CN111830250A - Preparation method of signal amplified enzyme-labeled secondary antibody - Google Patents
Preparation method of signal amplified enzyme-labeled secondary antibody Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to a preparation method of an enzyme-labeled secondary antibody with amplified signal, belonging to the technical field of enzyme-macromolecule conjugates; preparing a Dex-HRP-IgG conjugate by taking an enzyme-labeled antibody Horse Radish Peroxidase (HRP) -IgG which is widely applied in biochemical analysis as a system and taking glucan (Dex) as a carrier, wherein the aim is to improve the HRP/IgG molar ratio, so that the detection sensitivity of the enzyme-labeled antibody is improved; the combination of glucan, horseradish peroxidase and goat anti-mouse/rabbit IgG is carried out in an optimal mode, the second antibody enzyme labeling efficiency is improved, the second antibody signal amplification effect is enlarged, and the detection sensitivity is improved.
Description
Technical Field
The invention belongs to the technical field of enzyme-polymer conjugates, and particularly relates to a preparation method of an enzyme-labeled secondary antibody for signal amplification.
Background
The basic principle of the immunoassay is that firstly, an enzyme-labeled antibody reacts with tissues or cells, then a substrate of the enzyme is added to generate a colored insoluble product or particles with certain electron density, and various antigen components on the surface of the cells and in the cells are subjected to positioning research. The most commonly used technique is the immunoenzyme labeling technique. Compared with the immunofluorescence technology, the method has the main advantages that: accurate positioning, good contrast, long-term preservation of the stained specimen, and suitability for research on light and electron microscopes. The development of the immunoassay method is very rapid, a plurality of labeling methods are derived, and with the continuous improvement and innovation of the method, the specificity and the sensitivity of the immunoassay method are continuously improved, and the immunoassay method is more and more convenient to use.
The direct enzyme labeling method is widely used in scientific research, and the method is to label HRP molecules on a secondary antibody directly. Therefore, the enzyme-labeled secondary antibody is required to complete the process. The method has the advantages of simple experiment and low cost, but has low sensitivity and is easy to generate background because of no signal amplification effect.
Disclosure of Invention
The invention aims to provide a preparation method of an enzyme-labeled secondary antibody with amplified signals, which aims to solve the technical problems that the ordinary enzyme-labeled secondary antibody is labeled by directly combining HRP enzyme molecules with the secondary antibody, the immune signals are not high and the sensitivity is low.
In order to achieve the purpose, the specific technical scheme of the preparation method of the signal amplified enzyme-labeled secondary antibody is as follows:
firstly, preparing a Dex-HRP-IgG conjugate by taking an enzyme-labeled antibody Horse Radish Peroxidase (HRP) -IgG which is widely applied in biochemical analysis as a system and taking glucan (Dex) as a carrier, wherein the aim is to improve the HRP/IgG molar ratio, so that the detection sensitivity of the enzyme-labeled antibody is improved;
and secondly, the combination of glucan, horseradish peroxidase and goat anti-mouse/rabbit IgG is carried out in an optimal mode, so that the second antibody enzyme labeling efficiency is improved, the second antibody signal amplification effect is enlarged, and the detection sensitivity is improved.
According to the invention, a macromolecular compound is adopted for oxidation reaction or oxidative ammoniation reaction, and then horseradish peroxidase and a second antibody are combined, so that the enzyme labeling efficiency is improved, the signal amplification effect of an enzyme-labeled second antibody is improved, and the detection sensitivity is improved. The preparation process comprises the following steps:
the optimal scheme is as follows: the macromolecular compound is subjected to oxidation reaction or oxidative ammoniation reaction, and then is combined with horseradish peroxidase and a second antibody.
A preparation method of an enzyme-labeled secondary antibody for signal amplification comprises the following steps in sequence:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2, carrying out oDex-HRP synthesis, adding a carbonate buffer solution into HRP, immediately adding oxidized dextran in the step A1, mixing, and shaking in a dark place;
step A3, synthesizing oDex-HRP-IgG, adding goat anti-rabbit/mouse IgG into the polymer, mixing, stirring in the dark after fully mixing, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding equal volume of saturated ammonium sulfate under stirring; centrifuging and discarding the supernatant.
Further, the step A4, adding half saturated ammonium sulfate for washing, and finally dissolving the precipitate in PBS; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
Further, HRP oxidation is added between the steps a1 and a2, and the following steps are included and are performed sequentially:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2', oxidizing HRP, dissolving HRP in acetic acid buffer solution with pH4.4, adding sodium periodate solution, stirring at room temperature in a dark place, adding ethylene glycol, and standing uniformly; then dialyzing with acetic acid buffer solution at 4 ℃ overnight;
step A2, oDex-IgG synthesis, adding a part of oxidized dextran into goat anti-mouse/rabbit IgG, oscillating for reaction at room temperature, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dialyzing the reaction solution with carbonate buffer solution overnight; adding ethylenediamine hydrochloride, carrying out oscillation reaction, adding sodium cyanoborohydride, carrying out oscillation reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A3, synthesizing oDex-IgG-oHRP, taking the reaction solution, adding oxidized HRP and carbonate buffer solution, fully mixing uniformly, and stirring in the dark; adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding saturated ammonium sulfate with the same volume under stirring, standing, centrifuging, and removing supernatant; adding half saturated ammonium sulfate for cleaning, and finally dissolving the precipitate in PBS; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
Further, between said steps a1 and a2' dextran amination is added, comprising the following steps, and the following steps are performed in sequence:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2' of ammoniating glucan, wherein the dialyzed glucan obtained in the step A1 is added with ethylenediamine hydrochloride, and after oscillation reaction, sodium cyanoborohydride is added for oscillation reaction again; then dialyzing overnight by carbonate buffer solution;
step A2' and oxidizing HRP, dissolving HRP in acetic acid buffer solution, adding sodium periodate solution, stirring for a period of time in a dark place at room temperature, adding ethylene glycol, and uniformly standing; then dialyzed against acetate buffer overnight.
Step A2, synthesizing aDex-oHRP, adding carbonate buffer solution into the oxidized HRP obtained in the step A2', immediately adding ammoniated dextran, mixing, and shaking for a period of time at room temperature in a dark place; adding sodium cyanoborohydride for reduction, and carrying out oscillation reaction; the carbonate buffer was dialyzed overnight.
Step A3, synthesizing aDex-oHRP-IgG, taking half of the conjugate of the ammoniated glucan obtained in the step A2' and the HRP obtained in the step A2, adding goat anti-rabbit/mouse IgG for mixing, stirring in a dark place after fully mixing, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding saturated ammonium sulfate with the same volume under stirring, centrifuging, and removing supernatant; the mixture was washed with half-saturated ammonium sulfate and finally the precipitate was dissolved in PBS.
Further, the step A5, synthesizing aDex-oHRP-IgG, taking one half of the conjugate, adding glutaraldehyde, standing at room temperature overnight, gradually adding dropwise goat anti-mouse/rabbit IgG, and reacting at room temperature; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
The preparation method of the signal amplified enzyme-labeled secondary antibody has the following advantages: preparing a Dex-HRP-IgG conjugate by taking an enzyme-labeled antibody Horse Radish Peroxidase (HRP) -IgG which is widely applied in biochemical analysis as a system and taking glucan (Dex) as a carrier, wherein the aim is to improve the HRP/IgG molar ratio, so that the detection sensitivity of the enzyme-labeled antibody is improved; the combination of glucan, horseradish peroxidase and goat anti-mouse/rabbit IgG is carried out in an optimal mode, so that the second antibody enzyme labeling efficiency is improved, the second antibody signal amplification effect is enlarged, and the detection sensitivity is improved;
the method adopts a section of Polymer as a carrier chain, combines the Polymer with a second antibody, and then can label a plurality of HRP enzyme molecules, thereby realizing high sensitivity, low background, simple and convenient operation and no need of endogenous biotin labeling; a plurality of HRP enzyme molecules are marked by using a polymer as a carrier, and a secondary antibody and a primary antibody are synchronously marked by the polymer to carry out immunoreaction test, so that the aim of amplifying reaction signals is fulfilled.
Detailed Description
In order to better understand the purpose, structure and function of the present invention, the following will describe the preparation method of the signal-amplified enzyme-labeled secondary antibody of the present invention in detail with reference to the examples.
Preparing a Dex-HRP-IgG conjugate by taking an enzyme-labeled antibody Horse Radish Peroxidase (HRP) -IgG which is widely applied in biochemical analysis as a system and taking glucan (Dex) as a carrier, wherein the aim is to improve the HRP/IgG molar ratio, so that the detection sensitivity of the enzyme-labeled antibody is improved;
the combination of glucan, horseradish peroxidase and goat anti-mouse/rabbit IgG is carried out in an optimal mode, the second antibody enzyme labeling efficiency is improved, the second antibody signal amplification effect is enlarged, and the detection sensitivity is improved.
According to the invention, a macromolecular compound is adopted for oxidation reaction or oxidative ammoniation reaction, and then horseradish peroxidase and a second antibody are combined, so that the enzyme labeling efficiency is improved, the signal amplification effect of an enzyme-labeled second antibody is improved, and the detection sensitivity is improved. The preparation process comprises the following steps:
combining a high molecular compound with horseradish peroxidase and a second antibody, wherein the optimal scheme is as follows: the macromolecular compound is subjected to oxidation reaction or oxidative ammoniation reaction, and then is combined with horseradish peroxidase and a second antibody.
Example 1:
1: glucan oxidation (T500)
Dextran 2mg (T500) was dissolved well in 0.2ml 0.01mol/L pH7.4 PBS at room temperature and 100. mu.l 0.2M NaIO was added4Shaking at 4 ℃ in dark for 3 hours. 0.1ml of 0.69mol/L ethylene glycol is added to react, and the reaction is stopped by shaking for 15min at 4 ℃. The reaction solution was dialyzed overnight against 0.01mol/L of a carbonate buffer solution at pH9.5, during which the dialyzate was replaced three times.
2: glucan ammoniation (T500)
Mu.l of 150mg/ml ethylenediamine hydrochloride was added to the dialyzed dextran, and the mixture was reacted at 4 ℃ for 2 hours with shaking. 200ul of 10mg/ml sodium cyanoborohydride was added, and the reaction was stirred at 4 ℃ for 2 hours. Then dialyzed overnight against 0.01mol/L pH9.5 carbonate buffer, during which the dialysate was changed three times.
3: HRP Oxidation
10mg of HRP was dissolved in 2ml of 0.01mol/L pH4.4 acetic acid buffer solution, 0.4ml of 0.1mol/L sodium periodate solution was added thereto, and the mixture was stirred at room temperature for 20 minutes in the dark, 1ml of 0.16mol/L ethylene glycol was added thereto, and the mixture was stirred uniformly and allowed to stand for 30 minutes. Then dialyzed with pH 4.41 mmol/L acetate buffer overnight at 4 ℃ during which the dialysate was changed three times.
4: synthesizing the aDex-oHRP,
oxidized HRP was mixed with 400. mu.l of 0.2mol/L carbonate buffer pH9.5, then immediately mixed with 2mg of aminated dextran, and shaken at 4 ℃ in the dark for 2 hours at room temperature. Then 200. mu.l of 10mg/ml sodium cyanoborohydride is added for reduction, and the reaction is carried out for 2 hours at 4 ℃ with shaking. Dialysis was performed overnight with 0.01mol/L of a pH9.5 carbonate buffer solution, during which the dialysate was changed three times.
5: synthesizing aDex-oHRP-IgG,
half of the conjugate of the aminated dextran and the HRP is taken, 200ul of goat anti-rabbit/mouse IgG with the concentration of 5mg/ml is added for mixing, and after the mixture is fully mixed, the mixture is stirred for 2 hours at 4 ℃ in a dark place. To the solution was added 160. mu.l of 10mg/mL sodium cyanoborohydride, and the reaction was carried out at room temperature for 2 hours. An equal volume of saturated ammonium sulfate was added dropwise with stirring and left at 4 ℃ for 1 hour. Centrifuge at 5000rpm for 0.5 h and discard the supernatant. The precipitate was washed once with half-saturated ammonium sulfate and finally dissolved in 1ml of 0.01mol/L PBS, pH 7.4. Putting the above solution into dialysis bag, dialyzing with 0.01mol/L PBS (pH 7.4) overnight, changing the dialysate for three times, adding 1ml antibody diluent, mixing, packaging, and freezing for storage.
Example 2:
1: glucan oxidation (T500)
Dextran 2mg (T500) was dissolved well in 0.2ml 0.01mol/L pH7.4 PBS at room temperature and 100. mu.l 0.2M NaIO was added4Shaking at 4 ℃ in dark for 3 hours. 0.1ml of 0.69mol/L ethylene glycol is added to react, and the reaction is stopped by shaking for 15min at 4 ℃. The reaction solution was dialyzed overnight against 0.01mol/L of a carbonate buffer solution at pH9.5, during which the dialyzate was replaced three times.
2: the synthesis of oDex-HRP,
mu.l of HRP (5 mg/L) and 0.2mol/L of carbonate buffer pH9.5 were added, half of the oxidized dextran was immediately added and mixed, and the mixture was shaken at 4 ℃ in the dark for 2 hours.
3: the synthesis of oDex-HRP-IgG,
200 mu l of 2..5mg/ml goat anti-rabbit/mouse IgG is added into the polymer, mixed evenly and stirred for 2h at 4 ℃ in a dark place. To the solution was added 80. mu.l of 10mg/mL sodium cyanoborohydride, and the mixture was reacted at room temperature for 2 hours. An equal volume of saturated ammonium sulfate was added dropwise with stirring, and the mixture was left at 4 ℃ for 1 hour. Centrifuge at 5000rpm for 0.5 h and discard the supernatant. One wash with half-saturated ammonium sulfate was added and finally the pellet was dissolved in 500. mu.l of 0.01mol/L PBS pH 7.4. Putting the solution into a dialysis bag, dialyzing with 0.01mol/L PBS (pH 7.4) overnight, changing the solution for 3 times, adding 1ml antibody diluent, mixing, packaging, and freezing for storage.
Example 3:
1: glucan oxidation (T500)
Dextran 2mg (T500) was dissolved well in 0.2ml 0.01mol/L pH7.4 PBS at room temperature, 100. mu.l 0.2M NaIO4 was added, and the mixture was shaken at 4 ℃ in the dark for 3 hours. 0.1ml of 0.69mol/L ethylene glycol is added to react, and the reaction is stopped by shaking for 15min at 4 ℃. The reaction solution was dialyzed overnight against 0.01mol/L of a carbonate buffer solution at pH9.5, during which the dialyzate was replaced three times.
2: HRP Oxidation
10mg of HRP was dissolved in 2ml of 0.01mol/L pH4.4 acetic acid buffer solution, 0.4ml of 0.1mol/L sodium periodate solution was added thereto, and the mixture was stirred at room temperature for 20 minutes in the dark, 1ml of 0.16mol/L ethylene glycol was added thereto, and the mixture was stirred uniformly and allowed to stand for 30 minutes. Then dialyzed with pH 4.41 mmol/L acetate buffer overnight at 4 ℃ during which the dialysate was changed three times.
3: the synthesis of oDex-IgG,
quarter oxidized dextran was added to 200ul of 2.5mg/ml goat anti mouse/rabbit IgG and reacted with shaking at room temperature for 2 hours. To the solution was added 80. mu.l of 4mg/mL sodium cyanoborohydride, and the reaction was carried out at room temperature for 2 hours. The reaction solution was dialyzed overnight against 0.01mol/L of a carbonate buffer solution at pH9.5, during which the dialyzate was replaced three times. Adding 25ul of 150mg/mL ethylenediamine hydrochloride, carrying out shake reaction at 4 ℃ for 2 hours, adding 100 ul of 10mg/mL sodium cyanoborohydride, carrying out shake reaction for 2 hours, taking the reaction solution, dialyzing the reaction solution by using 0.01mol/L of pH9.5 carbonate buffer solution overnight, and replacing the dialyzate three times.
4: the synthesis of oDex-IgG-oHRP,
the reaction solution is taken, added with HRP oxide and 40 mu L of 0.2mol/L pH9.5 carbonate buffer solution, fully mixed and stirred for 2 hours at 4 ℃ in a dark place. To the solution was added 160. mu.l of 10mg/mL sodium cyanoborohydride, and the mixture was reacted at room temperature for 2 hours. An equal volume of saturated ammonium sulfate was added dropwise with stirring and left at 4 ℃ for 1 hour. Centrifuge at 5000rpm for 0.5 h and discard the supernatant. The precipitate was washed once with half-saturated ammonium sulfate and finally dissolved in 1ml of 0.01mol/L PBS, pH 7.4. Putting the solution into a dialysis bag, dialyzing with 0.01mol/L PBS (pH 7.4) overnight, changing the solution for 3 times, adding 1ml antibody diluent, mixing, packaging, and freezing for storage.
Example 4:
1: glucan oxidation (T500)
Dextran 2mg (T500) was dissolved well in 0.2ml 0.01mol/L pH7.4 PBS at room temperature and 100. mu.l 0.2M NaIO was added4Shaking at 4 ℃ in dark for 3 hours. 0.1ml of 0.69mol/L ethylene glycol is added to react, and the reaction is stopped by shaking for 15min at 4 ℃. The reaction solution was dialyzed overnight against 0.01mol/L of a carbonate buffer solution at pH9.5, during which the dialyzate was replaced three times.
2: glucan ammoniation (T500)
50ul of 150mg/ml ethylenediamine hydrochloride was added to the dialyzed dextran, and the mixture was reacted at 4 ℃ for 2 hours with shaking. Adding 200ul of 10mg/m sodium cyanoborohydride, and carrying out oscillation reaction at 4 ℃ for 2 hours; dialysis was performed overnight with 0.01mol/L of a pH9.5 carbonate buffer solution, during which the dialysate was changed three times.
3: HRP Oxidation
10mg of HRP was dissolved in 2ml of 0.01mol/L pH4.4 acetic acid buffer solution, 0.4ml of 0.1mol/L sodium periodate solution was added thereto, and the mixture was stirred at room temperature for 20 minutes in the dark, 1ml of 0.16mol/L ethylene glycol was added thereto, and the mixture was stirred uniformly and allowed to stand for 30 minutes. Then dialyzed with pH 4.41 mmol/L acetate buffer overnight at 4 ℃ during which the dialysate was changed three times.
4: aDex-oHRP Synthesis
Oxidized HRP was mixed with 400. mu.l of 0.2mol/L carbonate buffer pH9.5, then immediately mixed with 2mg of aminated dextran, and shaken at 4 ℃ in the dark for 2 hours at room temperature. Then 200. mu.l of 10mg/ml sodium cyanoborohydride is added for reduction, and the reaction is carried out for 2 hours at 4 ℃ with shaking. Dialysis was performed overnight with 0.01mol/L of a pH9.5 carbonate buffer solution, during which the dialysate was changed three times.
5: aDex-oHRP-IgG Synthesis
Taking one half of the conjugate, adding 1ml of 1.25% glutaraldehyde, standing at room temperature overnight, gradually adding dropwise 12ul of goat anti-mouse/rabbit IgG at 8.27mg/ml, and reacting at room temperature for 3 hours. Putting the solution into a dialysis bag, dialyzing with PBS (0.01 mol/LPH 7.4) overnight, changing the dialysate for three times, adding 1ml antibody diluent, mixing, packaging, and freezing for storage.
The enzyme-labeled secondary antibodies obtained in examples 1 to 4 were subjected to the following test methods:
enzyme-labeled secondary antibodies were diluted and subsequently subjected to elisa experiments using the corresponding generic IgG.
The results of the Elisa test are as follows:
| name (R) | Enzyme-labeled secondary antibody dilution ratio | OD value |
| Example 1 | 102400 times | 1.374 |
| Example 2 | 102400 times | 0.575 |
| Example 3 | 102400 times | 0.438 |
| Example 4 | 102400 times | 1.024 |
| Negative of | 0.045 |
Therefore, in example 1, the high molecular compound is oxidized and then ammoniated and combined with the oxidized horseradish peroxidase, and then the polymer is combined with goat anti-rabbit/mouse IgG, so that the secondary enzyme labeling efficiency is highest.
In example 1, the high molecular compound is oxidized and then aminated to bind with oxidized horseradish peroxidase, and then the polymer is bound with goat anti-rabbit/mouse IgG, so that the secondary enzyme labeling efficiency is highest. The results show that the labelling efficiency of the dextran after oxidation in example 1, example 4 was higher than in the experimental groups of example 2, example 3 without amination. Aminated dextrans react more readily with other aldehyde group containing materials. Also, the addition of glutaraldehyde in example 4 affects the direct binding of dextran complexes to IgG, and thus may affect an enzyme labeling efficiency.
It is to be understood that the present invention has been described with reference to certain embodiments, and that various changes in the features and embodiments, or equivalent substitutions may be made therein by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (5)
1. The preparation method of the signal amplified enzyme-labeled secondary antibody is characterized by comprising the following steps in sequence:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2, carrying out oDex-HRP synthesis, adding a carbonate buffer solution into HRP, immediately adding oxidized dextran in the step A1, mixing, and shaking in a dark place;
step A3, synthesizing oDex-HRP-IgG, adding goat anti-rabbit/mouse IgG into the polymer, mixing, stirring in the dark after fully mixing, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding equal volume of saturated ammonium sulfate under stirring; centrifuging and discarding the supernatant.
2. The method for preparing an enzyme-labeled secondary antibody for signal amplification according to claim 1, wherein step a4 is performed by adding half-saturated ammonium sulfate for washing, and finally dissolving the precipitate in PBS; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
3. The method for preparing an enzyme-labeled secondary antibody for signal amplification according to claim 1, wherein HRP oxidation is added between the steps A1 and A2, and the method comprises the following steps in sequence:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2', oxidizing HRP, dissolving HRP in acetic acid buffer solution with pH4.4, adding sodium periodate solution, stirring at room temperature in a dark place, adding ethylene glycol, and standing uniformly; then dialyzing with acetic acid buffer solution at 4 ℃ overnight;
step A2, oDex-IgG synthesis, adding a part of oxidized dextran into goat anti-mouse/rabbit IgG, oscillating for reaction at room temperature, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dialyzing the reaction solution with carbonate buffer solution overnight; adding ethylenediamine hydrochloride, carrying out oscillation reaction, adding sodium cyanoborohydride, carrying out oscillation reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A3, synthesizing oDex-IgG-oHRP, taking the reaction solution, adding oxidized HRP and carbonate buffer solution, fully mixing uniformly, and stirring in the dark; adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding saturated ammonium sulfate with the same volume under stirring, standing, centrifuging, and removing supernatant; adding half saturated ammonium sulfate for cleaning, and finally dissolving the precipitate in PBS; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
4. The method for preparing a signal-amplified enzyme-labeled secondary antibody according to claim 3, wherein dextran amination is added between the steps A1 and A2', and the method comprises the following steps in sequence:
step A1, oxidizing dextran, dissolving dextran in PBS at room temperature, adding NaIO4Shaking in dark; adding ethylene glycol for reaction, taking reaction liquid, and dialyzing with carbonate buffer solution overnight;
step A2' of ammoniating glucan, wherein the dialyzed glucan obtained in the step A1 is added with ethylenediamine hydrochloride, and after oscillation reaction, sodium cyanoborohydride is added for oscillation reaction again; then dialyzing overnight by carbonate buffer solution;
step A2' and oxidizing HRP, dissolving HRP in acetic acid buffer solution, adding sodium periodate solution, stirring for a period of time in a dark place at room temperature, adding ethylene glycol, and uniformly standing; then dialyzing with acetic acid buffer solution overnight;
step A2, synthesizing aDex-oHRP, adding carbonate buffer solution into the oxidized HRP obtained in the step A2', immediately adding ammoniated dextran, mixing, and shaking for a period of time at room temperature in a dark place; adding sodium cyanoborohydride for reduction, and carrying out oscillation reaction; dialyzing overnight with carbonate buffer solution;
step A3, synthesizing aDex-oHRP-IgG, taking half of the conjugate of the ammoniated glucan obtained in the step A2' and the HRP obtained in the step A2, adding goat anti-rabbit/mouse IgG for mixing, stirring in a dark place after fully mixing, adding sodium cyanoborohydride into the solution, and reacting at room temperature; dropwise adding saturated ammonium sulfate with the same volume under stirring, centrifuging, and removing supernatant; the mixture was washed with half-saturated ammonium sulfate and finally the precipitate was dissolved in PBS.
5. The method for preparing an enzyme-labeled secondary antibody for signal amplification according to claim 4, wherein the step A5 is a step of synthesizing aDex-oHRP-IgG, one half of the conjugate is taken, glutaraldehyde is added, the mixture is kept at room temperature overnight, goat anti-mouse/rabbit IgG is gradually added dropwise, and the reaction is carried out at room temperature; putting the solution into a dialysis bag, dialyzing with PBS overnight, adding antibody diluent, mixing, packaging, and freezing for storage.
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