An insulin fluorescence immunoassay chromatography determination kit and its preparation method

Abstract

An insulin fluorescence immunoassay kit provided by the present application and its preparation method use an insulin monoclonal antibody and a chicken IgY antibody as the antibodies for coating on the test line and the quality control line respectively, and a mixture of the insulin monoclonal antibody and a goat anti-chicken IgY antibody as the fluorescent microsphere-labeled antibody. Using the fluorescence immunoassay method, compared with the currently commonly used insulin detection methods such as enzyme-linked immunosorbent assay (ELISA) / radioimmunoassay (RIA), it not only greatly shortens the detection time and reduces the detection cost, but also improves the detection sensitivity. Compared with similar methods, it has the advantages of simple labeling process, high sensitivity, and accurate results. By designing experiments, suitable materials for preparing the test strip are selected. The preparation process is simple. The prepared kit has high detection sensitivity, accurate and stable results, low detection cost, easy use of the product, and low manufacturing cost, and is suitable for mass production.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic immunochromatography, and relates to an insulin fluorescence immunochromatography assay kit and a preparation method thereof. Specifically, it is a method for quantitatively detecting the insulin content in human blood by using the technical principle of fluorescence immunochromatography. Background Art

[0002] Insulin (INS) is a protein hormone secreted by pancreatic islet β cells in the pancreas in response to stimulation by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body that lowers blood sugar, and it can simultaneously promote glycogen, fat, and protein synthesis. Exogenous insulin is mainly used for the treatment of diabetes. Detection of blood insulin levels can evaluate islet function, be used for the diagnosis and classification of diabetes, and is also an indicator for the diagnosis, efficacy observation, and prognosis judgment of insulinoma.

[0003] Traditional methods for detecting insulin content usually require large, expensive instrument equipment and professional technical personnel, and the detection can only be carried out in the laboratory, with a long detection time, time-consuming and laborious.

[0004] Fluorescence immunochromatography has the advantages of low cost, simple operation, fast speed, and convenient to carry, and is an important part of the field of medical in vitro diagnosis. At the same time, in view of evaluating islet function and being used for the diagnosis and classification of diabetes, developing an INS immunofluorescence quantitative detection kit with high sensitivity and high precision has broad market value. There is an urgent need in the market for an insulin fluorescence immunochromatography assay kit with high sensitivity, high precision, and simple operation. Summary of the Invention

[0005] The purpose of the present invention is to provide an insulin fluorescence immunochromatography assay kit with high sensitivity, short detection time, strong specificity, and good accuracy.

[0006] The present application adopts the following scheme. An insulin fluorescence immunochromatography assay kit includes an insulin test card, a diluent, and a chip. The test card includes a card shell and a test strip. The test strip includes a PVC bottom plate, and a sample pad, a conjugate pad, an NC membrane, and a blotting paper are sequentially arranged on the PVC bottom plate. A detection line and a quality control line are provided on the NC membrane;

[0007] The detection line is coated with INS monoclonal antibody, and the coating concentration is 1.0 - 1.2 mg / ml;

[0008] The quality control line is coated with chicken IgY antibody, and the coating concentration is 1.0 mg / ml;

[0009] The binding pad is sprayed with a labeled antibody solution labeled with fluorescent microspheres. Specifically, the labeled antibody solution labeled with fluorescent microspheres is a mixture of an INS monoclonal antibody labeled with fluorescent microspheres and a goat anti-chicken IgY antibody containing fluorescently labeled microspheres.

[0010] Preferably, the fluorescent microspheres are time-resolved fluorescent microspheres with a hydroxylated particle size of 300 nm from Changsha Meiniu.

[0011] Preferably, the spraying amount of the labeled antibody solution is 4 μL / cm.

[0012] Preferably, the coating solution formula is: 0.01 M PBS with 1% sucrose at pH 7.4.

[0013] Preferably, the steps for labeling the INS antibody with fluorescent microspheres are as follows:

[0014] A. Take a 2 ml centrifuge tube and add 0.2 ml of the labeling buffer MES; take 20 μL of the fluorescent microspheres with a uniformly mixed solid content of 1% and add them to the labeling buffer, and vortex and mix again;

[0015] B. Add 20 μL of the labeling activation solution and mix well, and rotate and mix for 30 min;

[0016] C. Centrifuge at a centrifugal speed of 10,000 rpm for 30 min; discard the supernatant, add 200 μL of the labeling buffer and then ultrasonically oscillate;

[0017] D. Add 10 μg of the INS antibody and mix well, and rotate and mix for 30 min;

[0018] E. Add 20 μL of the labeling blocking solution and then ultrasonically vibrate, and rotate and mix for 2 h;

[0019] F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of the labeling preservation solution and ultrasonically vibrate;

[0020] G. Transfer the labeled substance into a new 2 ml centrifuge tube washed with 0.8 ml of the labeling preservation solution, and vortex and mix well.

[0021] Preferably, the steps for labeling the goat anti-chicken IgY antibody with fluorescent microspheres are as follows:

[0022] A. Take a 2 ml centrifuge tube and add 0.2 ml of the labeling buffer MES; take 20 μL of the fluorescent microspheres with a uniformly mixed solid content of 1% and add them to the labeling buffer, and vortex and mix again;

[0023] B. Add 20 μL of the labeling activation solution and mix well, and rotate and mix for 30 min;

[0024] C. Centrifuge at 10,000 rpm for 30 min; discard the supernatant, add 200 μL of labeling buffer and then perform ultrasonic oscillation;

[0025] D. Add 10 μg of goat anti-chicken IgY antibody and mix well, rotate and mix for 30 min;

[0026] E. Add 20 μL of labeling blocking solution and then perform ultrasonic treatment, rotate and mix for 2 h;

[0027] F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of labeling storage solution and perform ultrasonic treatment;

[0028] G. Transfer the labeled product into a new 2-ml centrifuge tube washed with 0.8 ml of labeling storage solution and vortex mix well.

[0029] Preferably, the dilution solution formula is: 0.01 M PBS pH 7.4 and 0.05% Proclin 300.

[0030] Another object of the present invention is to provide a preparation method of an insulin fluorescence immunoassay chromatography determination kit with high sensitivity, short detection time, strong specificity and good accuracy.

[0031] The technical solution of the present invention is as follows: A preparation method of the above insulin fluorescence immunoassay chromatography determination kit, including preparing a test strip by the following steps:

[0032] Step 1: Set a detection line and a quality control line on the NC membrane, prepare coating working solutions of INS monoclonal antibody and chicken IgY antibody at concentrations of 1.0 - 1.2 mg / ml and 1.0 mg / ml respectively, and use a gold-labeling scribing machine to scribe them onto the NC membrane at a coating amount of 1.0 - 1.2 mg / ml, and place the NC membrane in an electrothermal blast drying oven for drying for 16 - 24 h;

[0033] Step 2: Use a gold-spraying scribing machine to spray the antibody solution labeled with fluorescent microspheres onto the conjugate pad at a spraying amount of 4 μL / cm and then dry;

[0034] Step 3: After the sample pad is treated with the sample pad treatment solution, dry it;

[0035] Step 4: Stick the above-mentioned treated sample pad, conjugate pad, NC membrane and absorbent paper onto the PVC board in sequence to assemble into a test strip.

[0036] Preferably, the sample pad treatment solution is prepared from the following components: 10 mg of anti-human RBC antibody, 20 mg of blocker 001, 10 g of bovine serum albumin, 5 ml of Tween-20, and 1000 ml of 0.2 M pH 8.0 boric acid borax buffer solution.

[0037] Preferably, the labeled preservation solution is prepared from the following components: 20 g of bovine serum albumin, 20 g of sucrose, 5 g of casein, 50 g of trehalose, 0.5 ml of Proclin 300, and 1000 ml of 0.02 M Tris buffer with a pH of 8.0.

[0038] Principle of the determination of this kit: The double antibody sandwich method is adopted, utilizing the technical principle of fluorescence immunochromatography. During the test, the sample is mixed evenly with the diluent and dropped into the sample application hole of the test strip. Under capillary action, chromatography is carried out. Insulin in the sample binds to the fluorescently labeled insulin monoclonal antibody, diffuses to the test area, and is captured by the insulin monoclonal antibody coated on the test line, forming an "antibody - antigen - fluorescent antibody" complex; the insulin concentration in the sample is proportional to the fluorescence intensity of the complex. According to the standard curve in the chip, the insulin concentration in the sample can be calculated.

[0039] Compared with the prior art, this application has the following advantages:

[0040] The insulin fluorescence immunochromatography determination kit provided by this application uses insulin monoclonal antibody and chicken IgY antibody as the antibodies coated on the test line and the quality control line respectively, and the mixture of insulin monoclonal antibody and sheep anti-chicken IgY antibody is used as the fluorescent microsphere labeled antibody. Using fluorescence immunochromatography, compared with the currently commonly used insulin detection methods such as enzyme-linked immunosorbent assay (ELISA) / radioimmunoassay (RIA), it has the advantages of high sensitivity, short detection time, simple operation, strong specificity, and good accuracy. And the detection operation is simple, the cost is low, it is not limited to the laboratory, the detection time is short, and the detection result can be obtained within 15 minutes, which is convenient and fast.

[0041] The preparation method of the insulin fluorescence immunochromatography determination kit provided by this application selects suitable materials for the preparation of the test strip through experimental design. The preparation process is simple. The prepared kit has high detection sensitivity, accurate and stable results, low detection cost, simple product use, low manufacturing cost, and is suitable for mass production. Description of the Drawings

[0042] Figure 1 It is a comparison chart of the antibody preparation kits T / C from different manufacturers in the embodiment of this application;

[0043] Figure 2 It is a comparison chart of the fluorescent microsphere preparation kits T / C in the embodiment of this application;

[0044] Figure 3 It is a comparison chart of the coating solution preparation kits T / C in the embodiment of this application;

[0045] Figure 4It is the result of the combined gradient ratio of different coating concentrations of T lines and C lines in the embodiments of the present application;

[0046] Figure 5 It is the detection result of the linear range of the kit with batch number 1 in the embodiments of the present application;

[0047] Figure 6 It is the detection result of the linear range of the kit with batch number 2 in the embodiments of the present application;

[0048] Figure 7 It is the detection result of the linear range of the kit with batch number 3 in the embodiments of the present application;

[0049] Figure 8 It is the structural schematic diagram of the kit in the embodiments of the present application. Detailed implementation manners

[0050] The following combines the accompanying drawings and specific embodiments to illustrate the specific technical solutions of the present invention

[0051] Embodiment 1:

[0052] An insulin fluorescence immunoassay chromatography kit, comprising an insulin test card, a diluent and a chip. The test card includes a card shell and a test strip. The test strip includes a PVC bottom plate and a sample pad, a conjugate pad, an NC membrane and a blotting paper sequentially arranged on the PVC bottom plate. The NC membrane is provided with a test line and a quality control line;

[0053] The test line is coated with an INS monoclonal antibody, and the coating concentration is 1.0 - 1.2 mg / ml;

[0054] The quality control line is coated with a chicken IgY antibody, and the coating concentration is 1.0 mg / ml;

[0055] The conjugate pad is sprayed with a fluorescent microsphere-labeled labeled antibody solution, and the fluorescent microsphere-labeled labeled antibody solution is specifically a mixture of an INS monoclonal antibody labeled with fluorescent microspheres and a goat anti-chicken IgY antibody containing fluorescent-labeled microspheres.

[0056] Embodiment 2:

[0057] A preparation method of an insulin fluorescence immunoassay chromatography kit, comprising preparing a test strip by the following steps:

[0058] Step 1: Set a test line and a quality control line on the NC membrane. Prepare coating working solutions of an INS monoclonal antibody and a chicken IgY antibody at concentrations of 1.0 - 1.2 mg / ml and 1.0 mg / ml respectively, and use a gold-labeling scribing machine to draw them onto the NC membrane at a coating amount of 1.0 - 1.2 mg / ml, and place the NC membrane in an electrothermal blast drying oven for drying for 16 - 24 h;

[0059] Step 2: Spray the antibody solution labeled with fluorescent microspheres onto the cut binding site using a gold-spraying scribing machine at a spraying volume of 4 μL / cm, and then dry it;

[0060] Step 3: After the sample pad is treated with the sample pad treatment solution, dry it;

[0061] Step 4: Stick the above-treated sample pad, conjugate pad, NC membrane and absorbent paper onto the PVC board in sequence to assemble a test strip.

[0062] Among them, the steps for the INS monoclonal antibody labeled with fluorescent microspheres are as follows:

[0063] A. Take a 2 ml centrifuge tube and add 0.2 ml of the labeling buffer MES; take 20 μL of fluorescent microspheres with a uniformly mixed solid content of 1% and add them to the labeling buffer, and vortex and mix again;

[0064] B. Add 20 μL of the labeling activation solution and mix well, then rotate and mix for 30 min;

[0065] C. Centrifuge at 10000 rpm for 30 min; discard the supernatant, add 200 μL of the labeling buffer and then ultrasonically oscillate;

[0066] D. Add 10 μg of the INS antibody and mix well, then rotate and mix for 30 min;

[0067] E. Add 20 μL of the labeling blocking solution and then ultrasonically oscillate, rotate and mix for 2 h;

[0068] F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of the labeling preservation solution and ultrasonically oscillate;

[0069] G. Transfer the labeled product into a new 2 ml centrifuge tube washed with 0.8 ml of the labeling preservation solution, and vortex and mix well.

[0070] The steps for the goat anti-chicken IgY antibody labeled with fluorescent microspheres are as follows:

[0071] A. Take a 2 ml centrifuge tube and add 0.2 ml of the labeling buffer MES; take 20 μL of fluorescent microspheres with a uniformly mixed solid content of 1% and add them to the labeling buffer, and vortex and mix again;

[0072] B. Add 20 μL of the labeling activation solution and mix well, then rotate and mix for 30 min;

[0073] C. Centrifuge at 10000 rpm for 30 min; discard the supernatant, add 200 μL of the labeling buffer and then ultrasonically oscillate;

[0074] D. Add 10 μg of the goat anti-chicken IgY antibody and mix well, then rotate and mix for 30 min;

[0075] E. After adding 20 μL of the labeled blocking solution, perform ultrasonic treatment and rotate and mix evenly for 2 h;

[0076] F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of the labeled storage solution and perform ultrasonic treatment;

[0077] G. Transfer the labeled substance into a new 2-ml centrifuge tube washed with 0.8 ml of the labeled storage solution and vortex and mix evenly.

[0078] The labeled antibody solution labeled with fluorescent microspheres is specifically a 1:1 mixture of the INS antibody labeled with fluorescent microspheres and the goat anti-chicken IgY antibody containing fluorescently labeled microspheres.

[0079] Furthermore, for the coated antibody and the labeled antibody on the test line of the present application, the INS antibody of Hangzhou Longji Biotechnology Co., Ltd. is selected. The made test strip has the best linear gradient and better T / C precision. The selection process of the coated antibody and the labeled antibody on the test line is as follows:

[0080] Experimental design plan: Combine the use concentrations of the INS antibodies recommended by Beijing Protein Innovation Co., Ltd., Hangzhou Longji Biotechnology Co., Ltd. and Shanghai Lingchao Biotechnology Co., Ltd. with the concentrations commonly used in immunofluorescence method. After appropriately diluting the INS antibody with ultrapure water, measure the absorbance values of OD260 and OD280 by ultraviolet spectrophotometry, and then calculate the protein concentration. Make test strips to test the enterprise reference products. Take the enterprise reference products as the research materials, and screen the best coated antibody and labeled antibody for the test line based on the linear gradient of the enterprise reference products and the concentration measured by ultraviolet spectrophotometry.

[0081] The experimental method includes the following steps:

[0082] I. Antibody concentration determination

[0083] After appropriately diluting the INS antibody with ultrapure water, measure the absorbance values of OD260 and OD280 by ultraviolet spectrophotometry, and then calculate the protein concentration.

[0084] II. Use the same fluorescent microspheres to label the INS-labeled antibodies of each manufacturer. The steps are as follows:

[0085] A. Take a 2-ml centrifuge tube and add 0.2 ml of the labeling buffer MES; take 20 μL of the fluorescent microspheres with a uniformly mixed solid content of 1% into the labeling buffer and vortex and mix evenly again;

[0086] B. Add 20 μL of the labeling activation solution and mix evenly, rotate and mix evenly for 30 min;

[0087] C. Centrifuge for 30 min (centrifugation speed is 10,000 rpm); discard the supernatant, add 200 μL of the labeling buffer and perform ultrasonic treatment;

[0088] D. Add 10 μg of INS-labeled antibody from each manufacturer and mix well, then rotate and mix for 30 min;

[0089] E. Add 20 μL of labeled blocking solution and then perform ultrasonic treatment, followed by rotation and mixing for 2 h;

[0090] F. Centrifuge for 30 min; discard the supernatant, add 0.2 mL of labeled storage solution and perform ultrasonic treatment;

[0091] G. Transfer the labeled product to a new 2-mL centrifuge tube, pipette 0.8 mL of labeled storage solution to wash the 2-mL centrifuge tube used for labeling, and transfer the washed solution to the 2-mL centrifuge tube after washing, then vortex and mix well.

[0092] III. Preparation of conjugate pad

[0093] Load the fluorescent microsphere-labeled antibody solution prepared in step II onto a gold-spraying and scribing machine, and spray it onto the cut conjugate pad at a spraying volume of 4 μL / cm, then dry overnight for 17 h.

[0094] IV. Preparation of coated membrane

[0095] Prepare a coating solution with the INS-coated antibody at the common concentration of each manufacturer, and use a gold-labeling scribing machine to scribe it onto the NC membrane, then place the NC membrane in an electrothermal blast drying oven and dry for 17 h.

[0096] V. Preparation of sample pad

[0097] Set the height of the wheel shaft of the paper dipping and pressing pad machine, pour in the sample pad treatment solution, and place the treated sample pad on a drying rack and dry overnight for 17 h.

[0098] The performance comparison test results of antibody preparation kits from different manufacturers are shown in Table 1:

[0099] Table 1 Antibody detection concentrations (T line) of different manufacturers

[0100]

[0101] As can be seen from Table 1 above, the antibody concentrations of each manufacturer determined by ultraviolet spectrophotometry basically conform to the labeled antibody concentrations, and the relative deviations do not exceed ±10%, meeting the requirements.

[0102] Table 2 Performance comparison of antibody preparation kits from different manufacturers (T line)

[0103]

[0104]

[0105] From Figure 1As can be seen from Table 2, there are significant differences in the linear gradients of the test strips prepared with antibodies from different manufacturers for the enterprise reference products (the total gradient ranges from as low as 18.64 to as high as 28.04), where Manufacturer 2 > Manufacturer 3 > Manufacturer 1.

[0106] The antibody detection concentrations in Table 1 all meet the requirements. For Table 2, Figure 1 there are significant differences in the linear gradients (the total gradient ranges from as low as 18.64 to as high as 28.04). Manufacturer 2 is relatively the best. The INS antibody pair from Manufacturer 2, i.e., Hangzhou Longji Biotechnology Co., Ltd., is selected as the coated antibody and labeled antibody for the test line.

[0107] Furthermore, for the coated antibody and labeled antibody of the quality control line in this application, the chicken IgY and goat anti-chicken IgY antibodies from Ningbo Maiyue are selected. The selection process of the coated antibody and labeled antibody for the quality control line is as follows: Using the chicken IgY and rabbit anti-chicken IgY antibodies from Shanghai Lingchao Biotechnology Co., Ltd. and the chicken IgY and goat anti-chicken IgY antibodies from Ningbo Maiyue Biotechnology Co., Ltd. at the commonly used concentrations, after appropriately diluting the INS antibody with ultrapure water, the absorbance values of OD260 and OD280 are measured by ultraviolet spectrophotometry, and then the protein concentration is calculated. Then, test strips are made to test the enterprise reference products. Using the enterprise reference products as the research materials, the best coated antibody and labeled antibody for quality control are screened based on the linear gradient, precision, and concentration measured by ultraviolet spectrophotometry of the enterprise reference products.

[0108] The experimental method steps are similar to those of the experiment for the coated antibody and labeled antibody of the test line

[0109] The test results of the test strips prepared with the coated antibodies for the quality control line from different manufacturers are as follows:

[0110] Table 3 Antibody Detection Concentrations (C line) of Different Manufacturers

[0111]

[0112] As can be seen from the above Table 3, the antibody concentrations of each manufacturer measured by ultraviolet spectrophotometry basically meet the labeled antibody concentrations, and the relative deviations do not exceed ±10%, meeting the requirements.

[0113] Table 4 Test Results of Test Strips Prepared with Antibodies from Different Manufacturers (C line)

[0114]

[0115] The antibody detection concentrations in Table 3 all meet the requirements. As can be seen from Table 4, the linear gradient of Ningbo Maiyue (chicken IgY and goat anti-chicken IgY) is 45.16, and the T / C precision is < 7%, which is relatively the best. The chicken IgY and goat anti-chicken IgY from Ningbo Maiyue are selected as the coated antibody and labeled antibody for the quality control line.

[0116] Further, the fluorescent microspheres of the present application select the hydroxy - sized 300 - nm time - resolved fluorescent microspheres of Changsha Meiniu as the fluorescent microspheres for labeling. The test strip prepared therefrom has a linear gradient of 32.92, and the T / C precision is all <6%, with the best performance. The selection process of the fluorescent microspheres is as follows: The Eu - fluorescent nanometer microspheres with particle sizes of about 200 nm and 300 nm from Nanjing Micro - Detection Biotechnology Co., Ltd. and the hydroxy - time - resolved fluorescent microspheres with particle sizes of 200 nm and 300 nm from Changsha Meiniu are tested for particle size, fluorescence intensity, and PDI coefficient. Then, they are sequentially labeled with antibodies, the conjugate pads are prepared to make test strips, and they are detected with enterprise reference products.

[0117] The experimental method includes the following steps:

[0118] I. Four kinds of fluorescent microspheres are respectively labeled with INS - labeled antibody and goat anti - chicken IgY - labeled antibody. The steps are as follows:

[0119] A. Take a 2 - mL centrifuge tube and add 0.2 mL of labeling buffer MES; take 20 μL of fluorescent microspheres with a uniformly mixed solid content of 1% and place them in the labeling buffer, and vortex and mix again.

[0120] B. Add 20 μL of labeling activation solution and mix well, and rotate and mix for 30 min.

[0121] C. Centrifuge for 30 min (centrifugation speed is 10000 rpm); discard the supernatant, add 200 μL of labeling buffer, and ultrasonicate.

[0122] D. Respectively add 10 μg of INS - labeled antibody and 10 μg of goat anti - chicken IgY - labeled antibody and mix well, and rotate and mix for 30 min.

[0123] E. Add 20 μL of labeling blocking solution and then ultrasonicate, and rotate and mix for 2 h.

[0124] F. Centrifuge for 30 min; discard the supernatant, add 0.2 mL of labeling preservation solution and ultrasonicate.

[0125] G. Transfer the labeled matter into a new 2 - mL centrifuge tube, suck 0.8 mL of labeling preservation solution to wash the 2 - mL centrifuge tube used for labeling, and transfer it into the 2 - mL centrifuge tube after washing, and vortex and mix well.

[0126] H. Mix the labeled INS - labeled matter and goat anti - chicken IgY - labeled matter evenly according to a volume ratio of 1:1, and ultrasonicate sufficiently to obtain the fluorescent microsphere - labeled antibody solution.

[0127] II. Prepare the conjugate pad

[0128] The fluorescent microsphere - labeled antibody solution is loaded into a gold - spraying and scribing machine, and it is sprayed onto the cut conjugate pad at a spraying volume of 4 μL / cm; dry overnight for 18 h.

[0129] After assembling the experimental materials prepared from various raw materials into test strips, they were evaluated using the enterprise reference product.

[0130] The test results of test strips prepared with fluorescent microspheres from different manufacturers are shown in Table 5 below and Figure 2 as follows:

[0131] Table 5 Test Results of Test Strips Prepared with Fluorescent Microspheres from Different Manufacturers Concentration Unit: (mIU / L)

[0132]

[0133] As can be seen from Table 5 above, Changsha Meiniu's 300nm linear gradient of 32.92 is the best, and the T / C precision is all < 6%.

[0134] From Figure 2 it can be seen that there are certain differences in the linear trends of different fluorescent microspheres, and the linear trend of Changsha Meiniu's 300nm is relatively the best.

[0135] Combined with Table 5 and Figure 2 it can be known that for Changsha Meiniu's hydroxylated fluorescent microspheres at 300nm, the linear gradient is 32.92, and the T / C precision is all < 6%. The performance is the best, so Changsha Meiniu's hydroxylated fluorescent microspheres at 300nm are selected as the fluorescent microspheres for labeling.

[0136] Furthermore, the sample diluent formula is selected as: 0.01M PBS, pH 7.4 and Proclin 300 with a mass fraction of 0.05%. After making the reagent strip, it has a good linear gradient and high detection precision.

[0137] Furthermore, the sample pad treatment liquid is prepared according to 1000ml: 10mg anti-human RBC antibody, 20mg blocker 001, 10g bovine serum albumin, 5ml Tween-20 and 1000ml 0.2M pH 8.0 boric acid borax buffer solution. This formula has strong anti-interference ability. The process of determining the sample pad treatment liquid formula is as follows: Using 0.2M boric acid borax buffer solution (pH 8.0) as the buffer system, analyze the effect of anti-human RBC antibody on the interception of red blood cells and the influence of blocker 001 on specificity, so as to determine the formula of the sample pad treatment liquid. Prepare the sample pad treatment liquid using the formula table in Table 6 below:

[0138] Table 6 Formula Table of Sample Pad Treatment Liquids 1-4

[0139]

[0140] Table 7 Effects of Sample Pad Treatment Liquids 1-4 on Testing Whole Blood Samples

[0141]

[0142] Table 8 Effects of four sample pad treatment solutions on reference products with added interfering substances

[0143]

[0144]

[0145] As can be seen from Table 7, the anti-human RBC antibody can effectively intercept red blood cells under the conditions of 10 mg and 20 mg dosage;

[0146] As can be seen from Table 8, when the dosage of blocker 001 is 10 mg, the T / C of the reference product with added interfering substances shows a doubling growth, but it is significantly lower than that of the test strip with the untreated sample pad, indicating a certain anti-interference ability; when the dosage of blocker 001 is 20 mg, there is no significant difference in T / C between the reference product with added interfering substances and the reference product without added interfering substances, showing a strong anti-interference ability

[0147] Combining Table 7 and Table 8, it can be seen that the anti-human RBC antibody can effectively intercept red blood cells under the conditions of 10 mg and 20 mg dosage. When the dosage of blocker 001 is 20 mg, there is no significant difference in T / C between the reference product with added interfering substances and the reference product without added interfering substances, showing a strong anti-interference ability. Sample pad treatment solutions 2 and 3 both meet the requirements. Considering the comprehensive cost factor, sample pad treatment solution 3 is finally selected as the sample pad treatment solution formula.

[0148] Furthermore, the coating solution formula is 0.01 M PBS containing 1% sucrose at pH 7.4. The test strip made with this coating solution formula has a high linear gradient, and the ionic strength of the coating solution has basically no effect on the detection result. The selection process of the coating solution formula is as follows: the pH value, ionic strength, etc. of the coating buffer can affect the binding ability of the monoclonal antibody to the NC membrane. We referred to relevant literature to explore the pH and ionic strength of the coating buffer. The coating antibodies INS and chicken IgY were diluted to the coating concentration with 4 formulas of coating solutions respectively. After making the reagent strips, the coating solution with clear liquid, no crystal precipitation and good linear gradient was selected as the coating solution.

[0149] Table 9 Coating solution formula table

[0150] Type Sucrose (g) Buffer solution Coating solution 1 1.00 Dissolved in 100 mL of 0.01 M PBS buffer solution with pH 7.4 Coating solution 2 1.00 Dissolved in 100 mL of 0.01 M PBS buffer solution with pH 8.0 Coating solution 3 1.00 Dissolved in 100 mL of 0.02 M PBS buffer solution with pH 7.4 Coating solution 4 1.00 Dissolved in 100 mL of 0.02 M PBS buffer solution with pH 8.0

[0151] Table 10 Preservation status of different coating solutions

[0152] Type Appearance Coating solution 1 The solution is a clear liquid and no crystals precipitate after storing for 14 days Coating solution 2 The solution is a clear liquid and no crystals precipitate after storing for 14 days Coating solution 3 The solution is a clear liquid and no crystals precipitate after storing for 14 days Coating solution 4 The solution is a clear liquid and no crystals precipitate after storing for 14 days

[0153] As can be seen from Table 10, there is no obvious crystal precipitation in the 1% sucrose liquid, and the appearance of the solution is good.

[0154] Table 11 Detection results of test strips prepared with different coating solutions

[0155]

[0156]

[0157] As can be seen from Table 11, the linear gradient of Coating 1 is 35.96, and the precision of T / C is all < 6%.

[0158] From Tables 10 - 11, Figure 3 it can be seen that the pH value of the coating solution has a great influence on the linear gradient. When the pH of the coating solution is 7.4, the linear gradient is high. Therefore, the pH value of the coating solution is selected as 7.4; the ionic strength of the coating solution has basically no influence on the detection result. Considering comprehensively, 0.01M PBS containing 1% sucrose and pH 7.4 is selected as the final formula of the coating solution.

[0159] Furthermore, the final coating concentrations of the T line and C line are selected as 1.0 mg / mL for the INS - coated antibody and 1.0 mg / mL for the chicken IgY - coated antibody. The concentration gradient ratio of the test strip manufactured with this coating concentration is better than 64.16, with higher sensitivity, better linearity, and the precision of all < 6%. The selection process of the coating concentrations of the test line and quality control line is as follows: The INS - coated antibody is diluted to 0.8 mg / mL, 1.0 mg / mL, and 1.2 mg / mL respectively to prepare 3 kinds of T - line coating solutions, and the chicken IgY - coated antibody is diluted to 0.8 mg / mL, 1.0 mg / mL, and 1.2 mg / mL respectively to prepare 3 kinds of C - line coating solutions. They are combined in pairs and then scribed onto the NC membrane with a gold - label scribing machine. There are a total of 9 combinations, and the specific combinations are shown in Table 12 below:

[0160] Table 12 Coating Concentration Combinations of the T Line and C Line

[0161]

[0162] Table 13 T / C Precision Results of Different Coating Concentration Combinations of the T Line and C Line

[0163]

[0164]

[0165] From Table 13 and Figure 4 it can be known that the concentration gradient ratio of the reference products of each enterprise in Combination 5 is relatively the best at 64.16, with higher sensitivity, better linearity, and the precision of all < 6%. The coating concentrations of 1.0 mg / mL for the INS - coated antibody and 1.0 mg / mL for the chicken IgY - coated antibody are selected as the final coating concentrations of the T line and C line.

[0166] Furthermore, the reference product prepared with the insulin antigen of Beijing Fubo Biotechnology Co., Ltd. is an enterprise reference product. The prepared reference product has a long storage time and good stability, which can save time costs. The reference product numbers 1 to 5 are used to evaluate raw materials such as insulin antibodies and other R & D processes of test strips.

[0167] The usage method of an insulin fluorescence immunochromatography assay kit provided by this application is as follows:

[0168] 1. Use the Guangzhou Lanbo AFS-1000 dry fluorescence immunoassay analyzer, and the test is carried out at room temperature of 10 - 30°C.

[0169] 2. Turn on the instrument and insert the chip with the same reagent batch number;

[0170] 3. Use a pipette to aspirate 100 μL of the sample and add it to the diluent, then mix it well by inverting up and down;

[0171] 4. Open the aluminum foil bag, take out the test card, and place it horizontally on the table;

[0172] 5. Use a pipette to aspirate 100 μL of the diluted and mixed sample and add it to the sample addition hole of the test card;

[0173] 6. Select the sample type "plasma or serum" on the supporting instrument;

[0174] 7. Immediate test: After reacting at room temperature for 15 minutes, put the test card into the instrument card slot, select the "immediate test" mode, and click "test"; Standard test: Put the test card into the instrument card slot, select the "standard test" mode, click "test", the instrument automatically times, and after the timing ends, it automatically tests and displays the result;

[0175] Click "print" to print the test result report.

[0176] Perform the following performance tests on the kit prepared in the examples:

[0177] 1. Evaluation of the lowest detection limit of the analytical performance of the test strip:

[0178] Use the test strip prepared in the example and repeat the determination 20 times with the 0 mIU / L INS reference product. Calculate the average value (M) and standard deviation (SD) of the relative T / C, and obtain the corresponding value of M + 2SD. Substitute it into the linear equation obtained by two-point regression fitting of the dose-response curve or the T / C values of the zero-concentration calibrator and the adjacent calibrator concentration to obtain the corresponding concentration value, which is the lowest detection limit of the product.

[0179] According to the usage method, use this instrument to repeat the determination 20 times with the 0 mIU / L INS reference product;

[0180] The test results are as follows:

[0181] Table 14 Detection result (T / C) value

[0182]

[0183] It can be seen from the test data in Table 14 that the blank limit results of the insulin (INS) assay kits (fluorescent immunochromatography method) for three batches are 0.53 mIU / L, 0.54 mIU / L, and 0.49 mIU / L respectively, all of which meet the above design requirements. Therefore, not greater than 2.0 mIU / L is used as the minimum detectable limit performance index of this product.

[0184] 2. Linear evaluation of the analytical performance of the kit:

[0185] Use the kit in the examples to test the linear samples, and statistically analyze the test data according to "CLSI EP6-A Linear Evaluation of Quantitative Detection Methods: Statistical Methods". If it meets the acceptable criteria of the design experiment, it is used as the linear performance index of the INS kit.

[0186] The steps of linear analysis are as follows:

[0187] A. Preparation of linear samples:

[0188] B. Preparation of low-concentration samples: Collect human serum samples with a concentration of 2.0 mIU / L;

[0189] C. Preparation of high-concentration samples: Add an appropriate amount of insulin antigen to the treated negative serum matrix to prepare a high-concentration sample with a concentration of about 200 mIU / L.

[0190] D. Preparation methods for samples of each concentration:

[0191] Mix the high- and low-concentration samples into 11 concentrations according to Table 15, and use 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 to represent each concentration sample.

[0192] Establishment of linearity:

[0193] Take three batches of reagents to measure the linear concentration samples numbered 1-11 above, and measure each sample 4 times repeatedly. Calculate the relative deviation (B) and linear correlation coefficient (r) between the average value (M) of the 4 test results of each sample and the theoretical value according to the following formulas (1) and (2).

[0194] B = (M - T) / T × 100%.................... (Formula 1) Where:

[0195] B - Relative deviation; M - Average value of test results; T - Theoretical value.

[0196] In the formula:

[0197] r——regression coefficient; X i ——theoretical concentration; Y i ——the average value of the results of 4 repeated detections corresponding to the theoretical concentration;

[0198] i——1, 2, 3, …… n.

[0199] Verification of linearity:

[0200] Take three batches of reagents and measure the concentration points of 1.98 mIU / L, 10 mIU / L, 50 mIU / L, 100 mIU / L, and 160.15 mIU / L respectively. Each sample is measured 3 times repeatedly. Calculate the average value (M) and the linear correlation coefficient (r) of the 3 detection results of each sample according to the following formula (2).

[0201] Table 15 Concentrations of each sample

[0202]

[0203] Table 16 Test results of test strips of batch 1 Unit: mIU / L

[0204]

[0205]

[0206] Table 17 Test results of test strips of batch 2 Unit: mIU / L

[0207]

[0208] Table 18 Test results of test strips of batch 3 Unit: mIU / L

[0209]

[0210] As can be seen from Tables 16 - 18, the relative deviations of the average values of the test results of linear sample 10 (180.20 mIU / L) and linear sample 11 (200.00 mIU / L) both exceed 15%, not meeting the requirements, so these two concentration points are excluded; from Figures 5 - 7 it can be seen that the three batches of reagents show a linear relationship. In the linear range of [2, 160] mIU / L, the correlation coefficients r are 0.9983, 0.9973, and 0.9965 respectively. Thus, it can be known that the test results of linear samples 1 - 9 simultaneously meet the above acceptance conditions, that is, the linear range of this determination reagent is determined to be [2, 160] mIU / L.

[0211] Table 19 Correlation Coefficient of Test Strips for Batch 1 Unit: mIU / L

[0212]

[0213] Table 20 Correlation Coefficient of Test Strips for Batch 2 Unit: mIU / L

[0214]

[0215] Table 21 Correlation Coefficient of Test Strips for Batch 3 Unit: mIU / L

[0216]

[0217] As can be seen from Tables 19 - 21, in the range of [2, 160] mIU / L, the test results of the correlation coefficient (r) of the insulin (INS) test strips (fluorescence immunochromatography method) for the three batches are all greater than 0.9900.

[0218] The above data shows that both the linear establishment and verification meet the above design requirements. Therefore, in the range of [2, 160] mIU / L, the correlation coefficient r of linear regression ≥ 0.9900 is used as the linear performance index of this product.

[0219] 3. Accuracy Evaluation of the Analytical Performance of Test Strips:

[0220] Use the test strips with batch numbers 1, 2, and 3 prepared in the examples, and according to the set acceptable standards, test the accuracy of the test strips according to the detection method required by the technical requirements.

[0221] Table 22 Experimental Materials

[0222]

[0223] The detection of the accuracy reference product is carried out as follows:

[0224] Measure the insulin enterprise reference products at approximately 10.02 mIU / L and 100.10 mIU / L, and perform the detection according to the steps in the instruction manual. After each measurement is repeated 3 times, the average value result is recorded as M, and calculate the ratio of the average value of the measured value to the theoretical value.

[0225] B = M / T..................................(1)

[0226] Where: B - ratio; M - average value of the measured concentration; T - theoretical concentration.

[0227] The detection results are as follows:

[0228] Table 23 Accuracy Test Results

[0229]

[0230] As can be seen from Table 23, the accuracies all meet the above design requirements. Therefore, the ratio of the mean value of the measured values to the theoretical value between 0.85 and 1.15 is used as the accuracy performance index of this product.

[0231] 4. Precision evaluation of the analytical performance of the test strip:

[0232] Use the test strips with batch numbers 1, 2, and 3 prepared in the examples to test the repeatability reference product. Statistically analyze the test data according to the "CLSI EP5-A2 Document: Evaluation of Precision Performance of Quantitative Measurement Methods - Approved Guideline Second Edition", design the acceptable criteria, and verify the repeatability reference product according to the detection method required by the technical requirements. If the acceptable criteria are met, it is used as the within-run precision and between-batch precision performance index of the INS test strip.

[0233] Table 24 Experimental materials

[0234]

[0235]

[0236] Main analysis steps: Take three batches of this test strip with qualified quality inspection, and detect the INS enterprise reference products with concentrations of 10.02 mIU / L and 100.10 mIU / L by the same operator on the same instrument. Each sample is measured 2 times repeatedly for a total of 20 days. At the end of the evaluation, there are 60 pairs, that is, 120 test results. The within-run precision is calculated from 40 data in each batch measurement. The between-batch precision is calculated from all 120 data.

[0237] The test results are as follows:

[0238] Table 25 Results of within-run precision test data with the concentration unit of mIU / L

[0239]

[0240] Table 26 Results of between-batch precision test data with the concentration unit of mIU / L

[0241]

[0242]

[0243] Table 27 Summary table of precision analysis results

[0244]

[0245]

[0246] As can be seen from Tables 25 - 27, the within - batch coefficient of variation (CV) test results of the insulin (INS) assay test strips (fluorescence immunochromatography method) for three batches are all ≤ 15%. The between - batch coefficient of variation (CV) is all ≤ 20%, and the precision meets the above - mentioned design requirements. Therefore, the within - batch coefficient of variation (CV) should be ≤ 15%; the between - batch coefficient of variation (CV) should be ≤ 20%, which are used as the precision performance indicators of this product.

[0247] 5. Analytical specificity evaluation of the analytical performance of the test strip:

[0248] Use the test strips with batch numbers 1, 2, and 3 prepared in the examples. Refer to the "CLSI EP7 - A2 Clinical Biochemistry Interference Testing; Approved Guideline - Second Edition" to formulate the experiment, and conduct statistical analysis on the test results. If it meets the acceptable criteria of the designed experiment, it is used as the INS analytical specificity performance indicator.

[0249] Table 28 Experimental materials

[0250]

[0251] Experimental procedure:

[0252] A. Preparation and storage method of cross - samples:

[0253] Pro - Insulin: Dilute the Pro - Insulin purchased from Guangzhou Heyuan Biotechnology Co., Ltd. to 10 ng / mL with negative serum matrix;

[0254] C - Peptide: Dilute the C - Peptide purchased from Guangzhou Heyuan Biotechnology Co., Ltd. to 20 ng / mL with negative serum matrix. After aliquoting, seal and store at - 20 °C.

[0255] B. Preparation and storage method of endogenous and exogenous interfering substance samples:

[0256] Preparation of endogenous interference test samples:

[0257] Add bilirubin to the serum samples to make the final concentrations 7.5 mg / dL, 15 mg / dL, 30 mg / dL, and 60 mg / dL; add triglycerides to make the final concentrations 250 mg / dL, 500 mg / dL, 1000 mg / dL, and 2000 mg / dL; add ascorbic acid to make the final concentrations 125 mg / dL, 250 mg / dL, 500 mg / dL, and 1000 mg / dL; and the final concentration of INS is 10 mIU / L and 100 mIU / L.

[0258] Preparation of exogenous interference test samples:

[0259] Collect the blood of healthy volunteers using serum collection tubes, rapid serum collection tubes, lithium heparin anticoagulant tubes, EDTA-K2 anticoagulant tubes, and sodium citrate anticoagulant tubes. Add INS to a final concentration of 10 mIU / L and 100 mIU / L respectively, aliquot, seal, and store at -20°C.

[0260] The detection method is as follows:

[0261] a. Detect the specific reference products of pro-insulin at a concentration of 10 ng / mL and C-peptide at a concentration of 20 ng / mL, repeat each 3 times, and obtain the cross-reaction values with this reagent.

[0262] b. Detect the prepared endogenous and exogenous interfering substances, repeat the test 3 times for each concentration of the interfering substance, and calculate the relative deviation (%) of the test concentration.

[0263] The detection results are as follows:

[0264] Table 29 Cross-reaction test results of each batch

[0265]

[0266] As can be seen from Table 29, the cross-reaction values of the insulin (INS) test strips (fluorescence immunochromatography method) of the three batches with pro-insulin at 10 ng / mL and C-peptide at 20 ng / mL are not higher than 2.0 mIU / L.

[0267] Table 30 Test results of the endogenous interfering substance bilirubin test.

[0268]

[0269]

[0270] Table 31 Test results of the endogenous interfering substance triglyceride test

[0271]

[0272] Table 32 Test results of the endogenous interfering substance ascorbic acid test

[0273]

[0274] As can be seen from Tables 30 - 32, for the insulin (INS) test strips (fluorescence immunochromatography method) of the three batches, the bilirubin ≤ 30 mg / dL, ascorbic acid ≤ 500 mg / dL, and triglyceride ≤ 1000 mg / dL in the test samples, and the accuracy deviation is ≤ ±15%.

[0275] Table 33 Test Results of Exogenous Interference Substances in Serum

[0276]

[0277]

[0278] Table 34 Test Results of Exogenous Interference Substances in Plasma

[0279]

[0280] As can be seen from Tables 33 - 34, for the samples collected in serum collection tubes, rapid serum collection tubes, lithium heparin anticoagulant tubes, EDTA-K2 anticoagulant tubes, and sodium citrate anticoagulant tubes for testing insulin (INS) assay test strips (fluorescence immunochromatography) of three batches, the relative deviation does not exceed ±15%.

[0281] As can be seen from the above Tables 29 - 34: When bilirubin ≤ 30 mg / dL, ascorbic acid ≤ 500 mg / dL, triglyceride ≤ 1000 mg / dL, and the samples collected in serum collection tubes, rapid serum collection tubes, lithium heparin anticoagulant tubes, EDTA-K2 anticoagulant tubes, and sodium citrate anticoagulant tubes have no effect on the test results.

[0282] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An insulin fluorescence immunoassay chromatography kit, comprising an insulin test card, a diluent and a chip. The test card includes a card shell and a test strip. The test strip includes a PVC bottom plate and a sample pad, a conjugate pad, an NC membrane and a blotting paper sequentially arranged on the PVC bottom plate. The NC membrane is provided with a test line and a quality control line. It is characterized in that: The test line is coated with an INS monoclonal antibody, and the coating concentration is 1.0 - 1.2 mg / ml; The quality control line is coated with a chicken IgY antibody, and the coating concentration is 1.0 mg / ml; The conjugate pad is sprayed with a fluorescent microsphere-labeled labeled antibody solution. The fluorescent microsphere-labeled labeled antibody solution is specifically a mixture of an INS antibody labeled with fluorescent microspheres and a goat anti-chicken IgY antibody containing fluorescent-labeled microspheres; The fluorescent microspheres are time-resolved fluorescent microspheres with a hydroxylated particle size of 300 nm produced by Changsha Meiniu; The coating solution formula is: 0.01M PBS with 1% sucrose at pH 7.4; After the sample pad is treated with the sample pad treatment solution and dried, the coated antibody of the test line and the labeled antibody are selected as the INS antibody from Hangzhou Longji Biotechnology Co., Ltd., and the coated antibody and labeled antibody of the quality control line are selected as the chicken IgY and goat anti-chicken IgY antibodies from Ningbo Maiyue; The sample pad treatment solution is prepared from the following components: 10 mg of anti-human RBC antibody, 20 mg of blocker 001, 10 g of bovine serum albumin, 5 ml of Tween-20, and 1000 ml of 0.2M boric acid-borax buffer at pH 8.

0.

2. An insulin fluorescence immunoassay chromatography kit according to claim 1, It is characterized in that, The spraying amount of the labeled antibody solution is 4 μL / cm.

3. An insulin fluorescence immunoassay chromatography kit according to claim 1, It is characterized in that, The steps for labeling the INS antibody with fluorescent microspheres are as follows: A. Take a 2 ml centrifuge tube and add 0.2 ml of labeling buffer MES; take 20 μL of fluorescent microspheres with a solid content of 1% and mix well in the labeling buffer, and vortex again to mix well; B. Add 20 μL of labeling activation solution and mix well, and rotate and mix for 30 min; C. Under the condition of a centrifugal speed of 10000 rpm, centrifuge for 30 min; discard the supernatant, add 200 μL of labeling buffer and ultrasonically oscillate; D. Add 10 μg of INS labeled antibody and mix well, and rotate and mix for 30 min; E. Add 20 μL of labeling blocking solution and ultrasonically treat, and rotate and mix for 2 h; F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of labeling preservation solution and ultrasonically treat; G. Transfer the labeled product into a new 2 ml centrifuge tube washed with 0.8 ml of labeling preservation solution, and vortex to mix well.

4. An insulin fluorescence immunoassay chromatography kit according to claim 1, It is characterized in that, The steps for labeling the goat anti-chicken IgY antibody with fluorescent microspheres are as follows: A. Take a 2 ml centrifuge tube and add 0.2 ml of labeling buffer MES; take 20 μL of fluorescent microspheres with a solid content of 1% and mix well in the labeling buffer, and vortex again to mix well; B. Add 20 μL of labeling activation solution and mix well, and rotate and mix for 30 min; C. Centrifuge at 10,000 rpm for 30 min; discard the supernatant, add 200 μL of labeling buffer and then perform ultrasonic oscillation; D. Add 10 μg of goat anti - chicken IgY antibody and mix well, rotate and mix for 30 min; E. Add 20 μL of labeling blocking solution and then perform ultrasonic treatment, rotate and mix for 2 h; F. Centrifuge for 30 min; discard the supernatant, add 0.2 ml of labeling preservation solution and perform ultrasonic treatment; G. Transfer the labeled substance into a new 2 - ml centrifuge tube washed with 0.8 ml of labeling preservation solution, and vortex - mix well.

5. A kit for determining insulin by fluorescence immunochromatography according to claim 1, characterized in that the dilution solution formula is: 0.01 M PBS pH 7.4 and 0.05% Proclin 300.

6. A preparation method of a kit for determining insulin by fluorescence immunochromatography according to any one of claims 1 - 5, characterized in that it includes preparing a test strip by the following steps: Step 1: Set a detection line and a quality control line on the NC membrane. Prepare coating working solutions of INS monoclonal antibody and chicken IgY antibody at concentrations of 1.0 - 1.2 mg / ml and 1.0 mg / ml respectively, and use a gold - labeled scribing machine to scribe them onto the NC membrane at a coating amount of 1.0 - 1.2 mg / ml, and place the NC membrane in an electro - thermal blast drying oven for drying for 16 - 24 h; Step 2: Use a gold - spraying scribing machine to spray the antibody solution labeled with fluorescent microspheres onto the conjugate pad at a spraying amount of 4 μL / cm and then dry; Step 3: Treat the sample pad with the sample pad treatment solution and then dry; Step 4: Stick the treated sample pad, conjugate pad, NC membrane and absorbent paper onto the PVC board in sequence to assemble a test strip.

7. A preparation method of a kit for determining insulin by fluorescence immunochromatography according to claim 6, characterized in that the labeling preservation solution is prepared from the following components: 20 g of bovine serum albumin, 20 g of sucrose, 5 g of casein, 50 g of trehalose, 0.5 ml of Proclin 300, and 1000 ml of 0.02 M Tris buffer with a pH of 8.0.

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