A xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody and preparation method and kit thereof

Abstract

The invention discloses an HRP-labeled mouse anti-human troponin I monoclonal antibody based on xanthan gum, and a preparation method and a kit thereof. The preparation method comprises: step 1, oxidizing xanthan gum to obtain aldehyde-containing xanthan gum; step 2, polymerizing the aldehyde-containing xanthan gum with horseradish peroxidase to obtain a horseradish peroxidase polymer; step 3, purifying the horseradish peroxidase polymer; step 4, connecting the mouse anti-human troponin I monoclonal antibody to the purified horseradish peroxidase polymer; step 5, purifying the product of step 4 to obtain the HRP-labeled mouse anti-human troponin I monoclonal antibody. The mouse anti-human troponin I monoclonal antibody of the invention has high detection sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and relates to an HRP-labeled mouse anti-human troponin I monoclonal antibody based on xanthan gum and a preparation method thereof, and also relates to a kit for detecting acute myocardial infarction. Background Art

[0002] Troponin is a marker of myocardial injury and necrosis. Detection of troponin I in human serum has important clinical significance for the diagnosis and risk stratification of acute myocardial infarction. The content of cardiac troponin I in human serum is particularly low, and a highly sensitive method is required for detection.

[0003] At present, antibodies or antigen enzyme markers in enzyme immunoassays play an important role in the sensitivity and specificity of immunoassay analysis and are key reagents in enzyme immunoassay detection. Horseradish peroxidase (HRP) has high specific activity, stability, small molecular weight, and easy preparation of pure enzyme, so it is the most commonly used raw material for preparing protease markers. Traditional horseradish peroxidase-labeled antibodies use amino groups, sulfhydryl groups on enzymes and antibodies, or hydroxyl groups on glycoproteins as cross-linking groups, and directly connect antibodies and enzymes together through sodium periodate or glutaraldehyde method. Due to different molecular sizes, generally only 1 to 2 HRP molecules can be connected to one antibody molecule. The immune reaction is manifested by HRP catalytic substrate, and the detection sensitivity of the traditional HRP-labeled mouse anti-human troponin I monoclonal antibody is limited. Summary of the invention

[0004] In view of the above problems, the present invention provides a xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody with high detection sensitivity.

[0005] The present invention also provides a method for preparing an HRP-labeled mouse anti-human troponin I monoclonal antibody based on xanthan gum, the prepared antibody has high detection sensitivity and good application effect of the product.

[0006] The invention also provides a kit for detecting acute myocardial infarction, which has high detection sensitivity.

[0007] According to the first aspect of the present invention, a method for preparing a xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody comprises:

[0008] Step 1, oxidizing xanthan gum to obtain aldehyde-containing xanthan gum;

[0009] Step 2, polymerizing the aldehyde-containing xanthan gum with horseradish peroxidase to obtain a horseradish peroxidase polymer;

[0010] Step 3, purifying the horseradish peroxidase polymer;

[0011] Step 4: Mouse anti-human troponin I monoclonal antibody is linked to the purified horseradish peroxidase polymer;

[0012] Step 5: purify the product of step 4 to obtain the HRP-labeled mouse anti-human troponin I monoclonal antibody.

[0013] In a specific and preferred embodiment, the preparation method is specifically implemented as follows:

[0014] Step 1, 5-8 mg of xanthan gum reacts with 15-25 mg of sodium periodate to obtain aldehyde-containing xanthan gum;

[0015] Step 2: Add 5-15 mg of horseradish peroxidase to the aldehyde-containing xanthan gum obtained in step 1, mix thoroughly, then add 35-45 mg of sodium triacetylborohydride, and rotate at 3-5° C. to obtain a horseradish peroxidase polymer;

[0016] Step 3: dialyzing the horseradish peroxidase polymer obtained in step 2 with phosphate buffer to obtain a dialyzate;

[0017] Step 4: Add 2-4 mg of mouse anti-human troponin I monoclonal antibody to the dialysate obtained in step 3, mix thoroughly, then add sodium triacetylborohydride and rotate to react for 3-5 hours;

[0018] Step 5: dialyze the phosphate buffer solution, take out the dialyzate, and obtain the HRP-labeled mouse anti-human troponin I monoclonal antibody.

[0019] In a preferred embodiment, in step 1, xanthan gum is oxidized by sodium periodate.

[0020] More preferably, in step 1, 5-8 mg of xanthan gum reacts with 15-25 mg of sodium periodate to obtain aldehyde-containing xanthan gum.

[0021] More preferably, the xanthan gum is first dissolved in 10-100 mM carbonate buffer at pH 9-10, and then sodium periodate is added.

[0022] In a preferred embodiment, in step 2, 5-15 mg of horseradish peroxidase is added to the aldehyde-containing xanthan gum obtained in step 1, 35-45 mg of sodium triacetylborohydride is added after mixing, and the mixture is rotated at 3-5° C. overnight to obtain a horseradish peroxidase polymer.

[0023] In a preferred embodiment, in step 3, the purification is performed by dialyzing with phosphate buffer. Further, a dialysis bag with a molecular cutoff of 2000-4000D is selected, and the dialysis is performed at 3-5°C for 8-12 hours to obtain a dialyzed product.

[0024] In a preferred embodiment, in step 5, the purification is performed by dialyzing with phosphate buffer. Further, a dialysis bag with a molecular cutoff of 2000-4000D is selected, and the dialysis is performed at 3-5°C for 8-12 hours to obtain a dialyzed product.

[0025] In a preferred embodiment, in step 4, 2-4 mg of mouse anti-human troponin I monoclonal antibody is added to the dialysate obtained in step 3, mixed thoroughly, and then sodium triacetylborohydride is added and the reaction is carried out by rotation for 3-5 hours.

[0026] In one embodiment, the preparation method is specifically implemented as follows:

[0027] Step 1: Oxidation of xanthan gum by sodium periodate to form free aldehyde groups: 5-8 mg of xanthan gum is fully dissolved in 10-100 mM carbonate buffer with a pH of 9-10, and 15-25 mg of sodium periodate is added. The mixture is reacted for 30 minutes to obtain xanthan gum containing aldehyde groups.

[0028] Step 2, polymerization of xanthan gum skeleton and HRP: add 5-15 mg HRP to the aldehyde-containing xanthan gum obtained in step 1, mix thoroughly, add 35-45 mg sodium triacetylborohydride to the solution, and rotate at 3-5°C overnight to obtain HRP polymer;

[0029] Step 3, purification: The HRP polymer obtained in step 2 is dialyzed with 10 mM phosphate buffer for 3 times, using a dialysis bag with a molecular cutoff of 2000-4000D, and dialyzed at 3-5°C for 8-12 hours to obtain a dialyzate;

[0030] Step 4: Connecting mouse anti-human troponin I monoclonal antibody: Take out the dialyzate obtained in step 3, add 2-4 mg of mouse anti-human troponin I monoclonal antibody, mix well, add sodium triacetylborohydride, and rotate to react for 3-5 hours;

[0031] Step 5. Purification: Dialyze the solution three times with 10 mM phosphate buffer, use a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 3-5°C for 8-12 hours, take out the dialysate, and obtain the antibody and HRP polymer.

[0032] According to the second aspect of the present invention, a xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody is prepared according to the above-mentioned preparation method.

[0033] According to the third aspect of the present invention, a kit for detecting acute myocardial infarction comprises the xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody prepared according to the above-mentioned preparation method, or comprises the xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody prepared according to the above-mentioned preparation method.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0035] The invention uses xanthan gum as a skeleton, and couples mouse anti-human troponin I monoclonal antibody molecules and horseradish peroxidase on the xanthan gum skeleton. The number of xanthan gum skeleton repeating units is relatively large, and the number of graftable groups on each repeating unit is also relatively large. The amplified intensity is proportional to the number of coupled molecules. Each antibody and antigen reaction signal can be reflected by a multiple number of connected HRP catalytic substrate systems, thereby achieving the effect of amplifying the antibody and antigen reaction signals, greatly improving the sensitivity of troponin I detection, and having important clinical significance for the diagnosis and risk stratification of acute myocardial infarction. The preparation method is simple in steps, the obtained product has good application effect, and has better application prospects. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Traditional horseradish peroxidase-labeled antibodies use amino groups, sulfhydryl groups or hydroxyl groups on enzymes and antibodies or glycoproteins as cross-linking groups, and directly connect antibodies and enzymes together through sodium periodate or glutaraldehyde method. Due to different molecular sizes, generally only 1 to 2 HRP molecules can be connected to one antibody molecule. The immune response is manifested by HRP catalytic substrates. In this paper, the antibody and HRP are connected together using polymers as carriers. Due to the extension of the polymers, one antibody can be connected to more than 10 HRP molecules. Each antibody-antigen reaction signal can be reflected by the connected 10-fold number of HRP catalytic substrate systems, so the detection sensitivity is greatly improved. After searching, there are no reports on the literature and products of HRP-mouse anti-human troponin I labeled antibodies using xanthan gum as the skeleton.

[0038] In this article, the sodium periodate method is used to oxidize the ortho-hydroxyl groups on xanthan gum to form aldehyde groups, and then the oxidized xanthan gum skeleton is connected to the amino groups on HRP through the aldehyde groups to form an XG-HRP polymer. Finally, the mouse anti-human troponin I monoclonal antibody is connected to XG-HRP. After purification, dialysis and preservation treatment, the xanthan gum-coupled HRP-mouse anti-human troponin I labeled antibody is obtained. The mouse anti-human troponin I monoclonal antibody-HRP polymer obtained by this method has higher sensitivity and better application prospects than the normal labeled HRP-mouse anti-human troponin I monoclonal antibody of the commercial Medix in the prior art.

[0039] Example 1

[0040] ① Mix 15 mg of sodium periodate with 5 mg of xanthan gum (dissolved in 0.1 M carbonate buffer solution) and react at 37°C for 30 minutes to obtain an aldehyde-containing xanthan gum solution.

[0041] ② Add 5 mg HRP to the aldehyde-containing xanthan gum solution, mix thoroughly, add 35 mg sodium triacetoxyborohydride (dissolved in 0.6 mM carbonate buffer), and stir at 3°C ​​overnight to obtain HRP polymer.

[0042] ③ Purify the HRP polymer solution: Use 10 times the volume of 0.1mM PBS as the dialysate, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze the HRP polymer solution at 3°C ​​for 8 hours, change the dialysate 3 times, and obtain the dialyzed product.

[0043] ④ Add 2 mg of mouse anti-human troponin I monoclonal antibody to the dialyzed purified HRP polymer solution, mix thoroughly, then add 40 mg of sodium triacetylborohydride and rotate to react for 3 hours.

[0044] ⑤ Use 10 times the volume of 0.1mM PBS as the dialysate for the reaction solution, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 3°C ​​for 8 hours, change the dialysate 3 times, and obtain the dialyzate product, i.e., HRP-labeled mouse anti-human troponin I monoclonal antibody.

[0045] Example 2

[0046] ① Mix 20 mg of sodium periodate with 7.5 mg of xanthan gum (dissolved in 0.1 M carbonate buffer solution) and react at 37°C for 30 minutes to obtain an aldehyde-containing xanthan gum solution.

[0047] ② Add 10 mg of HRP to the aldehyde-containing xanthan gum solution, mix thoroughly, add 40 mg of sodium triacetoxyborohydride (dissolved in 0.6 mM carbonate buffer), and stir at 4°C overnight to obtain HRP polymer.

[0048] ③ Purify the HRP polymer solution: Use 10 times the volume of 0.1mM PBS as the dialysate, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 4°C for 10 hours, change the dialysis solution 3 times, and obtain the dialyzed product.

[0049] ④ Add 3 mg of mouse anti-human troponin I monoclonal antibody to the dialyzed purified HRP polymer solution, mix thoroughly, then add 45 mg of sodium triacetylborohydride and rotate to react for 3 hours.

[0050] ⑤ Use 10 times the volume of 0.1mM PBS as the dialysate for the reaction solution, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 4°C for 10 hours, change the dialysate 3 times, and obtain the dialysis product, i.e., HRP-labeled mouse anti-human troponin I monoclonal antibody.

[0051] Example 3

[0052] ① Mix 25 mg of sodium periodate with 8 mg of xanthan gum (dissolved in 0.1 M carbonate buffer solution) and react at 37°C for 30 minutes to obtain an aldehyde-containing xanthan gum solution.

[0053] ② Add 15 mg of HRP to the aldehyde-containing xanthan gum solution, mix thoroughly, add 45 mg of sodium triacetoxyborohydride (dissolved in 0.6 mM carbonate buffer), and stir at 5°C overnight to obtain the antibody and HRP polymer.

[0054] ③ Purify the HRP polymer solution: Use 10 times the volume of 0.1mM PBS as the dialysate, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 5°C for 12 hours, change the dialysis solution 3 times, and obtain the dialyzed product.

[0055] ④ Add 4 mg of mouse anti-human troponin I monoclonal antibody to the HRP polymer solution after dialysis purification, mix thoroughly, then add 50 mg of sodium triacetylborohydride and rotate to react for 3 hours.

[0056] ⑤ Use 10 times the volume of 0.1mM PBS as the dialysate for the reaction solution, select a dialysis bag with a molecular cutoff of 2000-4000D, dialyze at 5°C for 12 hours, change the dialysis solution 3 times, and obtain the dialyzed product.

[0057] Comparative Example

[0058] Normally labeled HRP-mouse anti-human troponin I monoclonal antibody was purchased from Medix. The amino group, sulfhydryl group or hydroxyl group on the HRP enzyme and the mouse anti-human troponin I monoclonal antibody or the glycoprotein were used as cross-linking groups to directly link the antibody and enzyme together by sodium periodate or glutaraldehyde method.

[0059] Evaluation experiment:

[0060] The xanthan gum-based HRP mouse anti-human troponin I monoclonal antibody prepared in Examples 1-3 and the commercial Medix normal labeled HRP-mouse anti-human troponin I monoclonal antibody of the comparative example were subjected to the following experiment:

[0061] ① Dilute the HRP mouse anti-human troponin I monoclonal antibody obtained in each embodiment and the commercialized Medix normal labeled HRP-mouse anti-human troponin I monoclonal antibody to a suitable concentration so that the blank detection signal value Δk / s is about 0.001-0.01;

[0062] ②Pipette 30-45ul of each diluted HRP-labeled antibody into the well of the enzyme-labeled solution in the MIA series fully automatic immunoassay test card (single-person test card, containing a solid phase carrier coated with anti-biotin antibody) of Oriental Innuo (Suzhou) Medical Technology Co., Ltd.;

[0063] ③ Dilute the commercial Medix biotinylated mouse anti-human troponin I monoclonal antibody to an appropriate concentration according to the dilution multiple specified by the merchant, and add 20-30ul to the reagent space in the MIA series fully automatic immunoassay analyzer special test card of Oriental Innuo (Suzhou) Medical Technology Co., Ltd.;

[0064] ④Add 140-160ul of MIA series fully automatic immunoanalyzer dedicated washing solution to the washing solution well, 40-70ul of MIA series fully automatic immunoanalyzer dedicated buffer to the buffer solution well, and 30-50ul of MIA series fully automatic immunoanalyzer dedicated TMB to the TMB solution well;

[0065] ⑤Select samples of each gradient concentration of troponin (cTnI) (20ng / ml, 10ng / ml, 2ng / ml, 0.5ng / ml, 0.1ng / ml, 0.05ng / ml, 0ng / ml), and add 20-40ul to the sample well of the test card;

[0066] ⑥Finally, the 28 test cards were automatically tested using the MIA series fully automatic immunoassay analyzer. The results are shown in Table 1 below.

[0067] Table 1

[0068]

[0069]

[0070] It can be seen from the data in Table 1 that compared with the comparative example, the signal values ​​of Examples 1, 2, and 3 at non-zero concentrations are greatly improved, and for the background value (i.e., the signal value at zero concentration), Examples 1, 2, and 3 are much lower than the comparative example. Therefore, the xanthan gum-based HRP mouse anti-human troponin I monoclonal antibody of the embodiment can effectively reduce the background value in the enzyme immunoreaction, and the signal value at non-zero concentration is greatly improved, and has a higher detection sensitivity.

[0071] As shown in the present invention, the terms "include" and "comprise" only indicate the inclusion of clearly identified steps and elements, and these steps and elements do not constitute an exclusive list, and the method or device may also include other steps or elements. The term "and / or" used herein includes any combination of one or more related listed items.

[0072] The above embodiment is only for illustrating the technical concept and features of the present invention, and is a preferred embodiment. Its purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting acute myocardial infarction, characterized in that: The invention comprises a xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody, wherein the HRP-labeled mouse anti-human troponin I monoclonal antibody is prepared by the following steps: Step 1, oxidizing xanthan gum to obtain aldehyde-containing xanthan gum; Step 2, polymerizing the aldehyde-containing xanthan gum with horseradish peroxidase to obtain a horseradish peroxidase polymer; Step 3, selecting a dialysis bag with a molecular cutoff of 2000-4000D, exchanging the liquid with a phosphate buffer, and dialyzing the horseradish peroxidase polymer at 3-5° C. for 8-12 hours for purification; Step 4: Mouse anti-human troponin I monoclonal antibody is linked to the purified horseradish peroxidase polymer; Step 5: Select a dialysis bag with a molecular cutoff of 2000-4000D, replace the liquid with phosphate buffer, and dialyze the product of step 4 at 3-5°C for 8-12 hours to purify the product to obtain the HRP-labeled mouse anti-human troponin I monoclonal antibody.

2. The kit according to claim 1, characterized in that In step 1, 5-8 mg of xanthan gum reacts with 15-25 mg of sodium periodate to obtain xanthan gum containing aldehyde groups; In step 2, 5-15 mg of horseradish peroxidase is added to the aldehyde-containing xanthan gum obtained in step 1, and after being fully mixed, 35-45 mg of sodium triacetylborohydride is added, and the mixture is rotated at 3-5° C. to obtain a horseradish peroxidase polymer; In step 4, 2-4 mg of mouse anti-human troponin I monoclonal antibody is added to the dialyzate obtained in step 3, mixed thoroughly, and then sodium triacetylborohydride is added, and the mixture is rotated to react for 3-5 hours.

3. The kit according to claim 1, characterized in that In step one, xanthan gum is oxidized by sodium periodate.

4. The kit according to claim 3, characterized in that In step 1, 5-8 mg of xanthan gum is reacted with 15-25 mg of sodium periodate to obtain aldehyde-containing xanthan gum.

5. The kit according to any one of claims 2 to 4, characterized in that Xanthan gum was first dissolved in 10-100 mM carbonate buffer at pH 9-10, and then sodium periodate was added.

6. The kit according to claim 1, characterized in that In step 2, 5-15 mg of horseradish peroxidase is added to the aldehyde-containing xanthan gum obtained in step 1, 35-45 mg of sodium triacetylborohydride is added after mixing, and the mixture is rotated at 3-5° C. overnight to obtain a horseradish peroxidase polymer.

7. The kit according to claim 1, characterized in that In step 4, 2-4 mg of mouse anti-human troponin I monoclonal antibody is added to the dialyzate obtained in step 3, mixed thoroughly, and then sodium triacetylborohydride is added, and the mixture is rotated to react for 3-5 hours.

Images