A hapten, antigen, antibody and enzyme-linked immunosorbent assay kit for detecting creatine content

Abstract

The present invention relates to the field of enzyme-linked immunosorbent assay technology, and more particularly to a hapten, antigen, antibody, and enzyme-linked immunosorbent assay kit for detecting creatine content. The present invention also discloses the use of the hapten in the antigen, antibody, and enzyme-linked immunosorbent assay kit. The enzyme-linked immunosorbent assay kit developed for the hapten used to detect creatine content can achieve rapid detection of large quantities of samples to be tested. It is easy to use, has low detection cost, and is efficient, accurate, and rapid. It can simultaneously detect large quantities of samples and is suitable for detecting creatine content.

Description

Technical Field

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

[0002] Creatine, a naturally occurring amino acid derivative, can rapidly increase muscle strength, accelerate fatigue recovery, and enhance explosive power. The more creatine stored in the body, the greater the strength and athletic ability. Creatine can also be obtained through food. It not only provides a rapid source of energy (human activities rely on ATP, or adenosine triphosphate, for energy, but the body's ATP reserves are very low. During exercise, ATP is quickly depleted, allowing creatine to rapidly resynthesize ATP to provide energy), but also increases strength, muscle growth, and accelerates fatigue recovery. The more creatine stored in the body, the more adequate the energy supply, the faster fatigue recovery, and the greater the energy for exercise.

[0003] Numerous studies have confirmed that appropriate creatine supplementation is safe and its effects are reversible. After reaching saturation levels, creatine levels typically return to baseline within four to six weeks of cessation. Therefore, long-term creatine supplementation does not inhibit endogenous creatine synthesis. However, excessive intake of nitrogen-containing foods may increase the burden on the kidneys and lead to impaired renal function. Studies have also shown that short-term and long-term creatine supplementation do not adversely affect renal function, muscle, or liver in athletes.

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

[0005] In view of the problems in the prior art, the present invention aims to provide a hapten, antigen, antibody and enzyme-linked immunosorbent assay kit for detecting creatine content, so as to meet the needs of rapid, simple and batch detection of creatine.

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

[0007] In a first aspect, the present invention provides a hapten for detecting creatine content, the chemical structure of which is:

[0008] .

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

[0010] .

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

[0012] 1) Dissolve creatine in carbonate buffer;

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

[0014] 3) separating and purifying the resulting reaction solution to obtain a 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 detecting creatine content, which is a conjugate obtained by coupling 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. The use of the above types of carrier proteins can effectively play the role of hapten detection.

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

[0020] The coupling method used in the present invention for the hapten and carrier protein can be a commonly used method for preparing an antigen from a hapten. For example, the coupling method using bovine serum albumin as the carrier protein can be as follows: the hapten and EDC are dissolved in an appropriate amount of PBS, stirred overnight at 2-8°C to activate the carboxyl group of the hapten, and the supernatant is centrifuged to obtain the activated creatine hapten derivative; the activated hapten derivative is slowly added dropwise to a coating buffer solution containing 5 mg / mL bovine serum albumin, and stirred at 2-8°C for 12 hours. The activated hapten derivative is dialyzed against PBS at 2-8°C for three days, with the dialysate changed three times daily, to obtain the antigen for creatine content detection, which is then aliquoted and stored at low temperature for future use. The specific reaction formula is:

[0021] .

[0022] In a fourth aspect, the present invention provides an antibody for detecting creatine content, which is an antibody obtained by immunizing an animal with the above antigen; the antibody undergoes a specific immune reaction with creatine.

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

[0024] The method for preparing antibodies from the above antigens used in this aspect can be a commonly used animal immunization method in the art, for example, comprising 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 is pH 7.2, containing 0.8% to 1.2% Tween-20 and 0.1 to 0.3 mol / L carbonate buffer;

[0034] The test kit prepared with the above components has high detection efficiency and good accuracy, and is suitable for testing large quantities of samples on site.

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

[0036] 1) The present invention provides a hapten for detecting creatine content. When the hapten is formulated into a kit for detecting creatine in a sample, the sensitivity is 1.56 μg / mL, indicating that the hapten has a high sensitivity for detecting creatine when formulated into a kit. When the kit is formulated according to existing methods, it can be used to effectively detect creatine content. When the hapten is formulated into a kit, the recovery rate for creatine is 100±10%, the intra-batch coefficient of variation is less than 10%, and the inter-batch coefficient of variation is less than 15%, which is accurate enough to meet the needs of detection. The hapten can retain the characteristic structure of creatine to the greatest extent. When an antibody is prepared using this hapten, the antibody has the characteristics of high sensitivity and strong specificity for creatine detection.

[0037] 2) The hapten provided by the present invention is used to detect creatine content. Antibodies prepared using the hapten as a raw material exhibit strong immunogenicity for creatine detection. Using the hapten as a raw material, an artificial antigen system suitable for animal immunization is prepared. When animals are immunized with the hapten as a raw material, the resulting antibodies have good titer, specificity, and affinity, and the detection sensitivity for creatine can reach 1.56 μg / mL.

[0038] 3) The antibodies and kit for detecting creatine content provided by the present invention are simple to operate and do not require the purchase of large and expensive instrumental analysis equipment. Sample detection can be completed using only the kit, making it suitable for large-scale testing.

[0039] 4) The enzyme-linked immunosorbent assay kit provided by the present invention is made with the obtained antibodies, is easy to use, has low detection cost, and the detection method is efficient, accurate, and rapid, and can simultaneously detect large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a four-parameter fitting curve diagram of the present invention;

[0041] Figure 2 This is the mass spectrum of compound 1 prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0042] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is 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 those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] Example 1: Creatine content detection Preparation of hapten for detection:

[0045] The preparation method of hapten for creatine content detection is to use creatine as raw material and react it with ethylenediamine carbonate buffer solution. The specific reaction process is as follows:

[0046] .

[0047] The method for preparing a hapten for detecting creatine content comprises the following steps:

[0048] 1) Add carbonate buffer to dissolve creatine;

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

[0050] 3) separating and purifying the resulting reaction solution to obtain a hapten for creatine content detection;

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

[0052] The prepared product was analyzed by hydrogen nuclear magnetic resonance (1H-NMR): 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 confirmed the correctness of the hapten structure.

[0053] Figure 2This is the mass spectrum of the prepared product. The figure shows a molecular weight of 173. The abundance at m / z 173 in the mass spectrum indicates the synthesis of compound 1. m / z 58, the highest column, corresponds to the guanidine group in the product; the guanidine group fragment is highly stable. m / z 70, 86, and 103, with relatively high column heights, are characteristic of fragmentation corresponding to the amide bond and McFadden rearrangement. This confirms the correct structure of the hapten.

[0054] Example 2: Preparation of antigen for detection of creatine content coupled to bovine serum albumin

[0055] The following steps are involved:

[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, pH 7.4, and stir overnight (10-16 hours) at 2-8°C to activate the carboxyl groups of the hapten creatine. Centrifuge to obtain 4.5 mL of the activated creatine hapten derivative. Slowly add 4.5 mL of the activated creatine hapten derivative dropwise to 2 mL of PBS, pH 7.4, containing 5 mg / mL bovine serum albumin. Stir at 2-8°C for 12 hours. Dialyze against 2 L of PBS, pH 7.4, at 2-8°C for three days, changing the dialysate three times daily to obtain the antigen for creatine content detection. Aliquot and store at low temperature until needed.

[0057] Example 3: Preparation of antigen for detection of creatine content coupled to ovalbumin

[0058] The following steps are involved:

[0059] The conjugation method using ovalbumin as a carrier protein can be as follows: dissolve 10 mg of creatine hapten and 15 mg of EDC in 5 mL of PBS, pH 7.4, and stir overnight (10-16 hours) at 2-8°C to activate the carboxyl groups of the hapten creatine. Centrifuge to obtain 4.5 mL of the activated creatine hapten derivative. Slowly add 4.5 mL of the activated creatine hapten derivative dropwise to 2 mL of PBS, pH 7.4, containing 5 mg / mL ovalbumin. Stir at 2-8°C for 12 hours. Dialyze the solution against 2 L of PBS, pH 7.4, at 2-8°C for three days, changing the dialysate three times daily to obtain the antigen for creatine content detection. Aliquot and store at low temperature until needed.

[0060] Example 4 Preparation of Monoclonal Antibodies for Creatine Content Detection

[0061] The following steps are involved:

[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 time. When thawing, remove the cryovials and immediately thaw them in a 37°C water bath. Centrifuge to remove the freezing 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, 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 measure the absorbance of each well at a wavelength of 450 nm.

[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) 6 bottles of creatine standard solution, 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 pH 7.2, containing 1.0% Tween-20 and 0.2 mol / L carbonate buffer;

[0077] The main reagents of this kit are provided in the form of working solution. The test method is convenient and easy to perform, with high specificity, high sensitivity, high precision and high accuracy, which facilitates rapid batch testing of large quantities of samples.

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

[0079] 1. Testing method of the kit obtained in Example 5

[0080] 1. Sample pretreatment:

[0081] ① Serum: Place the whole blood sample at room temperature for 1 hour or at 2-8°C overnight, then centrifuge at 2-8°C, 1000×g for 20 minutes, and remove the supernatant for testing.

[0082] ② Plasma: EDTA-Na2 is recommended as the anticoagulant. Centrifuge the sample at 1000×g for 15 minutes at 2-8°C within 30 minutes after collection, and remove the supernatant for testing.

[0083] ③Tissue homogenization: Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood, weigh and mince the tissue. Add the minced 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. The specific volume can be adjusted appropriately according to experimental needs and recorded. It is recommended to add protease inhibitors to PBS) into a glass homogenizer and grind thoroughly on ice. In order to further lyse tissue cells, the homogenate can be repeatedly frozen and thawed or ultrasonically disrupted. Finally, centrifuge the homogenate at 2-8°C, 5000×g for 5-10 minutes, and take the supernatant for detection.

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

[0085] 50 μL / well of the test solution was added to the microwells of an ELISA plate (coated with the ovalbumin-coupled creatine antigen obtained in Example 3), followed by the addition of 50 μL / well of the creatine monoclonal antibody working solution prepared in Example 4. The plate was covered with a cover film and reacted at 25°C in the dark for 30 minutes. The liquid in the wells was poured out, 250 μL of washing solution was added to each well, and after 30 seconds, the liquid in the wells was poured out. This washing operation was repeated 5 times, and the plate was patted dry with absorbent paper. 100 μL / well of horseradish peroxidase-labeled goat anti-mouse antibody was added, gently shaken to mix, and the plate was covered with a cover film. The plate was reacted at 25°C in the dark for 30 minutes, and the washing step was repeated.

[0086] Add 50 μL of substrate solution A (urea peroxide) and 50 μL of substrate solution B (tetramethylbenzidine (TMB)) to each well, shake gently to mix, cover the plate with a cover film and place it at 25°C in the dark for 15 minutes to develop the color, add 50 μL of the stop solution 2 mol / L sulfuric acid solution to each well, shake gently to mix, and use a microplate reader with the wavelength set at 450 nm to measure the absorbance (OD value) of each well.

[0087] 3. Detection and analysis of results

[0088] According to the above steps, the creatine standard solution with gradient concentration and the pre-treated sample were used as the test solution for detection. The logarithm of the creatine standard concentration (μg / mL) was used as the X-axis and the percentage absorbance value was used as the Y-axis. Using four-parameter fitting, a standard curve was drawn, as shown in the figure below. Figure 1 shown.

[0089] 2. Sensitivity determination of the kit obtained in Example 5:

[0090] As used herein, "sensitivity" refers to the lowest creatine concentration in the analyte that the kit can detect. The sensitivity of a quantitative kit can generally be understood as the lowest concentration of the standard sample in the kit, excluding the "0" standard, or the lowest standard concentration corresponding to the standard curve. Calculated using existing methods, the sensitivity of the kit provided in this application for creatine is 1.56 μg / mL.

[0091] 3. Determination of the detection limit of the kit obtained in Example 5:

[0092] Detection limit: This generally refers to the minimum detectable amount of a sample in a test kit. Its theoretical definition is: 20 negative (blank) samples are assayed using a reasonable pretreatment method, and the mean (X) and standard deviation (SD) are calculated. The result, calculated using the formula X-2SD, is the (lower) detection limit for that sample. Therefore, using the above-mentioned existing method, the detection limit of this kit for serum creatine is 1.56 μg / mL.

[0093] IV. Accuracy and precision determination of the kit obtained in Example 5:

[0094] Accuracy and Precision: The accuracy of ELISA assays is expressed as recovery rate, and precision as coefficient of variation. Serum samples were spiked with creatine at concentrations of 2, 8, and 30 μg / mL according to existing methods for recovery testing. The results showed that the sample recovery rate for the kit prepared using the hapten provided in this application was 100% ± 10%, with an intra-assay coefficient of variation of <10% and an inter-assay coefficient of variation of <15%.

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

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

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

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

[0099] VI. Test on the shelf life of the kit obtained in Example 5

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

[0101] It has been 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 from the hapten provided by the present invention and prepared into a kit has a relatively accurate detection effect in the detection of creatine content.

[0103] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A hapten for detecting creatine content, characterized in that: Its structural formula is: 。 2. A method for preparing a hapten for detecting creatine content as claimed in claim 1, characterized in that: Creatine is used as the raw material and reacted with ethylenediamine in a sodium carbonate buffer solution. The specific reaction process is as follows: 。 3. An antigen for detecting creatine content, characterized in that: The hapten according to claim 1 is coupled with a carrier protein, and its structural formula is: ； The carrier protein is at least one of serum albumin, ovalbumin, thyroglobulin, and keyhole limpet hemocyanin.

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