Joint detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 and preparation method thereof

Abstract

The invention relates to the technical field of biomedical examination, and discloses a joint detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 and a preparation method thereof.The kit comprises a joint detection reagent card and a diluent, and the joint detection reagent card comprises a card shell and a reagent strip; the reagent strip is composed of a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially pasted on the PVC bottom plate, the combination pad contains a quantum dot-labeled mouse anti-albumin monoclonal antibody, the reagent card further comprises a release pad, a quantum dot-labeled antibody mixed solution is sprayed on the release pad, and the release pad contains a quantum dot-labeled mouse anti-albumin monoclonal antibody. The quantum dot-labeled antibody mixed solution is obtained by mixing an HGH quantum dot-labeled antibody mixed solution, an IGF-1 quantum dot-labeled antibody mixed solution and an IGFBP-3 quantum dot-labeled antibody according to the mass ratio of 1: 1: 1. According to the scheme, three results can be obtained through one test, the error problem caused by the difference of operation conditions is avoided, and the detection accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical testing technology, and in particular to a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 and a preparation method thereof. Background Art

[0002] Human Growth Hormone (HGH) is a single-chain, non-glycosylated, hydrophilic globulin composed of 191 amino acids, with a molecular weight of 21,700 Da and an isoelectric point of 4.9. The cysteine ​​residues at positions 53 and 165 and 182 and 189 are linked by disulfide bonds; about 75% of the growth hormone secreted by the eosinophils of the anterior pituitary gland is secreted in this molecular structure, and 5%-10% is secreted in the form of 20 kDa. The difference between the 20 kDa form of growth hormone and the 22 kDa form is that the second coding exon of the former is excised from the RNA by 32 to 46 amino acid coding sequences. There are about 50 α-helices in the HGH conformation. From the three-dimensional structure, HGH includes four antiparallel α-helices, of which three α-helices play a key role. The cells that secrete HGH (in the posterior and lateral part of the adenohypophysis) contain round and dense secretory granules with a diameter of about 300-350 nm. HGH is a protein hormone that does not produce physiological effects through target glands. Its receptors are found throughout the body. Experimental studies using 125I-labeled HGH have found that HGH receptors are on the cell membrane. There are many receptors on the cell surfaces of the liver, kidney, spleen, pancreas, intestine, adrenal cortex, heart and skeletal muscle that bind to 125I-GH. The mechanism of action of HGH is generally believed to be through cAMP. After binding to target cells, it can change the flow of amino acids and metabolites and induce the synthesis of certain specific proteins and nucleic acids.

[0003] Insulin like growth factor-1 (IGF-1) is a polypeptide containing 70 amino acids, which has the effect of promoting cartilage growth. It is mainly produced by hepatocytes, and a small part is produced by osteoblasts and matrix cells themselves. It is a growth hormone-dependent cytokine with a wide range of effects. It can act on target cells through endocrine pathways, and can also participate in the growth regulation of local tissue cells through autocrine and paracrine methods. It has the effect of promoting mitosis of osteoblasts, and can also stimulate the AKP activity of osteoblasts to promote the synthesis of osteocalcin, and also has an effect on other differentiation functions of osteoblasts. The production of IGF-1 is affected by many factors such as age, gender, nutritional status and the release of growth hormone. Its concentration is low at birth, gradually increases in children and adolescence, and then decreases with age. In some children with dwarfism, growth hormone deficiency, dwarfism, etc., due to the different secretion of hormones by the pituitary gland, thyroid gland, etc., as well as other factors, their serum IGF-1 and IGFBP-3 levels can be affected and significantly reduced.

[0004] Insulin-like growth factor binding protein-3 (IGF-binding protein-3, IGFBP-3) is a macromolecular protein that mediates the synthesis of HGH. It is synthesized and secreted by a variety of cells and is the main carrier of IGF-1 in the blood. IGF-1 and IGFBP-3 exist in the form of a conjugate. IGF-1 can only undergo a series of biochemical reactions and exert its physiological effects after binding to its specific receptor. IGFBP-3 is the most abundant binding protein in the human body and has the strongest effect in circulation. It binds most of the free IGF-1 in the serum. After IGFBP-3 binds to IGF-1, it can reduce the concentration of free IGF-1, which is beneficial to enhance and exert the activity of IGF-1 receptors, prolong the half-life of IGF-1, and increase the level in serum.

[0005] At present, HGH, IGF-1, and IGFBP-3 mostly exist in the form of single detection kits. The detection methods include electrochemiluminescence, enzyme-linked immunosorbent assay, etc., which have the disadvantages of low detection efficiency and low detection sensitivity. In the clinical diagnosis process, only one single result of HGH, IGF-1, and IGFBP-3 cannot provide strong support for the diagnosis result. Testing the three indicators separately is not only time-consuming and labor-intensive, but also will cause certain experimental errors due to differences in detection conditions and detection time, which will have an adverse effect on the accuracy of the result judgment. Summary of the invention

[0006] The present invention aims to provide a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 and a preparation method thereof, so as to realize the combined detection of human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 while ensuring the sensitivity and accuracy of the detection.

[0007] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3, comprising a combined detection reagent card and a diluent, wherein the combined detection reagent card comprises a card shell and a reagent strip, wherein the reagent strip comprises a sample pad, a binding pad, a nitrocellulose membrane and an absorbent paper which are sequentially pasted on a PVC bottom plate, and the binding pad contains a mouse anti-albumin monoclonal antibody labeled with quantum dots, and the reagent card also comprises a release pad, wherein the release pad is sprayed with a quantum dot-labeled antibody mixed solution, and the quantum dot-labeled antibody mixed solution is obtained by mixing an HGH quantum dot-labeled antibody mixed solution, an IGF-1 quantum dot-labeled antibody mixed solution and an IGFBP-3 quantum dot-labeled antibody mixed solution in a mass ratio of (0.5-3):(0.5-3):(0.5-3).

[0008] Preferably, as an improvement, the mass ratio of the HGH quantum dot-labeled antibody mixture, the IGF-1 quantum dot-labeled antibody mixture and the IGFBP-3 quantum dot-labeled antibody mixture is (1-2): (1-2): (1-2); and the spraying amount of the quantum dot-labeled antibody mixture solution on the release pad is 1.8-2.2 μL / cm.

[0009] Preferably, as an improvement, a human growth hormone detection line, an insulin-like growth factor-1 detection line, an insulin-like growth factor binding protein-3 detection line and a quality control line are provided on the nitrocellulose membrane close to the sample addition hole, and each detection line is coated with a corresponding human growth hormone antibody, insulin-like growth factor-1 antibody, insulin-like growth factor binding protein-3 antibody, and the quality control line is coated with sheep anti-mouse IgG.

[0010] Preferably, as an improvement, the release pad and the sample pad are both pretreated, and the pretreatment method is: immersing the release pad / sample pad in a release pad / sample pad optimization buffer, and then drying and controlling the humidity to below 30%.

[0011] Preferably, as an improvement, the release pad optimization buffer comprises tris(hydroxymethyl)aminomethane at a concentration of 45-55 mmol / L, bovine serum albumin at a mass percentage of 0.8-1.2%, Tween 20 at a mass percentage of 0.4-0.6%, sucrose at a mass percentage of 1.8-2.2%, proclin300 at a mass percentage of 0.08-0.12%, and the remainder purified water.

[0012] Preferably, as an improvement, the sample pad optimization buffer comprises 45-55 mmol / L tris(hydroxymethyl)aminomethane, 0.4-0.6% by mass of Tween 20, 0.08-0.12% by mass of proclin 300, and the balance of purified water.

[0013] Preferably, as an improvement, the kit realizes the combined quantitative detection of human growth hormone, insulin-like growth factor-1, and insulin-like growth factor binding protein-3 based on quantum dot fluorescence immunochromatography, with a recovery rate of 90%-110%, a linear range of human growth hormone of 0.10-50.0 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%; a linear range of insulin-like growth factor-1 of 20-1500 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%; and a linear range of insulin-like growth factor binding protein-3 of 70-18000 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%.

[0014] Preferably, as an improvement, a method for preparing a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 comprises the following steps:

[0015] Step 1: Biotin labeling: label the antibodies with HGH biotin, IGF-1 biotin, and IGFBP-3 biotin respectively;

[0016] Step 2, preparation of release pad: dilute the quantum dot dilution buffer, and respectively react with the three labeled antibodies obtained in step 1 to obtain three quantum dot-labeled antibody mixed solutions, mix the three quantum dot-labeled antibody mixed solutions in a mass ratio of 1:1:1, spray them on the release pad, and dry them to obtain a release pad coated with quantum dot-labeled mouse anti-albumin monoclonal antibodies;

[0017] Step 3, dilute HGH antibody, IGF-1 antibody, IGFBP-3 antibody and goat anti-mouse IgG and spray them on the nitrocellulose membrane, dry them to obtain test line 1, test line 2, test line 3 and quality control line, and stick the nitrocellulose membrane to the PVC base plate;

[0018] Step 4: Assemble the bottom plate with the nitrocellulose membrane treated in step 3, the sample pad, the release pad treated in step 2, and the absorbent paper to obtain a detection reagent card.

[0019] Preferably, as an improvement, in step 2, the quantum dot dilution buffer includes tris(hydroxymethyl)aminomethane with a concentration of 90-110 mmol / L, bovine serum albumin with a mass percentage concentration of 0.8-1.2%, Tween 20 with a mass percentage concentration of 0.4-0.6%, sucrose with a mass percentage concentration of 1.8-2.2%, proclin300 with a mass percentage concentration of 0.08-0.12%, and the remainder purified water.

[0020] Preferably, as an improvement, in step 2, the quantum dot buffer is diluted at 1:10-1:15, and the mixed mass ratio of the diluted quantum dot dilution buffer and the three labeled antibodies is 1:0.1-1:0.3.

[0021] The principles and advantages of this scheme are: At present, HGH, IGF-1, and IGFBP-3 mostly exist in the form of single detection kits, and the detection methods include electrochemiluminescence, enzyme-linked immunosorbent assay, etc., which have the disadvantages of low detection efficiency and low detection sensitivity; and in the clinical diagnosis process, only one single result of HGH, IGF-1, and IGFBP-3 cannot provide strong support for the diagnosis result, and the detection of the three indicators separately is not only time-consuming and labor-intensive, but also will cause certain experimental errors due to differences in detection conditions and detection time, which will have an adverse effect on the accuracy of the result judgment. Based on this, this technical solution uses a combined detection method to concentrate the antibodies for detecting HGH, IGF-1, and IGFBP-3 on the same release pad. After the test object is processed by the sample pad, it is combined with the quantum dot-labeled antibody conjugate on the release pad respectively, and then captured by the corresponding coated antibodies on the nitrocellulose membrane and fixed to the nitrocellulose membrane to complete the combination and fluorescence. Then, a dry fluorescent immunoassay is used to detect the fluorescence intensity and calculate and output the corresponding concentration values ​​of the three substances to complete the quantitative detection, which not only ensures the compatibility of the detection, but also greatly improves the detection efficiency. Moreover, since this solution can obtain three results through one test, it avoids the error problem caused by differences in operating conditions and improves the accuracy of the detection.

[0022] During the technical research and development period, how to completely separate the three substances in the test object without interfering with each other is one of the difficulties in the research and development of this technical solution. After determining to use the fluorescent immunoassay, this solution first selects the coated antibodies, labeled antibodies and quantum dots determined in the HGH, IGF-1, and IGFBP-3 single fluorescent immunoassay detection projects that have been studied for combined experiments. In the first version of the scheme, the HGH, IGF-1, and IGFBP-3 labeled antibodies are combined with the corresponding quantum dots for each item, and then mixed together for a secondary reaction. After the labeled antibody-quantum dot mixture after the secondary reaction is sprayed onto the release pad, it is combined with the coated antibodies prepared for the three single tests to prepare the detection reagent strips, and each single quality control product is used for verification. The results show that the actual value of each single indicator deviates greatly from the theoretical value of the quality control product and the value is irregular. According to the analysis of the results, different batches of quantum dots will interfere with each other after mixing. In the second version of the experimental plan, each individual test item of HGH, IGF-1, and IGFBP-3 was redeveloped and a unified batch number of quantum dots was determined. After mixing the labeled antibodies together, they were combined with quantum dots. After the reacted labeled antibody-quantum dot mixture was sprayed onto the release pad, it was combined with the coated antibodies prepared for the three individual tests to prepare the test reagent strips, and each individual quality control product was used for verification. The results showed that the IGFBP-3 and IGF-1 index values ​​were smaller than the theoretical values ​​of the quality control products, and the HGH index value was larger than the theoretical value of the quality control products. According to the analysis of the results, the HGH, IGF-1, and IGFBP-3 labeled antibodies had different binding abilities with quantum dots, which interfered with the test results. In subsequent studies, HGH, IGF-1, and IGFBP-3 labeled antibodies were combined with quantum dots for reaction, mixed together for secondary reaction, and then the labeled antibody-quantum dot mixture after the secondary reaction was sprayed onto the release pad, and then combined with the coated antibodies prepared for the three single tests to prepare the test reagent strips and used the single quality control products for verification. The results showed that the indicators of the three items were smaller than the theoretical values ​​of the quality control products, but the trends were consistent. According to the analysis of the results, the scheme is feasible. The sensitivity and linearity of each specific project can be adjusted by continuing to adjust the ratio of labeled antibodies and quantum dots, the concentration of coated antibodies, and the treatment methods of the sample pad and the release pad. It was determined that Scheme 3 would be used for subsequent experiments.

[0023] In summary, the beneficial effects of this technical solution are: when preparing the sample pad, the sample pad is treated to prevent damage to the relatively easy-to-decompose IGFBP-3; when preparing the release pad, the release pad is treated and the composition of the optimized buffer when treating the release pad and the sample pad is fully optimized, and finally the quantum dot-labeled antibodies of the three substances are successfully mixed together without interfering with each other; and through multiple tests and verifications, the appropriate antibodies and corresponding ratios, as well as quantum dots that can meet the detection requirements, are found. Finally, the kit prepared by this solution can obtain three results through a single test, taking into account the efficiency and accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of the reagent strip in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0026] The reference numerals in the drawings of the specification include: reagent strip 1, PVC base plate 2, sample pad 3, nitrocellulose membrane 4, conjugate pad 5, absorbent paper 6, HGH antibody 7, IGF-1 antibody 8, IGFBP-3 antibody 9, goat anti-mouse IgG antibody 10.

[0027] Example 1

[0028] like Figure 1 As shown, a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 includes a human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 combined detection reagent card, a human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 diluent, a desiccant, an aluminum foil bag, an instruction manual and an SD card.

[0029] The human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 detection reagent card includes a card shell and a reagent strip 1;

[0030] The reagent strip 1 comprises a sample pad 3, a conjugation pad 5, a nitrocellulose membrane 4, an absorbent paper 6 and a PVC bottom plate 2; the sample pad 3, the conjugation pad 5, the nitrocellulose membrane 4 and the absorbent paper 6 are sequentially pasted on the PVC bottom plate 2; the conjugation pad 5 contains a mouse anti-albumin monoclonal antibody labeled with quantum dots; a human growth hormone detection line, an insulin-like growth factor-1 detection line, an insulin-like growth factor binding protein-3 detection line and a quality control line are sequentially arranged on the nitrocellulose membrane 4, and the human growth hormone detection line is close to the sample addition hole; the detection lines are respectively coated with human growth hormone antibodies (HGH antibodies 7), insulin-like growth factor-1 antibodies (IGF-1 antibodies 8), and insulin-like growth factor binding protein-3 antibodies (IGFBP-3 antibodies 9); and the quality control line is coated with sheep anti-mouse IgG antibodies 10.

[0031] Example 2

[0032] A method for preparing a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 comprises the following steps:

[0033] Step 1: Biotin labeling

[0034] S1, HGH biotin labeling:

[0035] (1) Weigh a certain amount of biotin, dissolve and dilute it with PBS solution (pH=7.2-7.4) to a final concentration of 5.56 mg / mL.

[0036] (2) Dilute the antibody to be labeled to 1 mg / mL using PBS solution (PH=7.2-7.4).

[0037] (3) The amount of biotin added is 3% of the labeled amount. The calculated corresponding volume of biotin is mixed with the HGH labeled antibody dilution solution, and the total volume is not less than 100 μL.

[0038] (4) Incubate the reaction in a dark place at room temperature for 1-1.5 h.

[0039] (5) After the reaction is completed, the reaction solution is transferred to a dialysis bag and dialyzed with a PBS solution (PH = 7.2-7.4) for 4-6 hours, and then replaced with a new PBS solution (PH = 7.2-7.4) for overnight dialysis. The next day, the PBS solution (PH = 7.2-7.4) is replaced again for another 4-6 hours of dialysis.

[0040] (6) After dialysis, collect and add 10% of the recovered BSA, mix well, and then divide into small portions and store at -20°C.

[0041] S2, IGF-1 biotin labeling:

[0042] (1) Weigh a certain amount of biotin, dissolve and dilute it with PBS solution (pH=7.2-7.4) to a final concentration of 5.56 mg / mL.

[0043] (2) Dilute the antibody to be labeled to 1 mg / mL using PBS solution (PH=7.2-7.4).

[0044] (3) The amount of biotin added is 3% of the labeled amount. The calculated corresponding volume of biotin is mixed with the IGF-1 labeled antibody dilution solution, and the total volume is not less than 100 μL.

[0045] (4) Incubate the reaction in a dark place at room temperature for 1-1.5 h.

[0046] (5) After the reaction is completed, the reaction solution is transferred to a dialysis bag and dialyzed with a PBS solution (PH = 7.2-7.4) for 4-6 hours, and then replaced with a new PBS solution (PH = 7.2-7.4) for overnight dialysis. The next day, the PBS solution (PH = 7.2-7.4) is replaced again for another 4-6 hours of dialysis.

[0047] (6) After dialysis, collect and add 10% of the recovered BSA, mix well, and then divide into small portions and store at -20°C.

[0048] S3, IGFBP-3 biotin labeling:

[0049] (1) Weigh a certain amount of biotin, dissolve and dilute it with PBS solution (pH=7.2-7.4) to a final concentration of 5.56 mg / mL.

[0050] (2) Dilute the antibody to be labeled to 1 mg / mL using PBS solution (PH=7.2-7.4).

[0051] (3) The amount of biotin added is 3% of the labeled amount. The calculated corresponding volume of biotin is mixed with the IGFBP-3 labeled antibody dilution solution, and the total volume is not less than 100 μL.

[0052] (4) Incubate the reaction in a dark place at room temperature for 1-1.5 h.

[0053] (5) After the reaction is completed, the reaction solution is transferred to a dialysis bag and dialyzed with a PBS solution (PH = 7.2-7.4) for 4-6 hours, and then replaced with a new PBS solution (PH = 7.2-7.4) for overnight dialysis. The next day, the PBS solution (PH = 7.2-7.4) is replaced again for another 4-6 hours of dialysis.

[0054] (6) After dialysis, collect and add 10% of the recovered BSA, mix well, and then divide into small portions and store at -20°C.

[0055] Step 2: Preparation of release pad

[0056] S1. Preparation of HGH quantum dot-labeled antibody mixture:

[0057] The quantum dot dilution buffer is diluted at a ratio of 1:10-1:15, and the diluted quantum dots are mixed with the HGH biotin-labeled antibody labeling solution prepared in step S1 at a mass ratio of 1:0.1-1:0.3, and reacted for 0.5-4 hours;

[0058] S2. Preparation of IGF-1 quantum dot-labeled antibody mixture:

[0059] The quantum dots are diluted with a quantum dot dilution buffer at a ratio of 1:10-1:15, and the diluted quantum dots are mixed with the IGF-1 biotin-labeled antibody labeling solution prepared in step S2 at a ratio of 1:0.1-1:0.3, and reacted for 0.5-4 hours;

[0060] S3, IGFBP-3 quantum dot-labeled antibody mixture preparation:

[0061] The quantum dots are diluted with a quantum dot dilution buffer at a ratio of 1:10-1:15, and the diluted quantum dots are mixed with the IGFBP-3 biotin-labeled antibody labeling solution prepared in step S3 at a ratio of 1:0.1-1:0.3, and reacted for 0.5-4 hours;

[0062] S4. Preparation of release pad:

[0063] The three quantum dot-labeled antibody mixtures prepared in the above steps S1 to S3 are mixed together in a ratio of 1:1:1, reacted for 0.5-4h, and then sprayed on a release pad in an amount of 1.8-2.2μL / cm, and dried to obtain a release pad coated with quantum dot-labeled mouse anti-albumin monoclonal antibody, wherein the quantum dot dilution buffer comprises tris(hydroxymethyl)aminomethane with a concentration of 90-110mmol / L, bovine serum albumin with a mass percentage concentration of 0.8-1.2%, Tween 20 with a mass percentage concentration of 0.4-0.6%, sucrose with a mass percentage concentration of 1.8-2.2%, proclin300 with a mass percentage concentration of 0.08-0.12%, and the balance of purified water.

[0064] Step 3, sticking the nitrocellulose membrane to the PVC base plate, diluting the HGH antibody to 0.5-1.0 mg / mL with a printing buffer, diluting the IGF-1 antibody to 0.5-1.5 mg / mL with a printing buffer, diluting the IGFBP-3 antibody to 0.5-1.0 mg / mL with a printing buffer, diluting the goat anti-mouse IgG to 0.8-1.5 mg / mL with a printing buffer, and then spraying them on the nitrocellulose membrane in an amount of 1.8-2 μL / cm, respectively, and drying to obtain test line 1, test line 2, test line 3 and quality control line, wherein the printing buffer includes a PBS buffer with a concentration of 0.01-0.03 mol / L and a pH of 7.0-7.4, a mass percentage concentration of trehalose of 0.8-1.2%, a concentration of 8-12 mmol / L of ethylenediaminetetraacetic acid, and a mass percentage concentration of 0.08-0.12% of proclin300;

[0065] Step 4: Assemble the PVC base plate with the nitrocellulose membrane attached thereto after the treatment in step 3, the sample pad, the release pad after the treatment in step 2, and the absorbent paper to obtain a detection reagent card.

[0066] The release pad is a pretreated release pad, and the pretreatment method is: immersing the release pad in a release pad optimization buffer, and then drying and controlling the humidity below 30%, wherein the release pad optimization buffer includes 50mmol / L tris(hydroxymethylaminomethane) (tris) with a pH of 8.2, 1% bovine serum albumin (BSA) with a mass percentage concentration, 0.5% Triton X-100 with a mass percentage concentration, 2% sucrose with a mass percentage concentration, 5% trehalose with a mass percentage concentration, 0.1% proclin300 with a mass percentage concentration, and the remainder purified water.

[0067] The sample pad is a pretreated sample pad, and the pretreatment steps are: immersing the sample pad in a sample pad optimization buffer, and then drying and controlling the humidity to below 30%, wherein the sample pad optimization buffer includes 50mmol / L tris (tris) with a pH of 8.2, 0.5% polyvinylpyrrolidone (PVP) with a mass percentage concentration of 0.5%, 0.2% casein sodium salt with a mass percentage concentration of 0.5%, and the remainder purified water.

[0068] Note: HGH, IGF-1, and IGFBP-3 antibodies were purchased from Beijing Jiufeng Runda Biotechnology Co., Ltd.

[0069] Quantum dots were purchased from SaiYa Hebei Biotechnology Co., Ltd.

[0070] Experimental Example Performance Evaluation

[0071] 1. Precision

[0072] 1.1 Human Growth Hormone (HGH) Precision

[0073] 30 reagent strips were randomly selected to test the company reference products with human growth hormone (HGH) concentrations of 1ng / mL, 10ng / mL, and 30ng / mL, respectively. 80μL of sample was added and tested using a dry fluorescent immunoassay after 10 minutes of reaction. The three levels of concentration were tested 10 times each. The test results are shown in Table 1:

[0074] Table 1 HGH intra-batch precision

[0075]

[0076]

[0077] 1.2 Precision of insulin-like growth factor-1 (IGF-1)

[0078] 30 reagent strips were randomly selected to test the company reference products with concentrations of 100 ng / mL, 400 ng / mL, and 800 ng / mL of insulin-like growth factor-1 (IGF-1). 80 μL of sample was added and tested using a dry fluorescent immunoassay after 10 minutes of reaction. The three levels of concentration were tested 10 times each. The test results are shown in Table 2:

[0079] Table 2 Intra-batch precision of IGF-1

[0080]

[0081] 1.3 Precision of insulin-like growth factor binding protein-3 (IGFBP-3)

[0082] 30 reagent strips were randomly selected to test the company reference products with concentrations of 800 ng / mL, 4000 ng / mL, and 8000 ng / mL of insulin-like growth factor binding protein-3, respectively. 80 μL of sample was added and tested using a dry fluorescent immunoassay after 10 minutes of reaction. The three levels of concentration were tested 10 times each. The test results are shown in Table 3:

[0083] Table 3 Intra-batch precision of IGFBP-3

[0084]

[0085] It can be seen from the above table that the maximum intra-batch precision of each individual item of HGH, IGF-1 and IGFBP-3 is 9.6%, which meets the requirement of precision within 15%.

[0086] 2. Accuracy

[0087] 2.1 Recovery experiment

[0088] 2.1.1 Human Growth Hormone (HGH) Recovery Experiment

[0089] The kit prepared in Example 1 was used for HGH recovery experiment. 80 μL of sample was added and the reaction was continued for 10 minutes before detection using a dry fluorescent immunoassay analyzer. The detection results are shown in Table 4:

[0090] Table 4 HGH recovery experimental results

[0091]

[0092] 2.1.2 Insulin-like growth factor-1 (IGF-1) recovery experiment

[0093] The kit prepared in Example 1 was subjected to an IGF-1 recovery experiment. 80 μL of the sample was added and the reaction was continued for 10 minutes before detection using a dry fluorescent immunoassay analyzer. The test results are shown in Table 5:

[0094] Table 5 IGF-1 recovery experiment results

[0095]

[0096] 2.1.3 IGFBP-3 recovery experiment

[0097] The kit prepared in Example 1 was subjected to an IGFBP-3 recovery experiment. 80 μL of the sample was added and the reaction was continued for 10 minutes before detection using a dry fluorescent immunoassay analyzer. The test results are shown in Table 6:

[0098] Table 6 IGFBP-3 recovery experiment results

[0099]

[0100]

[0101] It can be seen from the above table that the average recovery rates of HGH, IGF-1 and IGFBP-3 are between 97% and 102%, which meets the requirement of a recovery rate of 90% to 110%.

[0102] 2.2 Accuracy of enterprise reference products

[0103] 2.2.1 Accuracy of Human Growth Hormone (HGH) Corporate Reference Products

[0104] 9 reagent strips were randomly selected to test the company reference products with human growth hormone (HGH) concentrations of 1ng / mL, 10ng / mL, and 30ng / mL, respectively. 80μL of sample was added and tested using a dry fluorescent immunoassay after 10 minutes of reaction. The test results are shown in Table 7:

[0105] Table 7 Accuracy of HGH enterprise reference products

[0106]

[0107] 2.2.2 Accuracy of Insulin-like Growth Factor-1 (IGF-1) Corporate Reference Product

[0108] Nine reagent strips were randomly selected to test the company reference products with concentrations of 100 ng / mL, 400 ng / mL, and 800 ng / mL of insulin-like growth factor-1 (IGF-1), respectively. 80 μL of sample was added and the dry fluorescent immunoassay was used to test after 10 minutes of reaction. The test results are shown in Table 8:

[0109] Table 8 Accuracy of IGF-1 Enterprise Reference Products

[0110]

[0111]

[0112] 2.2.3 Accuracy of Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Corporate Reference Material

[0113] 9 reagent strips were randomly selected to test the company reference products with concentrations of 800 ng / mL, 4000 ng / mL, and 8000 ng / mL of insulin-like growth factor binding protein-3, respectively. 80 μL of sample was added and tested using a dry fluorescent immunoassay analyzer after a 10-minute reaction. The test results are shown in Table 9:

[0114] Table 9 Accuracy of IGFBP-3 Enterprise Reference Products

[0115]

[0116] It can be seen from the above table that the accuracy of the company's reference products HGH, IGF-1, IGFBP-3, each single test deviation is -5.2%-10.8%, which meets the requirement of relative deviation of ±20%.

[0117] 3. Linear range

[0118] 3.1 Human Growth Hormone (HGH) Linear Range

[0119] Human growth hormone (HGH) antigen diluent was used to dilute the human growth hormone (HGH) antigen to 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, and 50 ng / mL, and 80 μL was added for spotting. After reacting for 10 minutes, a dry fluorescent immunoassay was used for detection. Each concentration point was detected 3 times. The test results are shown in Table 10:

[0120] Table 10 HGH linear range

[0121]

[0122]

[0123] 3.2 Linear range of insulin-like growth factor-1 (IGF-1)

[0124] Insulin-like growth factor-1 (IGF-1) antigen diluent was used to dilute the insulin-like growth factor-1 (IGF-1) antigen to 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 1200 g / mL, and 1500 ng / mL, and 80 μL was added for spotting. After reacting for 10 minutes, the dry fluorescent immunoassay was used for detection. Each concentration point was detected 3 times. The detection results are shown in Table 11 below:

[0125] Table 11 IGF-1 linear range

[0126]

[0127] 3.3 Linear range of insulin-like growth factor binding protein-3 (IGFBP-3)

[0128] Insulin-like growth factor binding protein-3 (IGFBP-3) antigen diluent was used to dilute the insulin-like growth factor binding protein-3 (IGFBP-3) antigen to 70 ng / mL, 200 ng / mL, 600 ng / mL, 1500 ng / mL, 3000 ng / mL, 6000 ng / mL, 10000 ng / mL, and 18000 ng / mL, and 80 μL was added for spotting. After reacting for 10 minutes, the dry fluorescent immunoassay was used for detection. Each concentration point was detected 3 times. The detection results are shown in Table 12 below:

[0129] Table 12 IGFBP-3 linear range

[0130]

[0131]

[0132] As can be seen from the table above, HGH is within the linear range of 0.10-50.0ng / mL, the linear correlation coefficient r>0.9999, and the coefficient of variation within the linear range is less than 15%; IGF-1 is within the linear range of 20-1500ng / mL, the linear correlation coefficient r>0.9994, and the coefficient of variation within the linear range is less than 15%; IGFBP-3 is within the linear range of 70-18000ng / mL, the linear correlation coefficient r>0.9997, and the coefficient of variation within the linear range is less than 15%. This shows that this detection kit can meet the requirement of the preset target of research and development r≥0.990.

[0133] 4. Effect of sample pad and release pad optimization buffer on kit performance

[0134] In order to explore the effect of optimizing buffer treatment on the test results, the following study was conducted: HGH, IGF-1, and IGFBP-3 combined test strips prepared without sample pad treatment solution and release pad treatment solution were used to test the company's reference products. The test results are shown in Table 13: The results show that using sample pads and release pads that have not been treated with optimized buffer will lead to large deviations in the test results.

[0135] Table 13

[0136]

[0137] The results of the HGH, IGF-1, and IGFBP-3 combined test strips prepared after treatment with the sample pad treatment solution formula 1 and the release pad treatment solution formula 1 are as follows in Table 14:

[0138] The sample pad optimized buffer formula 1 includes 50 mmol / L tris(hydroxymethylaminomethane) (tris) with a pH of 8.2, 1% polyvinylpyrrolidone (PVP) by mass, 1% Tween 20 by mass, 0.5% casein sodium salt by mass, and the balance purified water.

[0139] Release pad optimized buffer formula 1 includes 50 mmol / L tris (tris) with a pH of 8.2, 0.2% bovine serum albumin (BSA) by mass, 0.1% Triton X-100 by mass, 1% sucrose by mass, 2% trehalose by mass, 0.1% proclin 300 by mass, and the remainder purified water.

[0140] Table 14

[0141]

[0142] The results of the HGH, IGF-1, and IGFBP-3 joint test strips prepared after treatment with the sample pad treatment solution formula 1 and the release pad treatment solution formula 2 for testing the company's reference products are as follows in Table 15:

[0143] The sample pad optimization buffer formula 1 is the same as above: including 50mmol / L tris(hydroxymethylaminomethane) (tris) with a pH of 8.2, 1% polyvinylpyrrolidone (PVP) with a mass percentage concentration, 1% Tween 20 with a mass percentage concentration, 0.5% casein sodium salt with a mass percentage concentration, and the balance purified water.

[0144] Release pad optimized buffer formula 2 includes 50 mmol / L tris (tris) with a pH of 8.2, 1% bovine serum albumin (BSA) by mass, 0.5% Triton X-100 by mass, 2% sucrose by mass, 5% trehalose by mass, 0.1% proclin 300 by mass, and the remainder purified water.

[0145] Table 15

[0146]

[0147] The results of the HGH, IGF-1, and IGFBP-3 joint test strips prepared after treatment with the sample pad treatment solution formula 2 and the release pad treatment solution formula 2 for testing the enterprise reference products are as follows in Table 16:

[0148] The sample pad optimized buffer formula 2 includes 50 mmol / L tris(hydroxymethylaminomethane) (tris) with a pH of 8.2, 0.5% polyvinylpyrrolidone (PVP) by mass, 0.5% Tween 20 by mass, 0.2% casein sodium salt by mass, and the balance purified water.

[0149] Release pad optimized buffer formula 2 includes 50 mmol / L tris (tris) with a pH of 8.2, 1% bovine serum albumin (BSA) by mass, 0.5% Triton X-100 by mass, 2% sucrose by mass, 5% trehalose by mass, 0.1% proclin 300 by mass, and the remainder purified water.

[0150] Table 16

[0151]

[0152] 5. Effect of Antibody Type and Ratio on Kit Performance

[0153] After optimizing the sample pad and release pad, the ratio of HGH, IGF-1, and IGFBP-3 antibodies was adjusted to Formula 1. The results of the test company reference products are as follows in Table 17:

[0154] Antibody formula 1 is: QD-3B21 (1:10 dilution): Bio-HGH-004 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGF-1-008 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGFBP-3-014 = 1:0.2 volume ratio mixed reaction for 30 minutes. After the reaction is completed, the three mixtures are mixed and reacted at a volume ratio of 1:1:1 for 30 minutes.

[0155] Among them: QD-3B21 is the batch number of quantum dots (the same below), and the purchase channel is Saiya Hebei Biotechnology Co., Ltd.; the HGH-004, IGF-1-008, 011, IGFBP-3-014, 012, 001 antibodies used are all purchased from Beijing Jiufeng Runda Biotechnology Co., Ltd.

[0156] Table 17

[0157]

[0158] The types of IGF-1 and IGFBP-3 antibodies were changed, and the ratio of HGH, IGF-1, and IGFBP-3 antibodies was adjusted to Formula 2. The results of the test of the enterprise reference product are as follows in Table 18:

[0159] Antibody formula 2 is: QD-3B21 (1:10 dilution): Bio-HGH-004 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGF-1-011 = 1:0.1 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGFBP-3-012 = 1:0.2 volume ratio mixed reaction for 30 minutes. After the reaction is completed, the three mixtures are mixed and reacted at a volume ratio of 1:1:1 for 30 minutes.

[0160] Table 18

[0161]

[0162]

[0163] The types of IGFBP-3 antibodies and the ratio of IGF-1 antibodies were changed, and the types and ratios of HGH, IGF-1, and IGFBP-3 antibodies were adjusted to Formula 3. The results of the test of the enterprise reference products are as follows in Table 19:

[0164] Antibody formula 3: QD-3B21 (1:10 dilution): Bio-HGH-004 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGF-1-011 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGFBP-3-001 = 1:0.2 volume ratio mixed reaction for 30 minutes. After the reaction is completed, the three mixtures are mixed and reacted at a volume ratio of 1:1:1 for 30 minutes.

[0165] Table 19

[0166]

[0167] The ratio of IGF-1 and IGFBP-3 antibodies was changed, and the ratio of HGH, IGF-1, and IGFBP-3 antibodies was adjusted to Formula 4. The results of the test of the enterprise reference product are as follows in Table 20:

[0168] Antibody formula 4 is: QD-3B21 (1:10 dilution): Bio-HGH-004 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:15 dilution): Bio-IGF-1-011 = 1:0.15 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:10 dilution): Bio-IGFBP-3-001 = 1:0.1 volume ratio mixed reaction for 30 minutes. After the reaction is completed, the three mixtures are mixed and reacted at a volume ratio of 1:1:1 for 30 minutes.

[0169] Table 20

[0170]

[0171]

[0172] The IGFBP-3 antibody ratio was changed, and the HGH, IGF-1, and IGFBP-3 antibody ratios were adjusted to Formula 5. The results of the test of the enterprise reference product are as follows in Table 21:

[0173] Antibody formula 5 is: QD-3B21 (1:10 dilution): Bio-HGH-004 = 1:0.2 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:15 dilution): Bio-IGF-1-011 = 1:0.15 volume ratio mixed reaction for 30 minutes; QD-3B21 (1:15 dilution): Bio-IGFBP-3-001 = 1:0.1 volume ratio mixed reaction for 30 minutes. After the reaction is completed, the three mixtures are mixed and reacted at a volume ratio of 1:1:1 for 30 minutes.

[0174] Table 21

[0175]

[0176] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A combined detection kit for measuring human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3, characterized in that: The invention comprises a combined detection reagent card and a diluent. The combined detection reagent card comprises a card shell and a reagent strip. The reagent strip consists of a sample pad, a binding pad, a nitrocellulose membrane and a wicking paper which are sequentially adhered to a PVC bottom plate. The binding pad contains a mouse anti-albumin monoclonal antibody labeled with quantum dots. The reagent card also comprises a release pad which is sprayed with a quantum dot-labeled antibody mixed solution. The quantum dot-labeled antibody mixed solution is obtained by mixing an HGH quantum dot-labeled antibody mixed solution, an IGF-1 quantum dot-labeled antibody mixed solution and an IGFBP-3 quantum dot-labeled antibody mixed solution according to a mass ratio of (0.5-3):(0.5-3):(0.5-3).

2. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 1, characterized in that: The mass ratio of the HGH quantum dot-labeled antibody mixture, the IGF-1 quantum dot-labeled antibody mixture and the IGFBP-3 quantum dot-labeled antibody mixture is (1-2): (1-2): (1-2); the spraying amount of the quantum dot-labeled antibody mixture solution on the release pad is 1.8-2.2 μL / cm.

3. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 2, characterized in that: The nitrocellulose membrane is provided with a human growth hormone detection line, an insulin-like growth factor-1 detection line, an insulin-like growth factor binding protein-3 detection line and a quality control line close to the sample addition hole, and each detection line is coated with a corresponding human growth hormone antibody, insulin-like growth factor-1 antibody, insulin-like growth factor binding protein-3 antibody, and the quality control line is coated with sheep anti-mouse IgG.

4. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 3, characterized in that: The release pad and the sample pad are both pretreated, and the pretreatment method is: immersing the release pad / sample pad in a release pad / sample pad optimization buffer, and then drying and controlling the humidity to be below 30%.

5. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 4, characterized in that: The release pad optimization buffer comprises 45-55 mmol / L tris(hydroxymethyl)aminomethane, 0.8-1.2% by mass of bovine serum albumin, 0.4-0.6% by mass of Tween 20, 1.8-2.2% by mass of sucrose, 0.08-0.12% by mass of proclin300, and the balance of purified water.

6. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 5, characterized in that: The sample pad optimization buffer comprises tris(hydroxymethyl)aminomethane with a concentration of 45-55 mmol / L, Tween 20 with a mass percentage concentration of 0.4-0.6%, proclin 300 with a mass percentage concentration of 0.08-0.12%, and the balance of purified water.

7. A combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 6, characterized in that: The kit realizes the combined quantitative detection of human growth hormone, insulin-like growth factor-1, and insulin-like growth factor binding protein-3 based on quantum dot fluorescence immunochromatography, with a recovery rate of 90%-110%, a linear range of human growth hormone of 0.10-50.0 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%; a linear range of insulin-like growth factor-1 of 20-1500 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%; and a linear range of insulin-like growth factor binding protein-3 of 70-18000 ng / mL, a correlation coefficient ≥0.990, and an intra-batch coefficient of variation of <15%.

8. A method for preparing a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to any one of claims 1 to 7, characterized in that: The steps include: Step 1: Biotin labeling: label the antibodies with HGH biotin, IGF-1 biotin, and IGFBP-3 biotin respectively; Step 2, preparation of release pad: dilute the quantum dot dilution buffer, and respectively react with the three labeled antibodies obtained in step 1 to obtain three quantum dot-labeled antibody mixed solutions, mix the three quantum dot-labeled antibody mixed solutions in a mass ratio of 1:1:1, spray them on the release pad, and dry them to obtain a release pad coated with quantum dot-labeled mouse anti-albumin monoclonal antibodies; Step 3, dilute HGH antibody, IGF-1 antibody, IGFBP-3 antibody and goat anti-mouse IgG and spray them on the nitrocellulose membrane, dry them to obtain test line 1, test line 2, test line 3 and quality control line, and stick the nitrocellulose membrane to the PVC base plate; Step 4: Assemble the bottom plate with the nitrocellulose membrane treated in step 3, the sample pad, the release pad treated in step 2, and the absorbent paper to obtain a detection reagent card.

9. The method for preparing a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 8, characterized in that: In step 2, the quantum dot dilution buffer includes tris(hydroxymethyl)aminomethane with a concentration of 90-110 mmol / L, bovine serum albumin with a mass percentage concentration of 0.8-1.2%, Tween 20 with a mass percentage concentration of 0.4-0.6%, sucrose with a mass percentage concentration of 1.8-2.2%, proclin 300 with a mass percentage concentration of 0.08-0.12%, and the balance purified water.

10. The method for preparing a combined detection kit for determining human growth hormone / insulin-like growth factor-1 / insulin-like growth factor binding protein-3 according to claim 9, characterized in that: In step 2, the quantum dot buffer solution is diluted at a ratio of 1:10-1:15, and the mixed mass ratio of the diluted quantum dot dilution buffer solution to the three labeled antibodies is 1:0.1-1:0.3.

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