Glutamic acid biological antigen, glutamic acid biological antibody and application thereof

Abstract

The invention discloses a glutamic acid biological antigen, a glutamic acid biological antibody and application of the glutamic acid biological antigen and the glutamic acid biological antibody. A glutamic acid biological antigen is used as a coating antigen and an immunogen, an antibody prepared from an immunogen immune animal is used as a detection antibody, and quantitative detection of glutamic acid is carried out; the structural formula of the glutamic acid biological antigen is shown as a formula I, and when the carrier protein is ovalbumin, a coating antigen is obtained; when the carrier protein is lactoferrin, the immunogen is obtained. An immunological detection method is applied to detect the content of glutamic acid and the activity of glutaminase, and the method has the advantages of being high in sensitivity, easy and convenient to operate, capable of achieving on-site rapid detection and the like, and a new method is provided for detecting the activity of glutamic acid and glutaminase. The method can better meet the field monitoring requirements of factory production workshops, and can also provide technical support for rapid detection of related scientific researchers.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a glutamate biological antigen, a glutamate biological antibody and their applications. Background Art

[0002] Soy sauce and the like are important condiments for enhancing umami flavor, and the umami components thereof mainly come from glutamate formed by microbial enzymatic hydrolysis of soybean protein. Soybean protein contains about 16% glutamate, and about 46% of the glutamate exists in the form of glutamine. Glutamine itself does not have umami flavor, but it can be converted into glutamate under the catalytic action of glutaminase. Therefore, detecting the content of glutamate and the activity of glutaminase during the soy sauce fermentation process is of great significance for soy sauce production and the like.

[0003] As a small molecule, glutamate can be detected by the most traditional instrumental methods, such as high performance liquid chromatography, amino acid analyzer method, etc., but these methods have limitations such as expensive instrument equipment and complex operation. In addition, glutamate has a maximum absorption wavelength at 280 nm, and its content can be detected by a spectrophotometer, but this method has poor anti-interference ability and low sensitivity. At present, the methods for glutaminase activity are mainly divided into two types. One is to react it with glutamine and detect the product glutamate or ammonia, thereby realizing the determination of glutaminase activity. For example, high performance liquid chromatography, amino acid analyzer method and spectrophotometer method can realize the determination of the product glutamate, but large instruments are required. For example, Nessler's method, ninhydrin method and phenol hypochlorite method can realize the determination of the product ammonia, but the detection time is long, it is easily interfered, the sensitivity is poor, and ammonia is volatile. The other is to detect the amount of glutaminase, which is to measure the number of enzymes and cannot truly reflect the enzyme activity. In view of this, it is necessary to establish a rapid and simple method for determining the content of glutamate and the activity of glutaminase during the soy sauce fermentation process.

[0004] The immunological detection method based on the specific binding of antigen and antibody has the advantages of simplicity, rapidity, high specificity, and meeting the on-site detection requirements, etc., but currently there are no biological antigens and biological antibodies available for detecting glutamate. Summary of the Invention

[0005] Based on this, it is necessary to provide a glutamate biological antigen, a glutamate biological antibody and their applications for the above technical problems.

[0006] In the first aspect, the present invention provides a glutamate biological antigen, which is a conjugate of N-p-aminobenzoyl-L-glutamate and a carrier protein, and the structural formula is as shown in Formula I,

[0007]

[0008] Formula I

[0009] When the carrier protein is ovalbumin, the coating antigen is obtained; when the carrier protein is lactoferrin, the immunogen is obtained.

[0010] In a second aspect, the present invention provides a method for preparing a glutamic acid biological antigen, which comprises the following steps:

[0011] (1-1) N-Benzoyl-L-glutamic acid is dissolved in water, concentrated hydrochloric acid is added until completely dissolved; an aqueous sodium nitrite solution is added under ice bath, and after stirring and reacting, an activation solution is obtained;

[0012] (1-2) The carrier protein is dissolved in a buffer solution, and the activation solution prepared in (1-1) is added dropwise under ice bath stirring. The pH is adjusted to neutral with sodium hydroxide solution, and the reaction is carried out overnight at room temperature. After dialysis, the glutamic acid biological antigen is obtained.

[0013] Furthermore, the concentration of N-benzoyl-L-glutamic acid in water is 0.02 g / mL, the concentration of hydrochloric acid in water is 0.02 g / mL; the concentration of the aqueous sodium nitrite solution is 0.1 g / mL, and the molar mass ratio of N-benzoyl-L-glutamic acid to sodium nitrite is 4:1; the molar mass ratio of N-benzoyl-L-glutamic acid to the carrier protein is 100:1.

[0014] In a third aspect, the present invention provides a glutamic acid biological antibody, which is prepared by immunizing an animal with the immunogen described above.

[0015] Furthermore, the glutamic acid biological antibody is a rabbit polyclonal antibody.

[0016] In a fourth aspect, the present invention provides a method for detecting glutamic acid by enzyme-linked immunosorbent assay, which comprises the following steps: Glutamic acid is fully reacted with benzoyl chloride to be converted into N-benzoylglutamic acid, and the molar ratio of glutamic acid to N-benzoylglutamic acid is 1:1. Using the biological antigen as the coating antigen and immunogen, and the antibody prepared by immunizing an animal with the immunogen as the detection antibody, the glutamic acid in the sample is quantitatively detected by the standard addition calibration curve method; the structural formula of the glutamic acid biological antigen is as shown in Formula I:

[0017] Formula I

[0018] When the carrier protein is ovalbumin, the coating antigen is obtained; when the carrier protein is lactoferrin, the immunogen is obtained.

[0019] In a fifth aspect, the present invention provides an enzyme-linked immunosorbent assay kit for detecting glutamic acid, which comprises the coating antigen described above, the glutamic acid biological antibody and benzoyl chloride.

[0020] Sixth aspect, the present invention provides a colloidal gold immunoassay kit for detecting glutaminase activity, which includes the above-mentioned coating antigen, the glutamic acid biological antibody, benzoyl chloride and the hydrolysis substrate glutamine.

[0021] The detection method of the colloidal gold immunoassay kit includes the following steps:

[0022] (2-1) For the sample to be tested, centrifuge to obtain the supernatant, dilute it and reserve it as the enzyme solution to be tested.

[0023] (2-2) After reacting the glutamine solution with the enzyme solution to be tested in a water bath, add sodium hydroxide solution and mix well, then add benzoyl chloride solution, shake and react at room temperature. After the reaction is completed, add sodium hydroxide solution for neutralization, dilute with buffer solution, and detect the glutaminase activity u1 on the colloidal gold immunochromatographic detection device as the experimental group.

[0024] (2-3) Mix the enzyme solution to be tested with sodium hydroxide solution, react in a water bath, and add glutamine solution. The rest is the same as step (2-2), and detect the glutaminase activity u2 on the colloidal gold immunochromatographic detection device as the control group.

[0025] (2-4) The calculation formula for the glutaminase activity in the sample to be tested is: u = (u1 - u2) × n.

[0026] (2-5) The definition of glutaminase activity is: for every 1 milliliter (or 1 gram) of sample, under certain temperature and pH conditions, catalyzing glutamine to generate 1 nmol of glutamic acid in 1 minute is defined as one enzyme activity unit, expressed as u / mL (or u / g).

[0027] Furthermore, the dilution multiple of the sample supernatant is determined according to the enzyme activity of the sample, and the recommended concentration is 0.1 - 4.7 u / mL; the concentration of the glutamine solution is 0.05 g / L, and the volume ratio of the glutamine solution to the enzyme solution to be tested is 1:1; the water bath temperature is 37 °C and the water bath time is 10 minutes; the room temperature is 25 °C and the room temperature shaking reaction time is 5 minutes; the concentration of the sodium hydroxide solution is 200 g / L, and the volume ratio of the sodium hydroxide solution used in the reaction to the total volume is 50:1; the volume ratio of the sodium hydroxide solution used for neutralization to the total volume is 1:(18 - 20); the buffer solution is 0.01 mol / L phosphate buffer with a pH of 8.4, and the dilution multiple is 4 times.

[0028] Seventh aspect, the present invention provides a colloidal gold chromatographic detection device for detecting glutaminase activity, which includes a test strip and a working solution. The test strip is sequentially provided with a sample pad, a chromatographic membrane and a water absorption pad from bottom to top; the detection line is coated with glutamic acid coating antigen, and the quality control line is sprayed with goat anti-mouse IgG; the working solution contains colloidal gold-labeled glutamic acid biological antibody.

[0029] Further, the colloidal gold-labeled glutamate biological antibody is prepared through the following steps:

[0030] (3-1) Mix the chloroauric acid solution and trisodium citrate solution under boiling conditions, continuously heat until the solution turns wine red and then heat for another 10 minutes, and cool to room temperature to obtain the colloidal gold solution;

[0031] (3-2) Adjust the pH value of the colloidal gold solution, add the glutamate biological antibody and mix evenly, and let it stand at room temperature for 10 ± 5 minutes;

[0032] (3-3) Add bovine serum albumin and mix evenly, let it stand at room temperature for 15 ± 5 minutes, and centrifuge to obtain a precipitate;

[0033] (3-4) Add a reconstitution solution with the same volume as the solution before centrifugation to the precipitate for reconstitution to obtain the colloidal gold-labeled glutamate biological antibody.

[0034] Further, the mass ratio of the added chloroauric acid to trisodium citrate is 1:(1-2); the pH value of the colloidal gold solution is adjusted to 7.0 ± 1, the mass ratio of the colloidal gold to the glutamate biological antibody is 1:(1-10); the mass ratio of the colloidal gold to bovine serum albumin is 1:(100-1000); the reconstitution solution is 0.01mol / L phosphate buffer solution (pH = 8.0) + 1wt% BSA + 5wt% sucrose + 0.1wt% NaN3.

[0035] A method for determining glutamyl enzyme activity by a colloidal gold chromatography detection device includes the following steps:

[0036] (4-1) Mix 200 μL of the test solution with 40 μL of the working solution evenly, react at 20-40 °C for 3 min to obtain a reaction solution;

[0037] (4-2) Insert the test strip into the reaction solution obtained in (4-1), after reacting at 20-40 °C for 3 min, take out the test strip, scrape off the sample pad at the lower end of the test strip, read the absorbance value with a reader, and calculate the glutaminase activity. Or compare with the color development of the standard solution, and read the glutaminase activity in the test solution according to the depth of the test line. If the control line does not show color, this detection is invalid and needs to be retested.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The method for detecting glutamic acid by enzyme-linked immunosorbent assay in the present invention uses a biological antigen as the coating antigen and immunogen, and a biological antibody prepared by immunizing an animal with the immunogen as the detection antibody for detection. N-benzoylglutamic acid is the reaction product of glutamic acid and benzoyl chloride. By adding an excessive amount of benzoyl chloride, glutamic acid can be converted into N-benzoylglutamic acid. An enzyme-linked immunosorbent assay for indirectly detecting glutamic acid in food is constructed using the antibody of the present invention. Based on the above experimental basis and the principle that glutaminase can specifically catalyze glutamine to release free glutamic acid, a colloidal gold immunochromatographic assay for detecting the activity of glutaminase is constructed. The present invention applies immunological detection methods to detect the content of glutamic acid and the activity of glutaminase, which has the advantages of high sensitivity, simple operation, and can achieve rapid on-site detection, providing a new method for the detection of glutamic acid and glutaminase activity. The present invention applies immunological detection methods to specifically detect glutamic acid, which has the advantages of high specificity, strong specificity, and simple operation. This method can better meet and be applicable to the needs of rapid on-line detection and monitoring in the grass-roots departments of factory production workshops, and can also provide technical support for rapid detection by relevant scientific research workers.

[0040] As a small molecule, glutamic acid is directly connected to the carrier protein, and it is difficult for the exposed sites to be insufficient, making it difficult to cause a response in the body. The preparation of its antibody is somewhat challenging. As an acylating reagent, benzoyl chloride has a benzene ring with a rigid structure in its structure and strong electrophilicity. The amino group (-NH2) in the glutamic acid molecule, as a nucleophile, can attack the carbonyl carbon (C=O) in benzoyl chloride, and a nucleophilic substitution reaction can occur within a short time in an alkaline environment, converting it into a derivative with a more complex and rigid structure, thereby enhancing the immunogenicity and increasing the success rate of antibody preparation. Therefore, the present invention selects N-p-aminobenzoyl-L-glutamic acid as a hapten to prepare an antibody that can recognize N-benzoylglutamic acid, and then realizes the detection of the content of glutamic acid and the activity of glutaminase. Brief Description of the Drawings

[0041] In order to more clearly illustrate the solutions in the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0042] Figure 1 It is the ultraviolet scanning diagram of glutamic acid, its biological antigen, bovine serum albumin, and lactoferrin in the embodiment;

[0043] Figure 2 It is the inhibition standard curve of the enzyme-linked immunosorbent assay for glutamic acid content;

[0044] Figure 3It is a schematic structural diagram of a colloidal gold chromatography detection device;

[0045] Figure 4 It is a schematic diagram of the inhibition standard curve and interpretation of the results of a colloidal gold chromatography detection device for glutaminase activity. Specific implementation manners

[0046] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0047] In order to enable those skilled in the art to better understand the solution of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0048] Example 1 Synthesis of biological antigen for glutamic acid immunity and biological antigen for coating

[0049] Add 4 mg of N-benzoyl-L-glutamic acid to 200 μL of water, and add 10 μL of concentrated hydrochloric acid until completely dissolved. Add 12.54 μL of a 0.1 g / mL sodium nitrite aqueous solution under ice bath, and stir and react at 4°C for 30 min to obtain an activation solution. Dissolve 10 mg of lactoferrin in 1 mL of carbonate buffer solution, and gradually add the above activation solution dropwise under ice bath stirring. Adjust the pH to neutral with 3 mol / L NaOH, and react at room temperature overnight. Dialyze with physiological saline at 4°C for 3 days, changing the dialysis solution 2 times a day during this period, to obtain a biological antigen for immunity.

[0050] Using the same preparation steps as those for preparing the antigen for glutamic acid immunity above, replace the bovine lactoferrin in the above preparation method with ovalbumin to prepare an antigen for coating glutamic acid.

[0051] The carbonate buffer solution (0.01 mol / L, pH = 9.6): 0.338 g of Na2CO3, 0.586 g of NaHCO3, and make up to 1000 mL with ultrapure water.

[0052] Example 2 Identification of antigen for immunity and antigen for coating

[0053] Perform ultraviolet scanning determination on glutamic acid, biological antigen for immunity, biological antigen for coating, and carrier proteins (LF, OVA), and the results are as Figure 1As shown, the ultraviolet absorption peaks of the biological antigen for immunization and the biological antigen for coating are blue-shifted to varying degrees compared with the ultraviolet absorption peak of the carrier protein, indicating the successful preparation of the biological antigen.

[0054] Example 3 Preparation of Glutamic Acid Rabbit Polyclonal Antibody

[0055] The biological antigen for immunization prepared in Example 1 was emulsified evenly with an equal amount of Freund's adjuvant, and then 2 female New Zealand white rabbits weighing 2 - 2.5 kg were immunized respectively. The immunization dose was 1000 μL / time / rabbit. Subcutaneous injection at multiple points on the back and abdomen was used for immunization. The interval between the first immunization and the second immunization was 4 weeks, and the interval between each subsequent immunization was 3 weeks. One week after the fifth booster immunization, blood was collected from the heart, and the supernatant was obtained by centrifugation to obtain rabbit polyclonal antibody.

[0056] Example 4 Derivatization Reaction of Glutamic Acid and Benzoyl Chloride

[0057] Take 500 μL of a 1 μg / mL glutamic acid solution prepared with phosphate buffer, add 20 μL of benzoyl chloride and 20 μL of NaOH solution (5 mol / L), and stir at room temperature for a derivatization reaction time of 5 min. The derivative is N-benzoylglutamic acid, and its structural formula is shown in Formula (II):

[0058]

[0059] The said phosphate buffer (0.02 mol / L, pH = 7.4): 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, 8.5 g of NaCl, and made up to 500 mL with ultrapure water.

[0060] Example 5 Drawing of Inhibition Standard Curve and Detection of Test Solution by Enzyme-Linked Immunosorbent Assay for Glutamic Acid Content

[0061] Using carbonate buffer (0.01 mol / L, pH = 9.6) as the dilution solution for the coating antigen, phosphate buffer (0.02 mol / L, pH = 7.4) as the dilution solution for the biological antibody and the standard product, and phosphate Tween buffer (0.01 mol / L, pH = 7.4, 0.05% Tween-20) as the dilution solution for the horseradish peroxidase-labeled goat anti-mouse solution.

[0062] Dilute the coating source to 15.6 ng / mL and add 100 μL / well to a 96-well plate. Incubate at 4°C for 12 hours. Wash the plate twice using a plate washer. Add 120 μL of 5% bovine serum albumin to each well and incubate at 37°C for 1.5 hours. Pat dry and dry in a 37°C oven for 0.5 hours. A commercially available glutamate standard was serially diluted to concentrations of 20,000, 4,000, 800, 160, 32, 6.4, 1.28, and 0 ng / mL. 500 μL of the sample was then derivatized. The solution was then neutralized with 55 μL of 5 mol / L sodium hydroxide solution and diluted with 1500 μL of phosphate buffer. Mix thoroughly and add 50 μL / well to a 96-well plate (blank wells contain phosphate buffer). The test solution was then added to the 96-well plate at 50 μL / well. Dilute the biological antibody to 62.5 ng / mL, add it to the above wells, 50 μL / well, incubate at 37°C for 40 minutes, and wash the plate 5 times. Dilute the goat anti-mouse 5000 times, 100 μL / well, incubate at 37°C for 30 minutes, and wash the plate 5 times. Add 100 μL of 3',5,5'-tetramethylbenzidine (TMB) substrate colorimetric solution to each well and incubate at 37°C for 10 minutes. Add 50 μL of 10% concentrated sulfuric acid to each well to terminate the reaction. Use an enzyme reader to measure the absorbance at a wavelength of 450 nm. The result of the standard well is B, the blank well is B0, and the well of the solution to be tested is B1. With B / B0 as the vertical coordinate and the logarithm of the glutamate concentration as the horizontal coordinate, the curve is fitted using Origin software, which is the inhibition standard curve. The results are as shown in the figure. Figure 2 As shown, IC 10 The detection limit is IC 20 ~IC 80 is the detection range, IC 50 For sensitivity. O Substitute it into the standard curve to get the glutamate concentration of the test solution.

[0063] Example 6 Preparation of a Colloidal Gold Chromatography Detection Device for Detecting Glutaminase Activity

[0064] 6.1 Preparation of colloidal gold

[0065] Weigh 100 mL of first-grade water into a 500 mL round-bottom flask and heat to a boil in a magnetic stirring oil bath (180°C, 700 rpm). Add 4 mL of 1 wt% chloroauric acid solution and continue heating and stirring until boiling again. Quickly add 4 mL of 1 wt% trisodium citrate. Once the solution turns a clear wine red, continue heating for 10 minutes. Then, stir and cool to room temperature.

[0066] 6.2 Preparation of colloidal gold-labeled rabbit polyclonal antibodies

[0067] Take 1 mL of colloidal gold solution, add 30 μL of 0.1 mol / mL potassium carbonate solution, stir evenly, then add 7.5 μg of antibody and mix well. Let it stand at room temperature for 10 min. Add 10 μL of 10 wt% BSA and mix well, then let it stand at room temperature for 15 min. Centrifuge for 8 min (4 °C, 8000 rpm) to obtain the precipitate of colloidal gold-labeled rabbit polyclonal antibody, and add an equal volume of reconstitution solution. The reconstitution solution is 0.01 mol / L phosphate buffer (pH = 8.0) + 1 wt% BSA + 5 wt% sucrose + 0.1 wt% NaN3.

[0068] 6.3 Preparation of colloidal gold detection device

[0069] The test strip is as Figure 3 shown. On the bottom plate, in the same direction, the sample pad, the nitrocellulose membrane sprayed with the antigen for glutamic acid coating (i.e., the test line) and goat anti-mouse IgG (i.e., the control line), and the absorbent paper are sequentially overlapped and adhered. Add 35 μL of colloidal gold-labeled polyclonal antibody into the reaction cup.

[0070] Example 7 Standard curve and interpretation schematic diagram of the results of the colloidal gold chromatography detection device for glutaminase activity

[0071] Dilute the commercially available standard glutaminase with phosphate buffer (0.01 mol / L, pH = 8.4) in gradients, and the concentrations are 1400, 140, 14, 1.4, 0.14, 0.014, 0.0014, 0 U / mL. Respectively take 500 μL and add it to 500 μL of glutamine solution with a concentration of 50 μg / mL. After reacting in a 37 °C water bath for 10 minutes, add 20 μL of 5 mol / L sodium hydroxide solution and benzoyl chloride. After reacting with shaking at room temperature for 5 minutes, add 55 μL of 5 mol / L sodium hydroxide solution to adjust the solution to neutral, and then add 3285 μL of phosphate buffer for dilution. Take 200 μL and add it to the reaction cup and mix well. After 3 minutes, insert the test strip. After five minutes, use a machine to read the absorbance value and take a photo with a smartphone. Use the ratio to the blank group as the ordinate and the logarithm of the concentration of the glutaminase standard solution as the abscissa to draw a standard curve, and use Origin software to fit the curve. The result is as Figure 4 shown. Take IC 10 as the detection limit, take IC 20 ~IC 80 as the detection range, and IC 50 as the sensitivity.

[0072] Example 8 Detection of actual samples of glutaminase activity

[0073] Take 20 mL of the fermented mash to be tested, centrifuge and take 1 mL of the supernatant, add 50 mL of phosphate buffer, and reserve it as the enzyme solution to be tested;

[0074] Take 500 μL of glutamine solution (50 μg / mL) into a 4 mL centrifuge tube, add 500 μL of the enzyme solution to be tested, react in a water bath for 10 minutes, add 20 μL of 5 mol / L sodium hydroxide solution and benzoyl chloride, shake and react at room temperature for 5 minutes, then add 55 μL of 5 mol / L sodium hydroxide solution to adjust the solution to neutral, and then add 3285 μL of phosphate buffer for dilution. Take 200 μL and add it to the reaction cup to mix well. After 3 minutes, insert the test strip. After five minutes, read the absorbance value as 0.45 (B1), which is used as the experimental group. Take 500 μL of the enzyme solution to be tested, add 20 μL of sodium hydroxide and mix well, react in a water bath for 10 minutes. After the reaction, add 500 μL of glutamine solution, and the rest is the same as the experimental group. Read the absorbance value as 1.23 (B2), which is used as the blank group. Take 500 μL of phosphate buffer, add 20 μL of sodium hydroxide and mix well, react in a water bath for 10 minutes. After the reaction, add 500 μL of glutamine solution, and the rest is the same as the experimental group. Read the absorbance value 1.24 (B0), which is used as the standard group. Substitute B1 / B0 and B2 / B0 into Figure 3 the standard curve, and calculate the glutaminase activity of the moromi to be 147 u / mL. Or compare with the color development of the standard test strip, and the glutaminase activity is between 50 - 500 u / mL.

[0075] Example 9 In the experimental steps described in Example 1, the coupling ratio of N - p - aminobenzoyl - L - glutamic acid to the carrier protein is 100:1. The present invention also explores the effects of different coupling ratios, different carrier proteins, and coating antigen concentrations on the antibody performance. The results are shown in Table 1. When the addition amount of N - p - aminobenzoyl - L - glutamic acid is 2 mg and the addition amount of sodium nitrite aqueous solution is 6.27 μL, the coupling ratio is 50:1; when the addition amount of N - p - aminobenzoyl - L - glutamic acid is 1 mg and the addition amount of sodium nitrite aqueous solution is 3.14 μL, the coupling ratio is 25:1. When the carrier protein is BSA, the coating antigen - BSA can be obtained. The results are shown in Table 1. When the carrier protein is OVA, the coupling ratio is 100:1, and the coating antigen concentration is 15.6 ng / mL, IC 10 and IC 50 values are the smallest, indicating the best effect under this condition.

[0076] Table 1 shows the effects of different carrier proteins, coupling ratios, and coating antigen concentrations on the antibody performance

[0077]

[0078]

[0079] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0080] As described above, the above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

[0081] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structure directly or indirectly using the content of the specification and drawings of the present application in other related technical fields shall be within the scope of the patent protection of the present application by the same token.

Claims

1. An enzyme-linked immunosorbent assay method for detecting glutamic acid, characterized in that, It includes the following steps: using glutamic acid biological antigen as the coating antigen and immunogen, using the antibody prepared by immunizing an animal with the immunogen as the detection antibody, and performing quantitative detection of glutamic acid; the structural formula of the glutamic acid biological antigen is as shown in Formula I: When the carrier protein is ovalbumin, the coating antigen is obtained; when the carrier protein is lactoferrin, the immunogen is obtained.

2. A glutamate biological antigen, characterized in that, The structural formula is as shown in Formula I, When the carrier protein is ovalbumin, the coating antigen is obtained; when the carrier protein is lactoferrin, the immunogen is obtained.

3. The glutamate biological antigen according to claim 1, wherein It is prepared by the following method: (1-1) N-Benzoyl-L-glutamic acid is dissolved in water, concentrated hydrochloric acid is added until completely dissolved; an aqueous sodium nitrite solution is added under ice bath, and after stirring and reacting, an activation solution is obtained; (1-2) The carrier protein is dissolved in a buffer solution, the activation solution prepared in (1-1) is added dropwise under ice bath stirring, the pH is adjusted to neutral with sodium hydroxide solution, and the reaction is carried out overnight at room temperature. After dialysis, the glutamic acid biological antigen is obtained.

4. The glutamate biological antigen according to claim 3, wherein, The concentration of N-benzoyl-L-glutamic acid in water is 0.02 g / mL, the concentration of hydrochloric acid in water is 0.02 g / mL; the concentration of the aqueous sodium nitrite solution is 0.1 g / mL, and the molar mass ratio of N-benzoyl-L-glutamic acid to sodium nitrite is 4:1; the molar mass ratio of N-benzoyl-L-glutamic acid to the carrier protein is 100:

1.

5. A glutamate biological antibody, characterized in that, It is prepared by immunizing an animal with the immunogen described in Claim 1 or 2.

6. The glutamate biological antibody according to claim 5, wherein The glutamic acid biological antibody is a rabbit polyclonal antibody.

7. An enzyme-linked immunosorbent assay kit for detecting glutamic acid, characterized in that, It includes the coating antigen described in Claim 2, the glutamic acid biological antibody described in Claim 5, and benzoyl chloride.

8. A colloidal gold immunoassay kit for detecting glutaminase activity, characterized in that, It includes the coating antigen described in Claim 2, the glutamic acid biological antibody described in Claim 5, benzoyl chloride, and the hydrolysis substrate glutamine.

9. A colloidal gold chromatography detection device for detecting glutaminase activity, characterized in that, It includes a test strip and a working solution, wherein the test strip is sequentially provided with a sample pad, a chromatographic membrane, and a water absorption pad from bottom to top; the detection line is coated with the coating antigen described in Claim 2, and the working solution contains the glutamic acid biological antibody described in Claim 5 labeled with colloidal gold.

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