Preparation method and application of HRP (horse radish peroxidase) enzyme-labeled antibody
Through the Staudings reaction to modify the peroxidase and combine with antibodies, the problem of large bidding complexes in the existing technology is solved, and the HRP markup antibody with high stability and small cluster structure is prepared, which improves the detection. Accuracy and reliability.
Patent Information
- Application Number
- CN202411645855.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-01-21
AI Technical Summary
In the existing preparation methods for HRP enzyme-labeled antibodies, the labeled complex is large, which can easily cause abnormal phenomena such as pore jumps, and the crosslinked aggregates are uneven after labeling, resulting in poor gradients and high background of the calibration product. .
Adopting the Staudinger reaction to combine nitrogen-PEG12-NHS modified spicy root peroxidase with antibodies to form a smaller cluster structure and high stability HRP markup antibody.
The HRP markup antibody prepared through this method has small polyette structure and high stability, which can effectively reduce the problem of high -value region difference and high background in the test process, and improve the accuracy of detection.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of immunological detection, and in particular to a method for preparing an HRP enzyme-labeled antibody and an application thereof. Background Art
[0002] Hepatitis B surface antigen (HBsAg) is the outer shell protein of the hepatitis B virus and is not contagious. . However, its appearance is often accompanied by the presence of hepatitis B virus, so it is a sign of hepatitis B virus infection. In magnetic particle chemiluminescence detection, the double antibody sandwich method is often used for detection, and enzyme-labeled HBs antibodies are required. Horseradish peroxidase (Horseradish Peroxidase, HRP) has high specific activity, stability, small molecular weight, and easy preparation of pure enzyme. It is now the most commonly used raw material for the preparation of protease markers.
[0003] The commonly used method for antibody labeling with HRP is NaIO 4 Oxidation method, using NaIO 4 HRP sugar groups are oxidized to aldehyde groups, which react with -NH 2 The reaction generates a Schiff base reaction, and the antibody molecule and the HRP molecule form a stable structure. This method uses the amino group on the antibody and the aldehyde group on the HRP. The number of amino groups and aldehyde groups is relatively large, which has the advantage of high coupling efficiency; the disadvantage is that the labeled complex is large, which is prone to abnormal phenomena such as hole skipping. Thiol labeling requires the introduction of thiol groups on the antibody, and the use of the amino group on HRP for cross-linking through SMCC. The labeling process is relatively complicated, and the antibody after the introduction of thiol groups is prone to self-polymerization. The cross-linked polymers after labeling are also large, and are not uniform HRP and antibody labeled complexes. Large labeled complexes are prone to poor gradients in the high value area of the calibration product and high background. Summary of the invention
[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing an HRP enzyme-labeled antibody and its application. The present invention provides an HRP-labeled antibody prepared by using the Staudinger reaction. The preparation method is simple, and the obtained antibody has a small polymer structure and high stability.
[0005] The present invention provides horseradish peroxidase, which is horseradish peroxidase modified with azide-PEG12-NHS.
[0006] Compared with other groups, the azide-PEG12-NHS used for coupling in the present invention has strong compatibility with horseradish peroxidase, and the modified enzyme effectively increases the cross-linking arm, which can reduce the steric hindrance of the binding of the enzyme-labeled antibody to the antigen, and the PEG chain can increase the hydrophilicity of the modified horseradish peroxidase or antibody and increase the stability of the complex, thereby obtaining a more accurate technical effect.
[0007] The invention also provides a method for preparing the horseradish peroxidase, which comprises mixing unmodified horseradish peroxidase with the azide-PEG12-NHS and reacting to obtain the product.
[0008] In some embodiments, in the reaction solution, the molar ratio of the unmodified horseradish peroxidase to the azide-PEG12-NHS is 1:(10-20), and the reaction time is 1-2 hours.
[0009] In some specific embodiments, the molar ratio of the unmodified horseradish peroxidase to the azide-PEG12-NHS is 1:10, 1:15 or 1:20, and the reaction time is 1 h, 1.5 h or 2 h.
[0010] The invention provides a horseradish peroxidase-labeled antibody, which is an antibody labeled with the horseradish peroxidase or the horseradish peroxidase prepared by the preparation method.
[0011] In some embodiments, the antibody comprises an HBs antibody.
[0012] The present invention also provides a method for preparing the antibody, comprising the following steps:
[0013] Step 1, mixing an unlabeled antibody with a phosphine compound and reacting to obtain an antibody modified with a phosphine group;
[0014] Step 2: Mix the antibody modified with the phosphine group in step 1 with the horseradish peroxidase or the horseradish peroxidase prepared by the preparation method, and react to obtain the horseradish peroxidase-labeled antibody.
[0015] The preparation method provided by the present invention is based on the Staudinger reaction, and utilizes the reaction of azide and phosphine to be applied to the labeling of HRP and antibody to prepare HRP-labeled antibody. Compared with other preparation methods, the preparation method of the present invention is simpler, and the prepared antibody has a small polymer structure and high stability, and can effectively reduce the problems of gradient difference and high background in the high value area of the calibration product caused in the detection process, thereby obtaining a more accurate technical effect.
[0016] In some embodiments, in step 1, the phosphine compound is NHS-phosphine, the molar ratio of the unlabeled antibody to the NHS-phosphine is 1:(10-15), and the reaction time is 1-1.5 h.
[0017] In some specific embodiments, the molar ratio of the unlabeled antibody to the NHS-phosphine is 1:10, 1:13 or 1:15, and the reaction time is 1 h or 1.5 h.
[0018] In some embodiments, in step 2, the molar ratio of the antibody modified with a phosphine group to the horseradish peroxidase or the horseradish peroxidase prepared by the preparation method is 1:(2-5), and the reaction time is 10-12 hours.
[0019] In some specific embodiments, the molar ratio of the antibody modified with a phosphine group to the horseradish peroxidase or the horseradish peroxidase prepared by the preparation method is 1:2, 1:3 or 1:5, and the reaction time is 10 hours, 11 hours or 12 hours.
[0020] The present invention provides a method for preserving the antibody or the antibody prepared by the preparation method, wherein the antibody is preserved by a preservation stabilizer, wherein the preservation stabilizer comprises 10-30 mM phosphate buffer at pH 7.4, 5-50 g / L sucrose, 2-30 g / L LADP, 2-10 g / L PLGA, 0.01 vol%-0.2 vol% TritonX-100, 0.02 vol%-0.1 vol% Proclin300 and 40 vol%-60 vol% ethylene glycol.
[0021] In some specific embodiments, the storage stabilizer includes 20 mM phosphate buffer at pH 7.4, 10 g / L sucrose, 10 g / L ADP, 5 g / L PLGA, 0.2 vol% TritonX-100, 0.1 vol% Proclin 300 and 50 vol% ethylene glycol.
[0022] The present invention provides use of the antibody or the antibody prepared by the preparation method in preparing a detection reagent or a kit.
[0023] In some embodiments, the detection reagent or kit is used to detect hepatitis B virus.
[0024] The present invention provides a detection reagent or a kit, comprising the antibody or the antibody prepared by the preparation method.
[0025] The present invention provides a detection method, comprising using the detection reagent or kit to detect a sample.
[0026] The present invention provides a method for preparing an HRP enzyme-labeled antibody and its application, which has the following beneficial effects compared with the prior art:
[0027] (1) The present invention applies the reaction of azide and phosphine to the labeling of HRP and antibodies. The labeling method is simple. Moreover, since the label is located at a relatively small amino site on HRP, the polymer structure of the labeled complex formed after labeling is small, which can effectively reduce the problems of gradient difference in the high value area of the calibrator and high background caused during the detection process.
[0028] (2) The azide-PEG12-NHS used for coupling in the present invention effectively increases the cross-linking arm and reduces the steric hindrance of the binding between the enzyme-labeled antibody and the antigen. The PEG chain can increase the hydrophilicity of the labeled complex and increase the stability of the complex;
[0029] (3) The storage buffer of the present invention uses PLGA instead of protein stabilizer to reduce nonspecific adsorption, and uses ethylene glycol instead of commonly used glycerol, so that it can be stored at a lower temperature without freezing. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The figure shows the results of HPLC analysis of three enzyme-labeled antibodies in Example 1 using a SEC500 column. DETAILED DESCRIPTION
[0031] The present invention provides the field of immunological detection technology, specifically to HRP enzyme-labeled antibody preparation method and application thereof, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.
[0032] The test materials used in the present invention are all common commercial products and can be purchased on the market. Among them:
[0033] Horseradish peroxidase (HRP) was purchased from Roche, catalog number 10121606103;
[0034] Azide-PEG12-NHS was purchased from Sigma with the product number QBD10505.
[0035] NHS-phosphine was purchased from Thermo Fisher Scientific with a catalog number of 88900.
[0036] HBs antibody was purchased from HyTest with the catalog number 3HB12.
[0037] The present invention will be further described below in conjunction with the embodiments.
[0038] Example 1
[0039] The preparation method of enzyme-labeled antibody is as follows:
[0040] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0041] 1) Dissolve HRP to 5 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0042] 2) Add the dissolved Azide-PEG12-NHS to the dissolved HRP; the molar ratio of Azide-PEG12-NHS to HRP is 20:1;
[0043] 3) Incubate the reaction at room temperature for 1 hour; then dialyze the sample into phosphate buffer.
[0044] Among them, the structure of azide-PEG12-NHS is shown below:
[0045] (Formula 1).
[0046] Step 2: The method of using NHS-phosphine to react with the amino group on the HBs antibody is as follows:
[0047] 1) Replace the HBs antibody buffer with pH 7.4 phosphate buffer at a concentration of 5 mg / mL; dissolve NHS-phosphine in DMSO to 5 mg / mL.
[0048] 2) Add the dissolved NHS-phosphine to the HBs antibody; the molar ratio of NHS-phosphine to HBs antibody is 15:1;
[0049] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0050] The NHS-phosphine structure is as follows:
[0051] (Formula 2).
[0052] Step 3: The molar ratio of the HRP and HBs reaction is 5:1, and the reaction is carried out at room temperature for 10 hours.
[0053] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0054] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0055] The enzyme-labeled antibody obtained in step 3 was added with sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), and ethylene glycol 50% (v / v), and the sample was stored at -20°C.
[0056] Example 2
[0057] The preparation method of enzyme-labeled antibody is as follows:
[0058] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0059] 1) Dissolve HRP to 8 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0060] 2) Add the dissolved Azide-PEG12-NHS to the dissolved HRP; the molar ratio of Azide-PEG12-NHS to HRP is 15:1;
[0061] 3) Incubate the mixture at room temperature for 1.5 h and dialyze the sample into phosphate buffer.
[0062] Step 2: The method of using NHS-phosphine to react with the amino group on the HBs antibody is as follows:
[0063] 1) The HBs antibody was replaced with pH 7.4 phosphate buffer at a concentration of 8 mg / mL; NHS-phosphine was dissolved in DMSO to a concentration of 5 mg / mL.
[0064] 2) Adding NHS-phosphine to the HBs antibody; the molar ratio of NHS-phosphine to the HBs antibody is 13:1.
[0065] 3) Incubate the mixture at room temperature for 1.5 h and dialyze the sample into phosphate buffer.
[0066] Step 3: The molar ratio of the HRP and HBs reaction is 3:1, and the reaction is carried out at room temperature for 11 hours.
[0067] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0068] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0069] Sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), ethylene glycol 50% (v / v) obtained in step 3, the sample was stored at -20°C, the sample was stored at -30°C.
[0070] Example 3
[0071] The preparation method of enzyme-labeled antibody is as follows:
[0072] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0073] 1) Dissolve HRP to 10 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0074] 2) Add the dissolved Azide-PEG12-NHS to the dissolved HRP; the molar ratio of Azide-PEG12-NHS to HRP is 10:1;
[0075] 3) React at room temperature for 2 hours; dialyze the reacted sample into phosphate buffer.
[0076] Step 2: The method of using NHS-phosphine to react with the amino group on the HBs antibody is as follows:
[0077] 1) The HBs antibody was replaced with pH 7.4 phosphate buffer at a concentration of 10 mg / mL; NHS-phosphine was dissolved in DMSO to 5 mg / mL.
[0078] 2) Adding NHS-phosphine to the HBs antibody; the molar ratio of NHS-phosphine to the HBs antibody is 10:1.
[0079] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0080] Step 3: The molar ratio of the HRP and HBs reaction is 2:1, and the reaction is carried out at room temperature for 12 hours.
[0081] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0082] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0083] Sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), ethylene glycol 50% (v / v) obtained in step 3, the sample was stored at -20°C, the sample was stored at -30°C.
[0084] Comparative Example 1
[0085] Conventional sodium periodate labeling:
[0086] Dissolve HRP in phosphate buffer to 10 mg / mL. Add 0.1 mL of freshly prepared 10 mM NaIO 4Solution, react at room temperature in the dark for 60 minutes; add glycerol to the oxidized HRP and react at room temperature in the dark for 30 minutes; put the above solution into a dialysis bag and dialyze with acetate buffer pH 4.4; dialyze the HBs antibody into carbonate buffer pH 9.5 solution; add the dialyzed antibody directly to the above aldehyde HRP, with the mass ratio of antibody to HRP being 3:1; react at room temperature for 4 hours; add 0.1mL sodium borohydride to terminate the reaction, and react at room temperature for 1-2 hours. The coupled enzyme-labeled antibody is dialyzed into phosphate buffer pH 7.4, and sucrose 10g / L, ADP 10g / L, PLGA 5g / L, TritonX-100 0.2% (v / v), Proclin 3000.1% (v / v), ethylene glycol 50% (v / v) are added and stored at -20℃.
[0087] Comparative Example 2
[0088] SATA thiol labeling method:
[0089] Step 1: Antibody sulfhydrylation
[0090] The antibody buffer was exchanged into PBS + 1 mM EDTA.
[0091] SATA was dissolved in DMSO to 5 mg / mL, and added at nAb / SATA = 1:20, and reacted at room temperature for 5 hours. Then, the antibody was added at nAb / hydroxylamine = 1 / 40, and reacted at room temperature for 5 hours. The excess SATA and hydroxylamine were removed with molecular sieves, and the buffer was PBS + 1mM EDTA. After passing through the molecular sieve, the antibody concentration was adjusted to 5 mg / mL.
[0092] Step 2: HRP activation with SMCC
[0093] HRP was dissolved in PBS to 5 mg / mL, and added according to nHRP / SMCC = 1 / 20, and reacted at room temperature for 5 hours. Excess SMCC was removed with a molecular sieve, and the buffer was PBS + 1mM EDTA. After passing through the molecular sieve, the antibody concentration was adjusted to 5 mg / mL.
[0094] Step 3: The molar ratio of HRP to HBs was 3:1, and the reaction was carried out at room temperature for 12 hours. Iodoacetamide was added to terminate the reaction. The reaction was dialyzed into PBS pH 7.4.
[0095] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0096] The enzyme-labeled antibody obtained in step 3 was added with sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), and ethylene glycol 50% (v / v), and the sample was stored at -20°C.
[0097] Comparative Example 3
[0098] The preparation method of enzyme-labeled antibody is as follows:
[0099] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0100] 1) Dissolve HRP to 10 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0101] 2) Add to the dissolved HRP; the molar ratio of azide-PEG12-NHS to HRP is 20:1;
[0102] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0103] Step 2: The method for reacting the above-mentioned NHS-phosphine with the HBs reagent and the amino group on the HBs antibody is as follows:
[0104] 1) Replace the buffer of HBs antibody with pH 7.4 phosphate buffer at a concentration of 10 mg / mL;
[0105] 2) adding NHS-phosphine to HBs antibody; the molar ratio of NHS-phosphine to HBs antibody is 15:1;
[0106] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0107] Step 3. The molar ratio of the HRP to HBs reaction is 5:1, and the reaction is carried out at room temperature for 12 hours.
[0108] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0109] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0110] The enzyme-labeled antibody obtained in step 3 was added with sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), and propylene glycol 50% (v / v), and the sample was stored at -30°C.
[0111] Comparative Example 4
[0112] The preparation method of enzyme-labeled antibody is as follows:
[0113] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0114] 1) Dissolve HRP to 10 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0115] 2) Add to the dissolved HRP; the molar ratio of azide-PEG12-NHS to HRP is 20:1;
[0116] 3) Incubate the reaction at room temperature for 1 hour; then dialyze the sample into phosphate buffer.
[0117] Step 2: The method for reacting the above-mentioned NHS-phosphine with the HBs reagent and the amino group on the HBs antibody is as follows:
[0118] 1) Replace the buffer of HBs antibody with pH 7.4 phosphate buffer at a concentration of 10 mg / mL;
[0119] 2) adding NHS-phosphine to HBs antibody; the molar ratio of NHS-phosphine to HBs antibody is 15:1;
[0120] 3) Incubate the reaction at room temperature for 1 hour; then dialyze the sample into phosphate buffer.
[0121] Step 3. The molar ratio of the HRP to HBs reaction is 5:1, and the reaction is carried out at room temperature for 12 hours.
[0122] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0123] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0124] The enzyme-labeled antibody obtained in step 3 was added with sucrose 10 g / L, ADP 10 g / L, BSA 5 g / L, Triton X-100 0.2% (v / v), Proclin 300 0.1% (v / v), and ethylene glycol 50% (v / v), and the sample was stored at -20°C.
[0125] Comparative Example 5
[0126] The preparation method of enzyme-labeled antibody is as follows:
[0127] The preparation method of enzyme-labeled antibody is as follows:
[0128] Step 1: The reaction method of azide-PEG12-NHS reagent with the amino group on HRP is as follows:
[0129] 1) Dissolve HRP to 5 mg / mL in pH 7.4 phosphate buffer; dissolve Azide-PEG12-NHS to 1 mg / mL in ultrapure water.
[0130] 2) Add the dissolved Azide-PEG12-NHS to the dissolved HRP; the molar ratio of Azide-PEG12-NHS to HRP is 20:1;
[0131] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0132] Among them, the structure of azide-PEG12-NHS is shown below:
[0133] (Formula 3).
[0134] Step 2: The method of using NHS-phosphine to react with the amino group on the HBs antibody is as follows:
[0135] 1) Replace the HBs antibody buffer with pH 7.4 phosphate buffer at a concentration of 5 mg / mL; dissolve NHS-phosphine in DMSO to 5 mg / mL.
[0136] 2) Add the dissolved NHS-phosphine to the HBs antibody; the molar ratio of NHS-phosphine to HBs antibody is 15:1;
[0137] 3) React at room temperature for 1 hour; dialyze the reacted sample into phosphate buffer.
[0138] Step 3: The molar ratio of the HRP and HBs reaction is 5:1, and the reaction is carried out at room temperature for 10 hours.
[0139] The resulting labeled complex was buffer exchanged into 20 mM PBS pH 7.4.
[0140] Step 4: Add a storage stabilizer to the enzyme-labeled antibody
[0141] The enzyme-labeled antibody obtained in step 3 was added with sucrose 10 g / L, ADP 10 g / L, PLGA 5 g / L, TritonX-100 0.2% (v / v), Proclin 300 0.1% (v / v), and ethylene glycol 50% (v / v), and the sample was stored at -20°C.
[0142] Gradient test of enzyme-labeled antibody calibrator
[0143] The enzyme-labeled antibodies prepared by the present invention and the enzyme-labeled antibodies prepared by the methods of comparative examples 1, 2, and 5 were used to detect the luminescence value of the calibration product by the magnetic microparticle chemiluminescence method, and the paired magnetic microparticle-coated antibodies were used for detection using the double antibody sandwich method principle. The enzyme-labeled antibody prepared by the present invention detects the calibration product while taking into account S1 / S0, and the gradient in the high value area (S5 / S4) is the best. The enzyme-labeled antibodies prepared by the three methods were sent to HPLC for analysis using a SEC500 chromatographic column. The more the spectrum is to the left, the larger the enzyme-labeled complex aggregate is. The peak that appears at 13min is free HRP, which can be ignored. The enzyme-labeled complex aggregate obtained by the present invention is the smallest.
[0144] Table 1 Luminescence values and gradient change data of enzyme-labeled antibodies prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 and 5
[0145]
[0146] Effect Example 2
[0147] Stability assessment of enzyme-labeled antibodies
[0148] The enzyme-labeled antibodies of Examples 1 to 3, Comparative Examples 3 and 4 were placed at -30°C for 14 days (1 day, 3 days, and 7 days were taken out and placed at room temperature and then placed at -30°C again), and the luminescence value of the calibration product was detected by magnetic microparticle chemiluminescence. S0 represents the background. The higher the S0, the more nonspecific adsorption; the greater the decrease in the luminescence value, the more unstable it is. The test results are shown in Table 2. The background (S0) and the decrease in the luminescence value of the embodiment are both optimal, indicating that the activity of the enzyme-labeled antibody with the addition of glycerol will decrease after being frozen and thawed at -30°C, and the addition of PLGA will reduce nonspecific adsorption.
[0149] Table 2
[0150]
[0151] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Horseradish peroxidase, characterized in that It is horseradish peroxidase modified with Azide-PEG12-NHS.
2. The method for preparing horseradish peroxidase according to claim 1, characterized in that: The method comprises taking unmodified horseradish peroxidase and the azide-PEG12-NHS, mixing and reacting them to obtain the product.
3. The preparation method according to claim 2, characterized in that: In the reaction solution, the molar ratio of the unmodified horseradish peroxidase to the azide-PEG12-NHS is 1:(10-20), and the reaction time is 1-2 hours.
4. A horseradish peroxidase-labeled antibody, characterized in that: The invention is an antibody labeled with the horseradish peroxidase according to claim 1 or the horseradish peroxidase prepared by the preparation method according to claim 2 or 3.
5. The method for preparing the antibody according to claim 4, characterized in that: The steps include: Step 1, mixing an unlabeled antibody with a phosphine compound and reacting to obtain an antibody modified with a phosphine group; Step 2: Mix the antibody modified with the phosphine group described in step 1 with the horseradish peroxidase described in claim 1 or the horseradish peroxidase prepared by the preparation method described in claim 2 or 3, and react to obtain the antibody labeled with the horseradish peroxidase.
6. The preparation method according to claim 5, characterized in that: In the step 1, the phosphine compound is NHS-phosphine, the molar ratio of the unlabeled antibody to the NHS-phosphine is 1:(10-15), and the reaction time is 1-1.5 hours.
7. The preparation method according to claim 5 or 6, characterized in that: In step 2, the molar ratio of the antibody modified with the phosphine group to the horseradish peroxidase or the horseradish peroxidase prepared by the preparation method is 1:(2-5), and the reaction time is 10-12 hours.
8. A method for storing the antibody according to claim 4 or the antibody prepared by the preparation method according to any one of claims 5 to 7, characterized in that: The antibody is stored in a storage stabilizer, wherein the storage stabilizer includes 10-30 mM phosphate buffer at pH 7.4, 5-50 g / L sucrose, 2-30 g / L ADP, 2-10 g / L PLGA, 0.01 vol%-0.2 vol% TritonX-100, 0.02 vol%-0.1 vol% Proclin 300 and 40 vol%-60 vol% ethylene glycol.
9. Use of the antibody according to claim 4 or the antibody prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a detection reagent or a kit.
10. A detection reagent or a kit, characterized in that: The invention comprises the antibody according to claim 4 or the antibody prepared by the preparation method according to any one of claims 5 to 7.