An enzyme-labeled anti-lysozyme gold antibody, an enzyme-linked immunosorbent assay kit for detecting lysozyme and its application

Abstract

The present invention discloses an enzyme-labeled anti-lysozyme gold antibody and an enzyme-linked immunosorbent assay kit for detecting lysozyme and their application. The enzyme-labeled anti-lysozyme gold antibody has both the ability to bind to lysozyme and the characteristics of horseradish peroxidase, and is applied to detect hen egg white lysozyme in enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay kit comprises: an enzyme-labeled anti-lysozyme gold antibody, an enzyme-labeled plate, a lysozyme standard, a standard diluent, an enzyme-labeled plate coated with a lysozyme-specific antibody, a color developing solution, a sample diluent, a washing solution and a stop solution. The present invention also discloses a method for performing sample detection using the kit. The kit adopts the direct method principle in enzyme-linked immunosorbent assay, and can quantitatively detect lysozyme in hen egg white. The kit uses a gold antibody instead of a natural antibody for detection, which not only reduces production costs, but also avoids cross-reactions, and has other advantages of an enzyme-linked immunosorbent assay kit.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to an enzyme-labeled anti-lysozyme gold antibody, an enzyme-linked immunosorbent assay kit for detecting lysozyme and its application, which are applied to the technical field of quantitative detection of lysozyme. Background Art

[0002] Lysozyme is an alkaline enzyme with strong bactericidal ability, and it achieves the bactericidal effect by hydrolyzing the β-1,4 bond between N-acetyl-d-glucosamine and N-acetylmuramic acid residues in the peptidoglycan layer of the bacterial cell wall. Hen Egg-White Lysozyme (HEWL) is one of the most widely studied and applied lysozymes, with a wide range of existence, and it is currently widely used in the fields of food, cosmetics and medicine. Moreover, as a common food allergen, it is statistically known that 1.6% of children are allergic to egg products.

[0003] Currently, the existing methods for measuring lysozyme include high performance liquid chromatography or liquid chromatography-mass spectrometry, etc. These methods all have the disadvantages of high price, complexity of the used instruments and high detection costs. The enzyme-linked immunosorbent assay has good sensitivity and accuracy, and also has the advantage of relatively low detection cost. However, the natural antibodies have the disadvantages of poor stability and high cost, which limit the application of the enzyme-linked immunosorbent assay to a certain extent. Summary of the Invention

[0004] In order to solve the problems of the existing technology, the purpose of the present invention is to overcome the deficiencies of the existing technology, and provide an enzyme-labeled anti-lysozyme gold antibody, an enzyme-linked immunosorbent assay kit for detecting lysozyme and its application. The enzyme-labeled anti-lysozyme gold antibody has the ability to bind lysozyme and the characteristics of horseradish peroxidase, and it is used in the enzyme-linked immunosorbent assay kit to detect the content of hen egg-white lysozyme, which has the advantages of high sensitivity, strong specificity and simple operation.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] An enzyme-labeled anti-lysozyme gold antibody is prepared by using gold nanoparticles to bind polypeptides to form an anti-lysozyme gold antibody, and simultaneously adding horseradish peroxidase (HRP) labeled with thiol polyethylene glycol (HS-PEG), and synthesizing the enzyme-labeled anti-lysozyme gold antibody by a one-step method.

[0007] Preferably, horseradish peroxidase (HRP) labeled with thiol polyethylene glycol (HS-PEG) is added simultaneously with the polypeptide, so that the -SH group on HRP-PEG forms an Au-S bond with the gold nanoparticles, and while forming the gold antibody, the gold antibody is combined with HRP to form an enzyme-labeled anti-lysozyme gold antibody, and the number of HRPs connected to a single gold nanoparticle is 1-20. Applied to the direct method of enzyme-linked immunosorbent assay, the enzyme-labeled anti-lysozyme gold antibody is used as a detection antibody, and it also has some advantages of the indirect method, that is, HRP-PEG is roughly equivalent to the enzyme-labeled secondary antibody in the indirect method, and the price is lower than the enzyme-labeled secondary antibody, which not only reduces the production cost, but also avoids cross-reactions. At the same time, a single enzyme-labeled anti-lysozyme gold antibody connected to multiple HRPs can improve the detection sensitivity.

[0008] Preferably, HS-PEG-COOH is reacted with horseradish peroxidase (HRP) via EDC-NHS to react the carboxyl group on PEG with the amino group of HRP to form HS-PEG-HRP.

[0009] The invention discloses an enzyme-linked immunosorbent assay kit for detecting lysozyme, comprising the enzyme-labeled anti-lysozyme gold antibody of the invention, a blank enzyme-labeled plate, a lysozyme standard, a diluent, a color developing solution, a washing solution and a stop solution.

[0010] Preferably, lysozyme is detected by enzyme-linked immunosorbent assay using an enzyme-labeled anti-lysozyme gold antibody. The specific method steps are as follows:

[0011] Lysozyme was pre-coated on a blank ELISA plate, and the ELISA-labeled anti-lysozyme gold antibody was added. After incubation for a set time, the lysozyme and the ELISA-labeled anti-lysozyme gold antibody were combined, and a color reaction was produced by adding a substrate of horseradish peroxidase. After the reaction time was set, the stop solution was added to terminate the reaction, and the ELISA plate was placed in a multifunctional ELISA reader for detection to obtain the OD values ​​corresponding to different lysozyme concentrations. 450nm value, and make a standard curve to determine the concentration of the lysozyme sample to be tested.

[0012] An application of the lysozyme detection enzyme-linked immunosorbent assay kit of the present invention is used to implement a method for detecting lysozyme, comprising the following steps:

[0013] (1) Coating: Add 20-200 μL of lysozyme of different concentrations to each well of the ELISA plate, use carbonate buffer solution as the diluent, and incubate at 4°C overnight. Wash the plate and pat dry.

[0014] (2) Blocking: 20-200 μL of BSA per well, using carbonate buffer solution or phosphate buffer as the diluent, shake at room temperature for 0.5-2 h, wash the plate and pat dry;

[0015] (3) Combine the enzyme-labeled gold antibody: Add 20 - 200 μL of the enzyme-labeled anti-lysozyme gold antibody to each well, shake at room temperature for 0.5 - 3 h, wash the plate and pat dry.

[0016] (4) Color reaction: Add 20 - 200 μL of the substrate chromogenic solution to each well, let it stand in the dark for 5 - 20 min, and then add the stop solution to terminate the reaction.

[0017] (5) Detection and analysis: Use a multifunctional microplate reader to measure the signal value OD of each well. 495nm , Analyze the detection results. Take the lysozyme concentration on the x-axis and the OD value at 495 nm on the y-axis to plot the standard curve for detecting lysozyme.

[0018] Preferably, the diluent is a buffer solution with a pH of 9 - 11.

[0019] Preferably, the substrate chromogenic solution is 3,3′,5,5′-tetramethylbenzidine (TMB) or o-phenylenediamine (OPD).

[0020] Preferably, the stop solution uses sulfuric acid or hydrochloric acid, and the acid concentration of the stop solution is not greater than 2 M.

[0021] Compared with the prior art, the present invention has the following obvious outstanding substantial features and remarkable advantages:

[0022] 1. The enzyme-linked immunosorbent assay kit for detecting lysozyme in the present invention uses a gold antibody instead of a natural antibody for detection. This not only avoids the disadvantage of poor stability of natural antibodies, but also, as a detection antibody, the enzyme-labeled gold antibody also has some advantages of the indirect method. That is, HRP-PEG is roughly equivalent to the enzyme-labeled secondary antibody in the indirect method, and its price is lower than that of the enzyme-labeled secondary antibody, which not only reduces the production cost but also avoids cross-reaction.

[0023] 2. The enzyme-labeled anti-lysozyme gold antibody of the present invention has good specificity and high sensitivity, and can perform accurate quantitative detection of results, with the advantages of simple operation, fast speed, and low detection cost.

[0024] 3. The present invention adopts the principle of the direct method in the enzyme-linked immunosorbent assay to quantitatively detect the lysozyme in egg white. The kit of the present invention uses a gold antibody instead of a natural antibody for detection, which not only reduces the production cost but also avoids cross-reaction, and also has other advantages of the enzyme-linked immunosorbent assay kit. Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the enzyme-labeled anti-lysozyme gold antibody of the preferred embodiment of the present invention.

[0026] Figure 2 It is a schematic diagram of the principle for detecting lysozyme of the preferred embodiment of the present invention.

[0027] Figure 3This is the standard curve for detecting lysozyme in the preferred embodiment of the present invention. Detailed implementation manners

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described 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 the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] In the preferred embodiment of the present invention, Pep1 designed according to the polypeptide fragment of the CDR3 of cAb-Lys3 disclosed in the patent document with the publication number of CN105884888A is grafted onto gold nanoparticles through S-Au bonds to prepare a gold antibody against lysozyme with the same specificity as the natural antibody. On this basis, as Figure 1 shown, horseradish peroxidase (HRP) labeled with mercapto polyethylene glycol and the polypeptide are added simultaneously, and are also grafted onto gold nanoparticles through Au-S bonds, thereby synthesizing an enzyme-labeled gold antibody against lysozyme in one step.

[0030] In the preferred embodiment of the present invention, the above-mentioned enzyme-labeled gold antibody against lysozyme is used to detect lysozyme according to the principle of the direct method in the enzyme-linked immunosorbent assay. As Figure 2 shown, its detection principle is: different concentrations of lysozyme are pre-coated on the well plate, such as Figure 2 part A in, after adding the enzyme-labeled gold antibody and incubating for a period of time, such as Figure 2 part B in, due to the different concentrations of lysozyme on the well plate, the number of enzyme-labeled gold antibodies retained on the well plate is also different. Using the HRP coupled with the gold antibody to react with the corresponding substrate, such as Figure 2 part C in, the absorbance OD 495nm on the well plate is positively correlated with the concentration of lysozyme, and thus a standard curve can be drawn.

[0031] The kit of the present invention includes: an enzyme-labeled gold antibody against lysozyme, a blank enzyme-labeled plate, a lysozyme standard, a diluent, a chromogenic solution, a washing solution, and a termination solution.

[0032] The steps for detecting lysozyme in the present invention are as follows:

[0033] (1) Coating: Add 20 - 200 μL of different concentrations of lysozyme to each well on the enzyme-labeled plate, and let it stand overnight at 4°C, wash the plate and pat it dry;

[0034] (2) Blocking: Add 20 - 200 μL of BSA to each well, oscillate at room temperature for 0.5 - 2 h, wash the plate and pat it dry;

[0035] (3) Add 20-200 μL of enzyme-labeled anti-lysozyme gold antibody to each well, shake at room temperature for 0.5-3 h, wash the plate and pat dry;

[0036] (4) Add 20-200 μL of freshly prepared OPD substrate to each well, incubate in dark for 5-20 min, and then add stop solution to terminate the reaction.

[0037] (5) Use a multifunctional microplate reader to measure the signal value OD of each well 495nm , analyze the test results.

[0038] In a preferred embodiment of the present invention, the diluent is a buffer solution with a pH of 9-11, the substrate color developing solution is tetramethylbenzidine (TMB) or o-phenylenediamine (OPD), and the stop solution is sulfuric acid or hydrochloric acid.

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] The above scheme is further described below in conjunction with specific implementation examples. The preferred embodiments of the present invention are described in detail as follows:

[0041] Embodiment 1:

[0042] In this embodiment, a method for preparing polyethylene glycol-coupled horseradish peroxidase is as follows:

[0043] Weigh 1.2 mg HS-PEG-COOH and dissolve it in 6 mL phosphate buffer (40 mM, pH 7.4), weigh 10 mg NHS and dissolve it in 50 μL ultrapure water, weigh 10 mg EDC and dissolve it in 50 μL ultrapure water, and weigh 8 mg HRP and dissolve it in 2 mL ultrapure water. Under stirring at 4 ° C, add 30 μL EDC solution to 5 mL HS-PEG-COOH solution, react in the dark for 10 minutes, add 30 μL NHS solution and react in the dark for 30 minutes, add 2 mL HRP solution to the mixed solution, and react in the dark overnight. After the reaction is completed, use an ultrafiltration tube with a molecular weight cutoff of 10 kDa to remove unreacted products by centrifugal ultrafiltration at 10 ° C 300g, wash three times with phosphate buffer (40 mM pH 7.4), and freeze-dry the resulting supernatant to obtain the final product, which is HRP-PEG-SH.

[0044] Embodiment 2:

[0045] This embodiment is basically the same as the first embodiment, except that:

[0046] In this example, the enzyme-labeled gold antibody was constructed as follows:

[0047] The 13-nm gold nanoparticles were synthesized as follows: 240 mL of ultrapure water and 25 mL of 39.47 mM trisodium citrate solution were added to a 500-mL round-bottom flask and placed in a 120 °C constant-temperature oil bath. After condensation and reflux, and after reacting for 20 min, 10 mL of 25 mM chloroauric acid solution was quickly added. After uniformly stirring for about 5 min, it was taken out and naturally cooled to room temperature, thus obtaining 13-nm gold nanoparticles.

[0048] Coupling of gold nanoparticles with polypeptides and horseradish peroxidase: After filtering the synthesized gold nanoparticles using a 0.22-μm filter membrane, 6 mL of the gold nanoparticle solution was added to a 10-mL glass bottle equipped with a 15×8-mm diamond-shaped polytetrafluoroethylene magnetic stirrer and uniformly stirred on a multi-site magnetic stirrer at a speed of 650 rpm to ensure that no bubbles were generated throughout the process. After the stirring was stable, 10 μL of 0.2 M trisodium citrate solution was added, and 1 mL of the polypeptide solution (containing 2 mM NaOH) was gradually added dropwise to the stirring solution and continuously stirred at room temperature for 1 h. 1 mL of the horseradish peroxidase solution labeled with HS-PEG(5K) was gradually added dropwise to the stirring solution and continuously stirred for 1 h. The prepared enzyme-labeled gold antibody was stored in a 4 °C refrigerator for later use.

[0049] Example 3:

[0050] This example is basically the same as the previous examples, with the special feature being that:

[0051] In this example, a standard curve of lysozyme was drawn:

[0052] The lysozyme standard was diluted with a diluent. The diluent used was 20 mM pH 10.7 carbonate buffer solution, and the final concentrations were 32, 16, 8, 4, 2, 1, 0.5 μg / mL. Then, the detection was started according to the following steps:

[0053] 1) Coating: 100 μL of lysozyme at different concentrations was added to each well. The diluent used was 20 mM pH 10.7 carbonate buffer solution, and it was left standing overnight at 4 °C. The plate was washed and patted dry, and this was repeated 4 times. Each time, the washing solution remained in the plate for 30 s;

[0054] 2) Blocking: 200 μL of 5% BSA was added to each well. The diluent used was 20 mM pH 7.4 carbonate buffer solution, and it was shaken at room temperature for 1 h. The plate was washed and patted dry, and this was repeated 4 times. Each time, the washing solution remained in the plate for 30 s;

[0055] 3) Binding of the enzyme-labeled gold antibody: 100 μL of the corresponding detection antibody was added to each well, and it was shaken at room temperature for 2 h. The plate was washed and patted dry, and this was repeated 4 times. Each time, the washing solution remained in the plate for 30 s);

[0056] 4) Color reaction: Add 100 μL of freshly prepared substrate OPD to each well, and let it react in the dark and stand still for 15 min. Then add 100 μL of 2 M H 2 SO 4 to terminate the reaction;

[0057] 5) Detection and analysis: Measure the signal value of each well using a multifunctional microplate reader. Take the lysozyme concentration on the x-axis and the OD value at 495 nm on the y-axis to plot the standard curve for detecting lysozyme, Figure 3 which is the obtained standard curve for detecting lysozyme.

[0058] Example 4:

[0059] This example is basically the same as the previous examples, with the special feature being:

[0060] In this example, to detect lysozyme in a complex environment, the method is as follows:

[0061] To test the effect of the enzyme-labeled gold antibody ELISA method for detecting lysozyme under complex solution conditions, positively charged RNase A with properties similar to HEWL and negatively charged albumin (BSA) that is abundant in blood are selected as representative interfering proteins. First, prepare pure solutions of HEWL, RNase A, and BSA separately, and determine their respective concentrations by measuring absorbance. Then mix them in a certain ratio to obtain spiked samples. The final concentrations of HEWL in the samples are 1.5 μg·mL -1 , 4.6 μg·mL -1 and 10 μg·mL -1 , the corresponding concentration of RNase A is 10 times the concentration of HEWL, i.e., 15 μg·mL -1 , 46 μg·mL -1 and 100 μg·mL -1 , and the corresponding BSA is set to 100 times the concentration of HEWL, i.e., 150 μg·mL -1 , 460 μg·mL -1 and 1000 μg·mL -1 . The three samples are subjected to spiked recovery determination by the traditional direct method and the enzyme-labeled gold antibody direct method respectively, and the measured values and spiked recovery rates are analyzed. Table 1 shows the detection results. For the three samples detected by the enzyme-labeled gold antibody, their recovery rates are all within 90%-110%, meeting the detection standards, and their accuracy is higher than that of the traditional ELISA direct method.

[0062] Table 1. Recovery rates of enzyme-labeled gold antibody ELISA and traditional direct method ELISA for different lysozyme-spiked samples

[0063]

[0064] Example 5:

[0065] This embodiment is basically the same as the foregoing embodiment, with the special feature being that:

[0066] In this embodiment, the method for detecting lysozyme in egg white is as follows:

[0067] Manually separate the egg white from fresh eggs. Use a diluent, which is a 20 mM pH 10.7 carbonate buffer solution. Dilute the egg white 500 and 1000 times, and then detect the diluted samples according to the steps for detecting lysozyme in Example 3. Substitute the obtained OD value into the standard curve to obtain the detection value, and the product of the detection value and the dilution factor is the content of lysozyme in the egg white. At the same time, use a traditional enzyme-linked immunosorbent assay (ELISA) direct method kit to detect the lysozyme in the egg white. The detection results are shown in Table 2. The consistency of the detection results of the enzyme-labeled gold antibody ELISA method for samples with different dilution factors is better than that of the traditional ELISA direct method.

[0068] Table 2. Detection results of lysozyme in egg white by enzyme-labeled gold antibody ELISA and traditional direct method ELISA

[0069]

[0070] The above embodiments relate to an enzyme-labeled anti-lysozyme gold antibody, a detection lysozyme enzyme-linked immunosorbent assay (ELISA) kit and its application. This enzyme-labeled anti-lysozyme gold antibody has both the ability to bind lysozyme and the characteristics of horseradish peroxidase, and it is applied to detect lysozyme in egg white by ELISA. This ELISA kit includes: an enzyme-labeled anti-lysozyme gold antibody, an ELISA plate, a lysozyme standard, a standard diluent, an ELISA plate coated with a lysozyme-specific antibody, a chromogenic solution, a sample diluent, a washing solution, and a termination solution. The present invention also discloses a method for detecting samples using this kit. The above embodiments of the kit adopt the principle of the direct method in the enzyme-linked immunosorbent assay (ELISA) method and can quantitatively detect the lysozyme in the egg white. The above embodiments of the kit use a gold antibody instead of a natural antibody for detection, which not only reduces the production cost but also avoids cross-reaction, and also has other advantages of an ELISA kit.

[0071] The above has described the embodiments of the present invention in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments and can be variously changed according to the purpose of the invention of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made based on the spirit and principle of the technical solution of the present invention shall be equivalent replacement methods. As long as they meet the invention purpose of the present invention and do not deviate from the technical principle and inventive concept of the present invention, they all fall within the protection scope of the present invention.

Claims

1. An enzyme-labeled anti-lysozyme gold antibody, characterized in that: Polypeptides are combined with gold nanoparticles to form anti-lysozyme gold antibodies, and horseradish peroxidase (HRP) labeled with mercapto polyethylene glycol (HS-PEG) is added simultaneously. The enzyme-labeled anti-lysozyme gold antibody is synthesized by a one-step method.

2. The enzyme-labeled anti-lysozyme gold antibody according to claim 1, characterized in that: Horseradish peroxidase (HRP) labeled with mercapto polyethylene glycol (HS-PEG) and polypeptides are added simultaneously, so that the -SH group on HRP-PEG forms an Au-S bond with gold nanoparticles. While forming the gold antibody, the gold antibody binds HRP to form an enzyme-labeled anti-lysozyme gold antibody, and the number of HRPs attached to a single gold nanoparticle is 1-20.

3. The enzyme-labeled anti-lysozyme gold antibody according to claim 1, characterized in that: HS-PEG-COOH and horseradish peroxidase (HRP) are reacted through an EDC-NHS reaction, so that the carboxyl group on PEG reacts with the amino group of HRP to form HS-PEG-HRP.

4. A lysozyme enzyme-linked immunosorbent assay kit, characterized in that: It includes the enzyme-labeled anti-lysozyme gold antibody according to claim 1, a blank enzyme-labeled plate, a lysozyme standard, a diluent, a chromogenic solution, a washing solution, and a terminating solution.

5. The lysozyme enzyme-linked immunosorbent assay kit according to claim 4, characterized in that: The enzyme-labeled anti-lysozyme gold antibody is used to detect lysozyme by the direct enzyme-linked immunosorbent assay method. The specific method steps are as follows: Pre-coat lysozyme on a blank ELISA plate, add enzyme-labeled anti-lysozyme gold antibody. After incubating for a set time, when lysozyme and the enzyme-labeled anti-lysozyme gold antibody are combined, a color reaction is generated by adding the substrate of horseradish peroxidase. After the reaction for the set time, add a stop solution to terminate the reaction, and place the ELISA plate into a multi-functional microplate reader for detection to obtain the OD 450nm values, and make a standard curve to determine the concentration of the lysozyme sample to be measured.

6. An application of the lysozyme enzyme-linked immunosorbent assay kit according to claim 4 for implementing a method for detecting lysozyme, characterized in that: It includes the following steps: (1) Coating: Add 20-200 μL of different concentrations of lysozyme to each well of the enzyme-labeled plate, use a carbonate buffer solution as the diluent, and let it stand overnight at 4 °C. Wash the plate and pat it dry; (2) Blocking: Add 20-200 μL of BSA to each well, use a carbonate buffer solution or a phosphate buffer solution as the diluent, oscillate at room temperature for 0.5-2 h, wash the plate and pat it dry; (3) Binding the enzyme-labeled gold antibody: Add 20-200 μL of the enzyme-labeled anti-lysozyme gold antibody to each well, oscillate at room temperature for 0.5-3 h, wash the plate and pat it dry; (4) Chromogenic reaction: Add 20-200 μL of the substrate chromogenic solution to each well. After reacting in the dark and standing for 5-20 min, add the terminating solution to terminate the reaction; (5)Detection and analysis: Use a multifunctional microplate reader to measure the signal value OD of each well. 495nm Analyze the detection results. Take the lysozyme concentration as the x-axis and the OD value at 495 nm as the y-axis to plot the standard curve for detecting lysozyme.

7. The application of the lysozyme enzyme-linked immunosorbent assay kit according to claim 6, characterized in that: The diluent is a buffer solution with a pH of 9-11.

8. The application of the lysozyme enzyme-linked immunosorbent assay kit according to claim 6, characterized in that: The substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine (TMB) or o-phenylenediamine (OPD).

9. The application of the lysozyme enzyme-linked immunosorbent assay kit according to claim 6, characterized in that: The terminating solution uses sulfuric acid or hydrochloric acid, and the acid concentration of the terminating solution is not greater than 2 M.

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