g / L–5 g / L), preservative (0.5 g / L–1 g / L), and purified alkaline phosphatase-labeled insulin antibody (0.5 μg / mL–2 μg / mL).

[0028] Optionally, in step 2, buffer 1 consists of 10 mM phosphate buffer, 9 g / L sodium chloride, and a pH value in the range of 7.40 ± 0.05; buffer 2 consists of 100 mM phosphate buffer, 9 g / L sodium chloride, 10 mM disodium ethylenediaminetetraacetate, and a pH value in the range of 8.00 ± 0.05.

[0029] Optionally, the execution process of step 3 includes the following steps:

[0030] Step 3.1: Take out the antibody at a ratio of 0.5 μg / mL to 2 μg / mL of biotin-labeled insulin monoclonal antibody;

[0031] Step 3.2: Add the biotin-labeled insulin monoclonal antibody to working solution Buffer A to prepare to the target concentration;

[0032] The working solution Buffer A is a PBS buffer with pH 7.40 ± 0.05, containing 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 20 g / L mannitol, 0.1 g / L 4-aminopyrine, 0.01 g / L biotin, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.5 g / L PC-300.

[0033] This invention provides a magnetic microparticle chemiluminescence detection kit for insulin and its preparation method. Specifically, the kit consists of three parts: reagent M, reagent R2, and reagent R1. Reagent M is a working solution containing streptavidin magnetic beads; reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody; and reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibody. Verification shows that the chemiluminescence detection kit for insulin provided by this invention achieves a detection sensitivity of 2.00 μIU / mL, with repeatability of <3% for both high and low value samples. Within a concentration range of 2.00–450 μIU / mL, the linear correlation with Roche is 0.98. This invention also provides a preparation method for the chemiluminescence detection kit for insulin, significantly shortening the production cycle, facilitating batch-to-batch variation control, and benefiting enterprises in their production operations. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a specific embodiment of the present invention, which compares reagent kit A with Roche's clinical testing and evaluation.

[0036] Figure 2 This is a specific embodiment of the present invention, showing reagent kit B and Roche's clinical test evaluation.

[0037] Figure 3 This is a specific embodiment of the present invention, showing the kit C and Roche's clinical testing and evaluation. Detailed Implementation

[0038] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0039] Please see Figure 1 This invention provides a magnetic microparticle chemiluminescence detection kit for insulin, comprising three parts: reagent M, reagent R2, and reagent R1. Reagent M is a magnetic microparticle working solution containing streptavidin magnetic beads; reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody; and reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibody.

[0040] The reagent M comprises 0.01–0.1 mol / L PBS buffer, 0.1 g / L–10 g / L protein stabilizer, 5–25 g / L carbohydrate molecules, 0.5 g / L–5 g / L nonionic surfactant, 0.5 g / L–1 g / L preservative, and 0.1 mg / mL–0.5 mg / mL inhibitor and streptavidin magnetic beads.

[0041] The reagent R2 consists of MES buffer (0.05–0.1 mol / L), zinc chloride, magnesium chloride, protein stabilizer (0.1 g / L–10 g / L), nonionic surfactant (0.5 g / L–5 g / L), preservative (0.5 g / L–1 g / L), and alkaline phosphatase-labeled insulin antibody (0.5 μg / mL–2 μg / mL).

[0042] The reagent R1 comprises PBS buffer, protein stabilizer 0.1 g / L to 10 g / L, carbohydrate molecules 5 to 25 g / L, nonionic surfactant 0.5 g / L to 5 g / L, preservative 0.5 g / L to 1 g / L, and biotin-labeled insulin monoclonal antibody 0.5 μg / mL to 2 μg / mL.

[0043] The PBS buffer has a pH range of 7.0±8.0 and contains 0.1 to 0.2 mol / L sodium chloride; the MES buffer has a pH range of 6.0±7.0 and contains 0.1 to 0.2 mol / L sodium chloride.

[0044] Furthermore, the present invention also proposes a method for preparing an insulin magnetic particle chemiluminescence detection kit, which includes the following steps:

[0045] Step 1: Prepare reagent M;

[0046] Step 2: Prepare reagent R2;

[0047] Step 3: Prepare reagent R1.

[0048] The following provides further explanation in conjunction with the specific implementation steps:

[0049] 1. Reagent M:

[0050] 1.1 Computational complexity

[0051] Take out the stock solution of magnetic beads at a ratio of 0.1 mg / mL to 0.5 mg / mL for streptavidin.

[0052] 1.2 Preprocessing

[0053] The extracted magnetic bead stock solution (using JSR's streptavidin magnetic beads MS160 / Streptavidin) was subjected to a liquid exchange pretreatment operation. Three times the volume of the magnetic bead stock solution of washing buffer B was added to the magnetic microparticles after the supernatant was discarded in the magnetic separator, and the mixture was placed on a mixer and mixed for 30 min to 1 h.

[0054] 1.3 Cleaning

[0055] Take Buffer B, which is 3 times the volume of the original magnetic bead solution, and add it to the magnetic microparticles after pretreatment and discarding the supernatant. Wash 3 times, place it on a magnetic separator, and discard the supernatant after the liquid becomes clear and transparent.

[0056] 1.4 Fixed volume

[0057] The above solution was added to the magnetic microparticle working solution Buffer A and brought to the target concentration. Then, 10–100 μg / mL of the blocking agent specifically interfering with the elimination protein was added.

[0058] The above magnetic microparticle working solution Buffer A is a PBS buffer with pH 7.40±0.05, with the following added: 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 20 g / L mannitol, 0.1 g / L 4-aminopyrine, 0.01 g / L biotin, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.5 g / L PC-300.

[0059] The washing buffer B mentioned above is a PBS buffer with pH 7.40±0.05, with the following added: 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 10 g / L sucrose, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.2 g / L PC-300.

[0060] All the above buffers were prepared and then filtered through a 0.22μm pore size nylon filter membrane.

[0061] 2. Reagent R2:

[0062] 2.1 Dissolve and dilute the heteroterminated bifunctional crosslinking agent with dimethyl sulfoxide, and react it with the antigen derivative diluted with buffer 1 at a molar ratio of 1:10 to 1:40 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours;

[0063] 2.2.2 Dissolve and dilute the thiol-modified crosslinking agent in buffer 2, and react it with alkaline phosphatase diluted in buffer 2 at a molar ratio of 1:10 to 1:30 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours.

[0064] 2.3 The activated antigen derivative and ALP modified with thiol groups were reacted at a mass ratio of 1:0.5 to 1:2 in the dark at a temperature of 20 to 37°C for 1 to 3 hours to obtain alkaline phosphatase-labeled insulin antibody.

[0065] 2.4 The alkaline phosphatase-labeled insulin antibody obtained above is purified using buffer 1. Purification methods include resin purification column and dialysis. The molecular weight cutoff should be less than 40 kDa.

[0066] 2.5 Preparation of enzyme-labeled working solution: MES buffer 0.05-0.1 mol / L, pH 6.0±7.0 (containing 0.1-0.2 mol / L sodium chloride), zinc chloride, magnesium chloride, protein stabilizer 0.1 g / L-10 g / L, nonionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and purified alkaline phosphatase-labeled insulin antibody 0.5 μg / mL-2 μg / mL.

[0067] The above buffer solution 1 consists of 10 mM phosphate buffer, 9 g / L sodium chloride, and a pH value within the range of 7.40 ± 0.05.

[0068] The above buffer solution 2 consists of 100 mM phosphate buffer, 9 g / L sodium chloride, and 10 mM disodium ethylenediaminetetraacetate, with a pH value in the range of 8.00 ± 0.05.

[0069] The aforementioned crosslinking agents include the heteroterminated bifunctional crosslinking agent 4-(N-maleimide)cyclohexane-1-carboxylic acid succinimide ester (SMCC) and its derivatives, such as Sulfo-SMCC, SM(PEG)4, SM(PEG)6, etc.; and also the thiol-modifying reagent 2-iminothione hydrochloride (Traut's reagent).

[0070] 3. Reagent R1:

[0071] 3.1 Computational complexity

[0072] Antibodies were extracted at a ratio of 0.5 μg / mL to 2 μg / mL of biotin-labeled insulin monoclonal antibody.

[0073] 3.2 Preparation of working solution

[0074] Biotin-labeled insulin monoclonal antibody was added to working solution Buffer A to prepare to the target concentration.

[0075] The above working solution Buffer A is a PBS buffer with pH 7.40±0.05, with the following added: 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 20 g / L mannitol, 0.1 g / L 4-aminopyrine, 0.01 g / L biotin, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.5 g / L PC-300.

[0076] Furthermore, the present invention provides several specific embodiments for auxiliary verification and illustration:

[0077] Example 1

[0078] Insulin Chemiluminescence Reagent Kit A

[0079] Preparation of reagent M:

[0080] 1. Take out the stock solution of magnetic beads according to the ratio of 0.4 mg / mL of streptavidin working solution.

[0081] 2. Perform a liquid exchange pretreatment operation on the extracted magnetic bead stock solution (using JSR's streptavidin magnetic beads MS160 / Streptavidin). Take 3 times the volume of the magnetic bead stock solution of washing buffer B according to the calculation, add it to the magnetic microparticles after the supernatant is discarded in the magnetic separator, and mix it on a mixer for 30 minutes.

[0082] 3. Take 3 times the volume of the original magnetic bead solution of the washing buffer B according to the calculation, add it to the magnetic microparticles after pretreatment and discarding the supernatant, wash 3 times, place it on a magnetic separator and wait for the liquid to become clear and transparent before discarding the supernatant.

[0083] 4. Add the above solution to the magnetic microparticle working solution Buffer A and bring the volume to the target concentration, and add 100 μg / mL of the blocking agent specifically interfering with the elimination protein.

[0084] Preparation of reagent R2:

[0085] 1. Dissolve and dilute the heteroterminated bifunctional cross-linking agent with dimethyl sulfoxide, and react it with the antigen derivative diluted with buffer 1 at a molar ratio of 1:10 at room temperature in the dark for 1 hour.

[0086] 2. Dissolve and dilute the thiol-modified crosslinking agent in buffer 2, and react it with alkaline phosphatase diluted in buffer 2 at a molar ratio of 1:10 at room temperature in the dark for 1 hour;

[0087] 3. The activated antigen derivative and ALP modified with thiol groups were reacted at a mass ratio of 1:1 at room temperature in the dark for 2 hours to obtain alkaline phosphatase-labeled insulin antibody.

[0088] 4. The reaction solution obtained above can be purified by dialysis using buffer 1.

[0089] 5. Preparation of enzyme-labeled working solution: MES buffer 0.05 mol / L, adjusted to pH=6.4 (containing 0.1 mol / L sodium chloride), freshly prepared zinc chloride (100mM) 0.1 mL / L, freshly prepared magnesium chloride (500mM) 10 mL / L, casein 1 g / L, sucrose 2 g / L, polyvinylpyrrolidone 30 (PVP K30) 2 g / L, PC-300 0.2 g / L, Tween 20 2 g / L, hydrolyzed albumin 1 g / L, alkaline phosphatase-labeled insulin antibody 0.6 μg / mL.

[0090] Preparation of reagent R1:

[0091] 1. Take out the antibody at a ratio of 1 μg / mL of biotin-labeled insulin monoclonal antibody.

[0092] 2. Add the biotin-labeled insulin monoclonal antibody to working solution Buffer A to prepare to the target concentration.

[0093] Example 2

[0094] Chemiluminescent reagent kit B for insulin

[0095] Preparation of reagent R2:

[0096] 1. Dissolve and dilute the heteroterminated bifunctional crosslinking agent in dimethyl sulfoxide in step 1 of Example 1, and react it with the antigen derivative diluted in buffer 1 at a molar ratio of 1:10 at room temperature in the dark for 0.5 h;

[0097] 2. Dissolve and dilute the thiol-modified crosslinking agent in step 2 of Example 1 with buffer 2, and react it with alkaline phosphatase diluted with buffer 2 at a molar ratio of 1:10 at room temperature in the dark for 0.5 h;

[0098] 3. The activated antigen derivative from step 3 of Example 1 and ALP modified with thiol groups were reacted at a mass ratio of 1:1 at room temperature in the dark for 1 hour to obtain alkaline phosphatase-labeled insulin antibody.

[0099] Without changing any other steps, we obtained the chemiluminescent insulin kit B.

[0100] Example 3

[0101] Chemiluminescent reagent kit for insulin C

[0102] Preparation of reagent R2:

[0103] 1. Dissolve and dilute the heteroterminated bifunctional crosslinking agent in dimethyl sulfoxide in step 1 of Example 1, and react it with the antigen derivative diluted in buffer 1 at a molar ratio of 1:10 at room temperature in the dark for 2 hours.

[0104] 2. Dissolve and dilute the thiol-modified crosslinking agent in step 2 of Example 1 with buffer 2, and react it with alkaline phosphatase diluted with buffer 2 at a molar ratio of 1:10 at room temperature in the dark for 2 hours.

[0105] 3. The activated antigen derivative from step 3 of Example 1 and ALP modified with thiol groups were reacted at a mass ratio of 1:1 at room temperature in the dark for 3 hours to obtain alkaline phosphatase-labeled insulin antibody.

[0106] Without changing any other steps, we obtained the chemiluminescent insulin kit C.

[0107] Comparison of detection results from different embodiments (see [link]) Figures 1 to 3 (Tables 1 to 7)

[0108] The detection system composed of reagent kits A, B, and C from this example and the Guilin Youlite IA260 chemiluminescence immunoassay analyzer was compared with the detection system composed of Roche's INS detection kit and the cobas e 411 instrument. The specific experiments are as follows:

[0109] 1. Sensitivity: For samples with concentrations of 2.00 μIU / mL, 2.05 μIU / mL, 2.10 μIU / mL, 2.15 μIU / mL, and 2.20 μIU / mL, the test was repeated 5 times, and the coefficient of variation (CV) of the test results should not exceed 10%.

[0110] Table 1. Sensitivity test results of kit A (unit: μIU / mL)

[0111]

[0112] Table 2. Sensitivity test results of kit B (unit: μIU / mL)

[0113]

[0114] Table 3. Sensitivity test results of kit C (unit: μIU / mL)

[0115]

[0116] The results showed that the coefficients of variation for all samples in kits A, B, and C were within 10%, indicating that the reagents maintained good sensitivity at a concentration of 2.00 μIU / mL. However, kit B had the shortest processing time, resulting in a slightly larger sensitivity deviation, which also demonstrates that the reaction time in the process has a significant impact on the performance of the kits.

[0117] 2. Repeatability: Test samples with different concentrations of 20.00 μIU / mL and 200.00 μIU / mL respectively. Each sample is tested 10 times. The coefficient of variation (CV) of the test results should not exceed 10%.

[0118] Table 4. Repeatability test results of kit A (unit: μIU / mL)

[0119]

[0120] Table 5. Repeatability test results of kit B (unit: μIU / mL)

[0121]

[0122] Table 6. Repeatability test results of kit C (unit: μIU / mL)

[0123]

[0124] The results showed that the coefficients of variation of the test results for high and low value samples of reagent kits A, B, and C were all within 10% and all below 3%, indicating that the reproducibility of the reagents was excellent.

[0125] 3. Clinical comparison: 40 clinical samples were tested using this reagent kit, and the correlation coefficient r with Roche's clinical results was calculated to be ≥0.975.

[0126] Table 7 Clinical testing of kits A / B / C and comparison reagents (unit: μIU / mL)

[0127]

[0128] The results showed that, within the concentration range of 2.00–450 μIU / mL, the linear correlation between kits A, B, and C and Roche was all above 0.98.

[0129] In summary, the present invention has the following beneficial effects.

[0130] 1. The present invention provides a chemiluminescent reagent kit for insulin, with a test sensitivity of 2.00 μIU / mL, a test repeatability of <3% for both high and low value samples, and a linear correlation of 0.98 with Roche in the concentration range of 2.00 to 450 μIU / mL.

[0131] 2. The method for preparing an insulin chemiluminescence reagent kit provided by the present invention greatly shortens the production cycle, makes it easy to control batch-to-batch differences, and facilitates production work for enterprises.

[0132] The above description discloses only one or more preferred embodiments of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A magnetic microparticle chemiluminescence detection kit for insulin, characterized in that, It consists of three parts: reagent M, reagent R2, and reagent R1. Reagent M is a magnetic microparticle working solution containing streptavidin magnetic beads; reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody; and reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibody. The reagent M comprises 0.01–0.1 mol / L PBS buffer, 0.1 g / L–10 g / L protein stabilizer, 5–25 g / L carbohydrate molecules, 0.5 g / L–5 g / L nonionic surfactant, 0.5 g / L–1 g / L preservative, and 0.1 mg / mL–0.5 mg / mL inhibitor and streptavidin magnetic beads. The reagent R2 consists of MES buffer (0.05–0.1 mol / L), zinc chloride, magnesium chloride, protein stabilizer (0.1 g / L–10 g / L), nonionic surfactant (0.5 g / L–5 g / L), preservative (0.5 g / L–1 g / L), and alkaline phosphatase-labeled insulin antibody (0.5 μg / mL–2 μg / mL). The reagent R1 comprises PBS buffer, protein stabilizer 0.1 g / L to 10 g / L, carbohydrate molecules 5 to 25 g / L, nonionic surfactant 0.5 g / L to 5 g / L, preservative 0.5 g / L to 1 g / L, and biotin-labeled insulin monoclonal antibody 0.5 μg / mL to 2 μg / mL.

2. The chemiluminescence immunoassay kit for insulin using magnetic microparticles as described in claim 1, characterized in that, The PBS buffer has a pH range of 7.0±8.0 and contains 0.1 to 0.2 mol / L sodium chloride; the MES buffer has a pH range of 6.0±7.0 and contains 0.1 to 0.2 mol / L sodium chloride.

3. A method for preparing a magnetic microparticle chemiluminescence detection kit for insulin, used to prepare the magnetic microparticle chemiluminescence detection kit for insulin as described in any one of claims 1 and 2, characterized in that, Includes the following steps: Step 1: Prepare reagent M; Step 2: Prepare reagent R2; Step 3: Prepare reagent R1.

4. The method for preparing the insulin magnetic microparticle chemiluminescence detection kit as described in claim 3, characterized in that, The execution process of step 1 includes the following steps: Step 1.1: Take out the magnetic bead stock solution at a ratio of 0.1 mg / mL to 0.5 mg / mL for streptavidin magnetic beads; Step 1.2: Perform a liquid exchange pretreatment operation on the extracted magnetic bead stock solution. Take 3 times the volume of the magnetic bead stock solution of washing buffer B according to the calculation, add it to the magnetic microparticles after the supernatant is discarded in the magnetic separator, and place it on a mixer to mix for 30 min to 1 h. Step 1.3: Take 3 times the volume of the original magnetic bead solution of the washing buffer B according to the calculation, add it to the magnetic microparticles after pretreatment and discarding the supernatant, wash 3 times, place it on the magnetic separator and wait for the liquid to become clear and transparent before discarding the supernatant. Step 1.4: Add the magnetic microparticle working solution Buffer A to the magnetic microparticles after discarding the supernatant, adjust the volume to the target concentration, and add 10-100 μg / mL of the blocking agent specifically interfering with and eliminating protein to obtain reagent M.

5. The chemiluminescence immunoassay kit for insulin using magnetic microparticles as described in claim 4, characterized in that, The magnetic microparticle working solution Buffer A in step 1 is a PBS buffer with pH 7.40±0.05, with the addition of 0.15 mol NaCl, casein 5 g / L, bovine serum albumin 1 g / L, mannitol 20 g / L, 4-aminopyrine 0.1 g / L, biotin 0.01 g / L, Tween-20 1 g / L, TX-100 0.5 g / L, and PC-300 0.5 g / L. Washing buffer B is a PBS buffer with pH 7.40 ± 0.05, with the following added: 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 10 g / L sucrose, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.2 g / L PC-300. All buffers were prepared and then filtered through a 0.22 μm nylon filter membrane.

6. The method for preparing the insulin magnetic microparticle chemiluminescence detection kit as described in claim 5, characterized in that, The execution process of step 2 includes the following steps: Step 2.1: Dissolve and dilute the heteroterminated bifunctional cross-linking agent with dimethyl sulfoxide, and react it with the antigen derivative diluted with buffer 1 at a molar ratio of 1:10 to 1:40 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours. Step 2.2: Dissolve and dilute the thiol-modified crosslinking agent with buffer 2, and react it with alkaline phosphatase diluted with buffer 2 at a molar ratio of 1:10 to 1:30 at a temperature of 20 to 37°C in the dark for 0.5 to 2 hours. Step 2.3: The activated antigen derivative and ALP modified with thiol groups are reacted at a mass ratio of 1:0.5 to 1:2 in the dark at a temperature of 20 to 37°C for 1 to 3 hours to obtain alkaline phosphatase-labeled insulin antibody. Step 2.4: Purify the alkaline phosphatase-labeled insulin antibody obtained above using buffer 1; Step 2.5 Prepare enzyme-labeled working solution using MES buffer (0.05–0.1 mol / L), zinc chloride, magnesium chloride, protein stabilizer (0.1 g / L–10 g / L), nonionic surfactant (0.5 g / L–5 g / L), preservative (0.5 g / L–1 g / L), and purified alkaline phosphatase-labeled insulin antibody (0.5 μg / mL–2 μg / mL).

7. The method for preparing the insulin magnetic microparticle chemiluminescence detection kit according to claim 6, characterized in that, In step 2, buffer 1 consists of 10 mM phosphate buffer, 9 g / L sodium chloride, and a pH value within the range of 7.40 ± 0.05; buffer 2 consists of 100 mM phosphate buffer, 9 g / L sodium chloride, 10 mM disodium ethylenediaminetetraacetate, and a pH value within the range of 8.00 ± 0.

05.

8. The method for preparing the insulin magnetic microparticle chemiluminescence detection kit according to claim 7, characterized in that, The execution process of step 3 includes the following steps: Step 3.1: Take out the antibody at a ratio of 0.5 μg / mL to 2 μg / mL of biotin-labeled insulin monoclonal antibody; Step 3.2: Add the biotin-labeled insulin monoclonal antibody to working solution Buffer A to prepare to the target concentration; The working solution Buffer A is a PBS buffer with pH 7.40 ± 0.05, containing 0.15 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 20 g / L mannitol, 0.1 g / L 4-aminopyrine, 0.01 g / L biotin, 1 g / L Tween-20, 0.5 g / L TX-100, and 0.5 g / L PC-300.

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