Hapten, antigen, antibody and enzyme-linked immunosorbent assay kit for creatine content detection

Abstract

The invention relates to the technical field of enzyme-linked immunosorbent assay, in particular to a hapten for creatine content detection, an antigen, an antibody and an enzyme-linked immunosorbent assay kit, and further discloses application of the hapten in the aspects of the antigen, the antibody and the enzyme-linked immunosorbent assay kit. The enzyme linked immunosorbent assay kit established by aiming at the hapten for creatine content detection can realize rapid detection of a large batch of samples to be detected, is convenient to use, low in detection cost, efficient, accurate and rapid in detection method, can simultaneously detect a large batch of samples, and is suitable for creatine content detection.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme-linked immunosorbent assay, and particularly relates to a hapten, an antigen, an antibody for detecting creatine content and an enzyme-linked immunosorbent assay kit. Background Art

[0002] Creatine is an amino acid derivative naturally produced in the body. It can rapidly increase muscle strength, accelerate fatigue recovery, and improve explosive power. The more creatine stored in the body, the stronger the strength and athletic ability. It can also be ingested from food. It can not only quickly provide energy (all activities of the human body rely on ATP, that is, adenosine triphosphate to provide energy, and the storage amount of ATP in the body is very small. During exercise, ATP is quickly consumed, and at this time, creatine can quickly resynthesize ATP to supply energy). It can also increase strength, build muscle, and accelerate fatigue recovery. The more creatine stored in the body, the more sufficient the energy supply, the faster the fatigue recovery, and the stronger the exercise energy.

[0003] Numerous studies have confirmed that reasonable supplementation of creatine is safe and its effects are reversible. After supplementing creatine to the saturation level, generally stop supplementing for 4 to 6 weeks, and the creatine level will return to the baseline level. Therefore, long-term supplementation of creatine will not inhibit endogenous creatine synthesis. However, excessive intake of nitrogen-containing foods may increase the burden on the kidneys and cause kidney function damage. Studies have also shown that short-term and long-term supplementation of creatine will not have an adverse impact on the kidney function, muscles and liver of athletes.

[0004] Currently, the detection of blood creatine content mainly uses mass spectrometry. The equipment used in mass spectrometry is expensive, the sample pretreatment before detection is complex, the cycle is long, and it is not convenient for batch detection. Summary of the Invention

[0005] Aiming at the problems in the prior art, the purpose of the present invention is to provide a hapten, an antigen, an antibody for detecting creatine content and an enzyme-linked immunosorbent assay kit, so as to meet the need for rapid, simple and batch detection of creatine.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a hapten for detecting creatine content, and its chemical structural formula is:

[0008] .

[0009] In the second aspect, the present invention provides a preparation method of a hapten for detecting creatine content, which uses creatine as a raw material and reacts with ethylenediamine in a carbonate buffer solution. The specific reaction process is as follows:

[0010] .

[0011] Preferably, the method for preparing the hapten for creatine content detection comprises the following steps:

[0012] 1) Dissolve creatine with carbonate buffer solution;

[0013] 2) Add ethylenediamine and react at 60 °C for 4 h, then stop the reaction to obtain a reaction solution;

[0014] 3) Separate and purify the obtained reaction solution to obtain the hapten for creatine content detection;

[0015] The molar ratio of creatine to ethylenediamine is 1:1.

[0016] In a third aspect, the present invention provides an antigen for creatine content detection, which is a conjugate obtained by conjugating the above-mentioned hapten with a carrier protein, and its structural formula is:

[0017] .

[0018] Preferably, the carrier protein is at least one of serum albumin, ovalbumin, thyroglobulin, and keyhole limpet hemocyanin. Using the above types of carrier proteins can effectively play the detection role of the hapten.

[0019] More preferably, the carrier protein is ovalbumin, human serum albumin or hemocyanin.

[0020] The method for conjugating the hapten and the carrier protein used in the present invention can be the commonly used methods for preparing antigens from haptens in the prior art. For example, the conjugating method using bovine serum albumin as the carrier protein can be: dissolve the hapten and EDC in an appropriate amount of PBS, activate the carboxyl group of the hapten by stirring overnight at 2-8 °C, and centrifuge to take the supernatant, which is the activated derivative of the creatine hapten; slowly add the activated hapten derivative drop by drop to the coating buffer solution containing 5 mg / mL bovine serum albumin, and stir and react at 2-8 °C for 12 h. Dialyze with PBS at 2-8 °C for three days, changing the dialysis solution three times a day, to obtain the antigen for creatine content detection, aliquot and store at low temperature for later use. The specific reaction formula is:

[0021] .

[0022] In a fourth aspect, the present invention provides an antibody for creatine content detection, which is an antibody obtained by immunizing an animal with the above-mentioned antigen; the antibody specifically immunologically reacts with creatine.

[0023] Preferably, the antibody is a creatine monoclonal antibody.

[0024] The method for preparing the antibody from the above-mentioned antigen used in this aspect can be the commonly used animal immunization method in the art. For example, it includes the following steps:

[0025] (1) Animal immunization: Inject creatine antigen coupled with bovine serum albumin into Balb / c mice at an immunization dose of 150 μg / mouse to produce polyclonal antibody serum. (2) Cell fusion and cloning: After the mouse serum test results are high, the spleen cells are taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1. The cell supernatant is measured by indirect competitive ELISA to screen the positive wells. The positive wells are cloned by limiting dilution method until a hybridoma cell line secreting monoclonal antibodies is obtained. (3) Cell freezing and thawing: The monoclonal hybridoma cell line of creatine is prepared into 1×10 9 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryovials were removed and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture bottles for culture. (4) Production and purification of monoclonal antibodies: Balb / c mice were intraperitoneally injected with 0.5 mL of sterilized paraffin oil. Seven days later, 6×10 monoclonal hybridoma cells of creatine were intraperitoneally injected. 5 Each rat was treated with ascites collected after 7 days. The ascites was purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C. (5) Determination of monoclonal antibody titer: The antibody titer was determined to be 1:216,000 using the indirect competitive ELISA method.

[0026] The antibody is put into use in the form of a monoclonal antibody and has a high titer.

[0027] In a fifth aspect, the present invention provides an enzyme-linked immunosorbent assay kit for detecting creatine content, comprising: an ELISA plate and an antibody, wherein the ELISA plate is coated with the antigen for detecting creatine content as described above; and the antibody is the antibody for detecting creatine content as described above.

[0028] Preferably, the enzyme-linked immunosorbent assay kit for detecting creatine content further comprises: gradient concentrations of creatine standard solutions, enzyme-labeled secondary antibodies, substrate solution A, substrate solution B, stop solution, and washing solution.

[0029] Preferably, the gradient concentrations of the creatine standard solution are 0 μg / mL, 1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL respectively;

[0030] The enzyme-labeled secondary antibody was horseradish peroxidase-labeled goat anti-mouse antibody;

[0031] Substrate liquid A is carbamide peroxide, and substrate liquid B is tetramethylbenzidine;

[0032] The stop solution is 2 mol / L sulfuric acid solution;

[0033] The washing solution has a pH of 7.2 and contains 0.8% - 1.2% Tween - 20 and 0.1 - 0.3 mol / L carbonate buffer solution;

[0034] The kit prepared with the above - mentioned components has high detection efficiency and good accuracy, and is suitable for detecting a large number of samples on - site.

[0035] The beneficial effects that the present invention can produce include:

[0036] 1) The hapten for detecting creatine content provided by the present invention. When the kit prepared with this hapten is used to detect creatine contained in a sample, the sensitivity is 1.56 μg / mL, indicating that after the kit is prepared with this hapten, the detection sensitivity for creatine is relatively high; after being prepared into a kit according to the existing method, it can be used for effective detection of creatine content; after the kit is prepared with this hapten, the recovery rate of creatine is 100 ± 10%, the within - batch coefficient of variation < 10%, and the between - batch coefficient of variation < 15%, with good accuracy and can meet the detection requirements. This hapten can retain the characteristic structure of creatine to the greatest extent. After preparing an antibody with this hapten, this antibody has the characteristics of high detection sensitivity and strong specificity for creatine.

[0037] 2) The hapten for detecting creatine content provided by the present invention. The antibody prepared with this hapten as the raw material shows strong immunogenicity for creatine detection because it uses this hapten as the raw material; using this hapten as the raw material to prepare an artificial antigen system suitable for animal immunization to immunize animals, the titer, specificity, and affinity of the obtained antibody are all relatively good, and the detection sensitivity for creatine can reach 1.56 μg / mL.

[0038] 3) The antibody and kit for detecting creatine content provided by the present invention have simple detection operations, do not require purchasing large and expensive instrument analysis equipment, and only need to complete the detection of samples through the kit, which is suitable for large - batch detection.

[0039] 4) The enzyme - linked immunosorbent assay kit provided by the present invention is prepared with the obtained antibody, is easy to use, has low detection cost, high - efficiency, accurate, fast detection methods, and can detect a large number of samples simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the four - parameter fitting standard curve graph of the present invention;

[0041] Figure 2 is the mass spectrometry graph of compound 1 prepared in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] Example 1: Detection of Creatine Content Preparation of the hapten for detection:

[0045] The preparation method of the hapten for creatine content detection uses creatine as the raw material and reacts with an ethylenediamine carbonate buffer solution. The specific reaction process is as follows:

[0046] 。

[0047] The preparation method of the hapten for creatine content detection includes the following steps:

[0048] 1) Dissolve creatine in a carbonate buffer solution;

[0049] 2) Add ethylenediamine and react at 60 °C for 4 h, then stop the reaction to obtain a reaction solution;

[0050] 3) Separate and purify the obtained reaction solution to obtain the hapten for creatine content detection;

[0051] The molar ratio of creatine to ethylenediamine is 1:1.

[0052] Perform hydrogen nuclear magnetic resonance (1H-NMR) measurement on the prepared product: 1H NMR (500 MHz, Chloroform-d): δ (ppm) 7.83 (br, 2H), 6.61 (br, 2H), 4.07 (s, 2H), 3.66 (t, J = 10.8 Hz, 2H), 3.05 (s, 3H), 2.76 (t, J = 10.8 Hz, 2H), 1.5 (br, 2H). The presence of these peaks proves that the hapten structure is correct.

[0053] Figure 2It is the mass spectrum of the prepared product. It can be seen from the figure that the molecular weight of the product is 173, and the abundance at the position of m / z 173 in the mass spectrum indicates that the compound 1 is synthesized. For m / z 58, the column is the highest, corresponding to the guanidine group in the product, and the guanidine group fragment has high stability. For m / z 70, 86, and 103, the column heights are relatively high, which are the cleavage characteristics of the corresponding amide bond and McLafferty rearrangement in the product. Thus, it is confirmed that the hapten structure is correct.

[0054] Example 2: Preparation of an antigen for detecting creatine content conjugated with bovine serum albumin

[0055] It includes the following steps:

[0056] The conjugation method using bovine serum albumin as the carrier protein can be as follows: Dissolve 10 mg of creatine hapten and 15 mg of EDC in 5 mL of PBS with a pH of 7.4, stir overnight (10 - 16 h) at 2 - 8 °C to activate the carboxyl group of the creatine hapten, and centrifuge to obtain 4.5 mL of solution, which is the activated derivative of the creatine hapten; Slowly add 4.5 mL of the activated creatine hapten derivative drop by drop to 2 mL of PBS buffer solution with a pH of 7.4 containing 5 mg / mL of bovine serum albumin, and stir and react at 2 - 8 °C for 12 h. Dialyze with 2 L of PBS with a pH of 7.4 at 2 - 8 °C for three days, changing the dialysis solution three times a day, to obtain the antigen for detecting creatine content, aliquot and store at low temperature for later use.

[0057] Example 3: Preparation of an antigen for detecting creatine content conjugated with ovalbumin

[0058] It includes the following steps:

[0059] The conjugation method using ovalbumin as the carrier protein can be as follows: Dissolve 10 mg of creatine hapten and 15 mg of EDC in 5 mL of PBS with a pH of 7.4, stir overnight (10 - 16 h) at 2 - 8 °C to activate the carboxyl group of the creatine hapten, and centrifuge to obtain 4.5 mL of solution, which is the activated derivative of the creatine hapten; Slowly add 4.5 mL of the activated creatine hapten derivative drop by drop to 2 mL of PBS buffer solution with a pH of 7.4 containing 5 mg / mL of ovalbumin, and stir and react at 2 - 8 °C for 12 h. Dialyze with 2 L of PBS with a pH of 7.4 at 2 - 8 °C for three days, changing the dialysis solution three times a day, to obtain the antigen for detecting creatine content, aliquot and store at low temperature for later use.

[0060] Example 4: Preparation of a monoclonal antibody for detecting creatine content

[0061] It includes the following steps:

[0062] (1) Animal immunization: The creatine antigen coupled to bovine serum albumin prepared in Example 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce polyclonal antibody serum.

[0063] (2) Cell fusion and cloning: After the mouse serum test results are high, the spleen cells are taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1. The cell supernatant is measured by indirect competitive ELISA to screen the positive wells. The positive wells are cloned by limiting dilution method until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0064] (3) Cell freezing and recovery: The monoclonal hybridoma cell line of creatine was prepared into 1×10 9 Cell suspensions of 100 cells / mL can be stored in liquid nitrogen for a long term. When thawing, remove the cryovials and immediately thaw in a 37°C water bath. Centrifuge to remove the cryopreservation solution and transfer to a culture flask for culture.

[0065] (4) Production and purification of monoclonal antibodies: Balb / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, 6×10 monoclonal hybridoma cells containing creatine were intraperitoneally injected. 5 After 7 days, the ascites was collected and purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C.

[0066] (5) Determination of monoclonal antibody titer: The antibody titer was determined to be 1:256,000 by indirect competitive ELISA.

[0067] Indirect competitive ELISA method: Coat an ELISA plate with the ovalbumin-coupled creatine antigen prepared in Example 3, add a creatine standard solution and a monoclonal antibody working solution, react at 4°C for 30 minutes, pour out the liquid in the wells, wash 3-5 times with PBST washing solution, and pat dry with absorbent paper; add goat anti-mouse anti-antibody labeled with horseradish peroxidase, react at 25°C for 30 minutes, remove and repeat the plate washing step; add substrate solution, react at 25°C for 15 minutes, and then add stop solution to terminate the reaction; set the microplate reader to a wavelength of 450 nm to measure the absorbance of each well.

[0068] Example 5 Preparation of ELISA kit for creatine content detection

[0069] The kit includes:

[0070] (1) An ELISA plate coated with creatine antigen coupled to ovalbumin;

[0071] (2) Creatine monoclonal antibody working solution;

[0072] (3) horseradish peroxidase-labeled goat anti-mouse antibody;

[0073] (4)Six bottles of creatine standard solutions with concentrations of 0 μg / mL, 1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL respectively;

[0074] (5)The substrate solution consists of Solution A and Solution B. Solution A is urea peroxide, and Solution B is tetramethylbenzidine;

[0075] (6)The stop solution is 2 mol / L sulfuric acid solution;

[0076] (7)The washing solution is a carbonate buffer solution with a pH of 7.2, containing 1.0% Tween-20 and 0.2 mol / L;

[0077] The main reagents of this kit are provided in the form of working solutions. The test method is convenient and easy to perform, with the characteristics of high specificity, high sensitivity, high precision, high accuracy, etc., facilitating the rapid batch detection of a large number of samples.

[0078] Application of the enzyme-linked immunosorbent assay kit for creatine content detection obtained in Example 6 in detection and its performance detection

[0079] I. Test method of the kit obtained in Example 5

[0080] 1. Sample pretreatment:

[0081] ① Serum: The whole blood sample is placed at room temperature for 1 hour or overnight at 2 - 8°C, then centrifuged at 1000×g for 20 minutes at 2 - 8°C, and the supernatant can be used for detection.

[0082] ② Plasma: EDTA-Na2 is recommended as the anticoagulant. After sample collection, centrifuge at 1000×g for 15 minutes at 2 - 8°C within 30 minutes, and the supernatant can be used for detection.

[0083] ③ Tissue homogenate: Rinse the tissue with pre-cooled PBS (0.01M, pH = 7.4) to remove residual blood, weigh it, and then cut the tissue into small pieces. Add the cut tissue and the corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, for example, 1 g of tissue sample corresponds to 9 mL of PBS, and the specific volume can be appropriately adjusted according to experimental needs and records should be made. Protease inhibitors are recommended to be added to PBS) into a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be repeatedly frozen and thawed or ultrasonically disrupted. Finally, centrifuge the homogenate at 5000×g for 5 - 10 minutes at 2 - 8°C, and take the supernatant for detection. <s

[0084] 2. Detection operation steps of the kit obtained in Example 5:

[0085] Add 50 μL / well of the test solution to the micro-wells of the enzyme-linked immunosorbent assay (ELISA) plate (coated with the creatine antigen conjugated with ovalbumin obtained in Example 3), then add 50 μL / well of the working solution of the creatine monoclonal antibody prepared in Example 4. After covering the plate with a cover membrane, react at 25 °C in the dark for 30 min; pour out the liquid in the wells, add 250 μL of washing solution to each well, pour out the liquid in the wells after 30 s, and repeat this operation 5 times to wash the plate. Pat dry with absorbent paper; add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse antibody, gently mix well, cover the plate with a cover membrane, and react at 25 °C in the dark for 30 min. Repeat the plate washing step;

[0086] Add 50 μL of urea peroxide in Substrate Solution A and 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) in Substrate Solution B to each well, gently mix well, cover the plate with a cover membrane, and develop color at 25 °C in the dark for 15 min. Add 50 μL of 2 mol / L sulfuric acid solution as the stop solution to each well, gently mix well, and measure the absorbance value (OD value) of each well with an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm.

[0087] 3. Detection and result analysis

[0088] According to the above operation steps, use creatine standard solution and pretreated samples with gradient concentrations as the test solutions for detection respectively. Take the logarithm of the creatine standard concentration (μg / mL) as the X-axis and the percentage absorbance value as the Y-axis, and use four-parameter fitting to draw a standard curve, as Figure 1 shown.

[0089] II. Determination of the sensitivity of the kit obtained in Example 5:

[0090] Sensitivity in this application: refers to the lowest content of creatine in the analyte that can be detected by this kit. The sensitivity of a quantitative kit itself can generally be understood as the concentration value of the standard product with the smallest concentration among the kit standard products except the "0" standard product, or the lowest standard product concentration corresponding to when establishing the standard curve. According to the existing method, the sensitivity of the kit provided in this application for creatine is 1.56 μg / mL.

[0091] III. Determination of the detection limit of the kit obtained in Example 5:

[0092] Detection limit: usually refers to the minimum detectable amount of the actual sample measured by the kit product. Its theoretical definition is: measure 20 negative (blank) samples according to a reasonable pretreatment method, calculate the average value X and the standard deviation (SD), and the result obtained according to the formula X - 2SD is the detection (lower) limit of the sample. Therefore, according to the above existing method, the detection limit of this kit for creatine in serum is 1.56 μg / mL.

[0093] IV. Determination of the accuracy and precision of the kit obtained in Example 5:

[0094] Accuracy and precision: The accuracy of the ELISA assay is expressed as the recovery rate, and the precision is expressed as the coefficient of variation. Serum samples were taken, and spiking recovery tests were performed on them with creatine at three concentrations of 2, 8, and 30 μg / mL according to the existing method. Experimental results: The recovery rate of the kit prepared with the hapten provided in this application for the samples was 100% ± 10%, the within-batch coefficient of variation < 10%, and the between-batch coefficient of variation < 15%.

[0095] It shows that the kit prepared with the hapten provided in this application has good accuracy and precision and can accurately detect the creatine contained in the samples.

[0096] V. Determination of the antibody cross-reactivity of the kit obtained in Example 5:

[0097] Sarcosine, creatine phosphate, inositol, creatine kinase, and creatine hemisulfate, which have structures similar to creatine, were respectively determined by the existing indirect competitive ELISA method. The results showed that except for creatine being 100%, the others were all < 1%.

[0098] It shows that the antibody prepared with the hapten provided in this application has good specificity for creatine, can accurately detect the creatine contained in the samples, and is not affected by other structural analogs.

[0099] VI. Test on the storage period conditions of the kit obtained in Example 5

[0100] After storing the kit at 2 - 8°C for 12 months, its maximum absorbance value (zero standard), 50% inhibition concentration, and actual creatine determination value were detected, and each value was within the normal range.

[0101] It was determined that the kit provided in this application can be stored for at least 12 months.

[0102] From the above test results, it can be seen that the antibody prepared with the hapten provided by the present invention, after being made into a kit, has a relatively accurate detection effect in the detection of creatine content.

[0103] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A hapten for detecting creatine content, characterized in that, Its structural formula is: 。 2. A preparation method of a hapten for detecting creatine content as described in claim 1, characterized in that, It is prepared by reacting creatine with ethylenediamine in a carbonate buffer solution, and the specific reaction process is as follows: 。 3. An antigen for detecting creatine content, characterized in that, It is obtained by conjugating the hapten as described in claim 1 with a carrier protein, and its structural formula is: 。 4. The antigen for detecting creatine content according to claim 3, wherein The carrier protein is at least one of serum albumin, ovalbumin, thyroglobulin, and keyhole limpet hemocyanin.

5. An antibody for detecting creatine content, characterized in that, It is an antibody obtained by immunizing an animal with the antigen as described in claim 3.

6. The antibody for detecting creatine content according to claim 5, wherein The antibody has a specific immune reaction with creatine.

7. The antibody for detecting creatine content according to claim 5, characterized in that, The antibody is a creatine monoclonal antibody.

8. An enzyme-linked immunosorbent assay kit for detecting creatine content, characterized in that, It includes: An enzyme-linked immunosorbent assay (ELISA) plate and an antibody. The ELISA plate is coated with the antigen for detecting creatine content as described in claim 3 or 4; the antibody is the antibody for detecting creatine content as described in any one of claims 5-7.

9. The enzyme-linked immunosorbent assay kit for detecting creatine content according to claim 8, characterized in that, The kit further includes: creatine standard solutions with gradient concentrations, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, termination solution, and washing solution; The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody; The substrate solution A is urea peroxide, and the substrate solution B is tetramethylbenzidine; The termination solution is 2 mol / L sulfuric acid solution; The washing solution is a carbonate buffer solution with a pH of 7.2, containing 0.8% - 1.2% Tween-20 and 0.1 - 0.3 mol / L.

10. The enzyme-linked immunosorbent assay kit for detecting creatine content according to claim 9, wherein, [[ID=

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