A method for improving the signal-to-noise ratio of directional coupling labeling of antibodies and fluorescent proteins
By using crosslinking agents such as Sulfo-S-HyNic and S-4FB, reacting with phycoerythrin and antibodies, and combining hydrazone-based catalysts and cysteine blocking, the problem of low signal-to-noise ratio in traditional labeling methods is solved, significantly improving the The positive signal strength and detection effect of phycoerythrin labeling method were obtained.
Patent Information
- Application Number
- CN202011368522.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-11-30
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2040-11-30
AI Technical Summary
In traditional phycoerythrin labeling method, amine-mercapto crosslinking agents bind to amino and mercapto groups when activated by protein, resulting in poor crosslinking effect, low signal-to-noise ratio, weak positive signal and strong background signal.
Sulfo-S-HyNic or S-4FB is used as the first crosslinking agent to react with phycoerythrin, Sulfo-S-4FB or S-HyNic is used as the second crosslinking agent to react with the antibody, and is treated by crosslinking reaction and desalting. Combining hydrazone-based catalyst and cysteine blocking improves cross-linking efficiency and signal-to-noise ratio.
The signal-to-noise ratio of the directed coupling marker between antibodies and fluorescent proteins is significantly improved, the positive signal intensity is enhanced, and the negative signal is reduced. The obtained fluorescent protein probe has higher fluorescence intensity and better detection effect.
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Figure CN112630422B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of immunology, and in particular relates to a method for labeling immunofluorescent proteins such as phycoerythrin. Background Art
[0002] Phycoerythrin (PE) is a new type of fluorescent labeling reagent that is isolated and purified from red algae and is currently widely used. Under specific wavelength excitation, phycobiliprotein can emit strong fluorescence, and its fluorescence intensity is 30-100 times that of fluorescein. It has good light absorption performance and high quantum yield, and has a wide excitation and emission range in the visible spectrum.
[0003] Phycoerythrin has advantages over traditional chemical fluorescent dyes in fluorescence analysis. For example: (1) It has a wide absorption spectrum in a wide pH range, making it easier to select a suitable excitation wavelength to obtain efficient fluorescence emission, and has a specific fluorescence emission peak when excited; (2) It has high absorbance and fluorescence quantum yield, strong and stable fluorescence, and high sensitivity; (3) It has a small fluorescence background, is not easy to quench, and has a long fluorescence shelf life; (4) It has excellent water solubility, is easy to cross-link with other molecules, and has little non-specific adsorption; (5) It is extracted from pure natural marine organisms, has no toxic side effects, does not contain radioactivity, and is very safe to operate and use.
[0004] In the prior art, PE labeling is often used to combine phycoerythrin with antibodies, biotin, avidin, immune proteins and other substances to make fluorescent probes. By detecting the fluorescence emitted by it, it can be used for fluorescence microscopy, fluorescence immunoassay, two-color or multi-color fluorescence analysis, cancer cell surface antigen detection, analysis of biological macromolecules such as proteins and nucleic acids; it can also be used for clinical diagnosis and bioengineering technology such as immunoassay, fluorescence microscopy and flow cytometry.
[0005] Traditional PE labeling methods (such as AnaTag TM The implementation steps of the R-PE Labeling Kit are roughly as follows: (1) thiolation of the target protein; (2) activation of PE using SMCC; and (3) cross-linking of the thiolated target protein with the activated PE.
[0006] The implementation steps of the PE labeling method developed by the present invention (other protein labeling methods are similar) are roughly as follows: (1) reacting PE with Sulfo-S-4FB; (2) reacting the antibody with Sulfo-S-HyNic; (3) cross-linking the PE reacted with Sulfo-S-4FB and the antibody reacted with Sulfo-S-HyNic. (Alternatively, (1) reacting PE with Sulfo-S-HyNic; (2) reacting the antibody with Sulfo-S-4FB; (3) cross-linking the PE reacted with Sulfo-S-HyNic and the antibody reacted with Sulfo-S-4FB.)
[0007] Since the target protein and phycoerythrin carry both amino groups and thiol groups, when using traditional amine-thiol crosslinkers such as SMCC for activation, the amine-thiol crosslinkers will not only bind to the amino groups, but also to the thiol groups. This results in that when the protein activated by the amine-thiol crosslinker is cross-linked with the thiol-modified protein, the crosslinkers that have been bound to the thiol groups on the protein activated by the amine-thiol crosslinker are difficult to cross-link with the thiol-modified protein again, resulting in less phycoerythrin bound to the target protein and poor labeling effect. The positive signal generated by the obtained immunofluorescence probe is weak, the background signal is strong, and the signal-to-noise ratio is low during detection. The method developed by Zhengxi Bio achieves directional coupling of antibodies and fluorescent proteins, and there are no complexes between antibodies and complexes between fluorescent proteins. Summary of the invention
[0008] The present invention provides a method for improving the signal-to-noise ratio of directional coupling labeling of an antibody and a fluorescent protein, the method comprising the following steps: step 1) reacting PE with a first crosslinking agent; step 2) reacting the antibody with a second crosslinking agent; step 3) crosslinking the PE crosslinked in step 1) with the antibody crosslinked in step 2);
[0009] Wherein, the first cross-linking agent is Sulfo-S-HyNic or S-HyNic, and the second cross-linking agent is Sulfo-S-4FB or S-4FB; or the first cross-linking agent is Sulfo-S-4FB or S-4FB, and the second cross-linking agent is Sulfo-S-HyNic or S-HyNic; when performing immunoassay, this method has a strong positive signal and a high signal-to-noise ratio.
[0010] In some embodiments, in step 2), the second cross-linking agent binds to free amino groups in the antibody molecule.
[0011] In some embodiments, the amino group on the PE in step 1) reacts with the first cross-linking agent.
[0012] In some embodiments, in step 3), a nucleophilic catalyst is added to the PE cross-linked in step 1) and the antibody cross-linked in step 2) to carry out a cross-linking reaction; preferably, the nucleophilic catalyst is a hydrazone catalyst; in some embodiments, the hydrazone catalyst is selected from 2,4-dimethoxyaniline, aniline, aniline derivatives, 5-aminoindole, phenylenediamine derivatives, 3,5-diaminobenzoic acid, and o-aminobenzoic acid derivatives.
[0013] In some embodiments, in the step 3), an excess of a second cross-linking agent is added to the reaction mixture to block the excess PE that reacts with the first cross-linking agent, and after blocking, excess cysteine is added and unreacted PE is removed by an ultrafiltration tube; through the above method, the method reduces the negative signal of the reaction and improves the signal-to-noise ratio.
[0014] In some embodiments, in step 1) and step 2), the first cross-linking agent and the second cross-linking agent that have not participated in the reaction are removed by desalting.
[0015] In some embodiments, the specific method of step 2) is: adding a second cross-linking agent solution to the antibody, mixing and reacting. In this step, the second cross-linking agent molecules bind to the free amino groups in the antibody molecules to prepare a second cross-linking agent-antibody; transferring the obtained reaction solution to an ultrafiltration centrifuge tube, adding sodium phosphate buffer, centrifuging and removing the filtrate, then adding sodium phosphate buffer, mixing and centrifuging, repeating this operation multiple times and fixing the antibody to a fixed volume.
[0016] In some embodiments, the specific method of step 1) is: add the first cross-linking agent solution to PE, mix and react. In this step, the first cross-linking agent molecules combine with the free amino groups in the phycoerythrin molecules to prepare the first cross-linking agent-PE; transfer the obtained reaction solution to an ultrafiltration centrifuge tube, add sodium phosphate buffer, centrifuge and remove the filtrate, then add sodium phosphate buffer, mix and centrifuge, repeat this operation multiple times and make the PE volume constant.
[0017] In some embodiments, in step 3), the cross-linked antibody obtained in step 2) and the cross-linked PE obtained in step 1) are mixed and reacted to prepare antibody-PE, wherein the molar ratio of the antibody to PE is 1:1 to 5, preferably 1:3.
[0018] The invention also provides a method for preparing a fluorescent protein probe, which comprises reacting PE with Sulfo-S-4FB or Sulfo-S-HyNic or -S-4FB or S-HyNic.
[0019] In some embodiments, Sulfo-S-4FB or Sulfo-S-HyNic or -S-4FB or S-HyNic solution is added to PE, mixed and reacted. In this step, Sulfo-S-4FB or Sulfo-S-HyNic or -S-4FB or S-HyNic molecules bind to free amino groups in phycoerythrin molecules; the obtained reaction solution is transferred to an ultrafiltration centrifuge tube, sodium phosphate buffer is added, centrifuged and the filtrate is removed, sodium phosphate buffer is added, mixed and centrifuged, this operation is repeated multiple times and PE is fixed to volume.
[0020] The present invention obtains a fluorescent protein probe according to the method.
[0021] The PE-antibody of the present invention has a higher positive signal than the traditional labeling method, and further reduces the negative signal and improves the signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1-Figure 3 The flow cytometry scatter plot of mouse spleen cells co-stained with the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC prepared in Example 1 and the histogram of the PE fluorescence signal intensity detection results;
[0023] Figure 4-6 The flow cytometry scatter plot of mouse spleen cells co-stained with the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC prepared in Example 2 and the histogram of the PE fluorescence signal intensity detection results;
[0024] Figure 7-9 The flow cytometry scatter plot of mouse spleen cells co-stained with the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC prepared in Experimental Example 3 and the histogram of the PE fluorescence signal intensity detection results;
[0025] Figure 10-12 The flow cytometry scatter plot of the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC co-stained mouse spleen cells prepared in Comparative Example 1 and the histogram of the PE fluorescence signal intensity detection results. DETAILED DESCRIPTION
[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.
[0027] In the present invention, the structure of Sulfo-S-4FB is shown below:
[0028]
[0029] The structure of Sulfo-S-HyNic is shown below:
[0030]
[0031] Example 1
[0032] This example uses anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody) as the target protein to introduce a method for preparing a phycoerythrin immunofluorescent probe. The preparation method includes the following steps:
[0033] (1) Reaction of the amino groups on the target protein with Sulfo-S-HyNic;
[0034] Specifically include:
[0035] Add 0.7ul 60mM Sulfo-S-HyNic solution to 200ug CD4 antibody, mix well, and place at room temperature above 25℃ for 1.5h;
[0036] In this step, Sulfo-S-HyNic molecules bind to free amino groups in CD4 antibody molecules to prepare Sulfo-S-HyNic-CD4;
[0037] Transfer the reaction solution obtained in step (1.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5min and remove the filtrate, then add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of antibody to 46ul;
[0038] In this step, desalting is performed to remove the unreacted Sulfo-S-HyNic residues in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-HyNic residues in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0039] (2) reacting the amino group on the fluorescent protein (phycoerythrin) with Sulfo-S-4FB;
[0040] Specifically include:
[0041] (2.1) Add 2.3 μl of 60 mM Sulfo-S-4FB solution to 600 μg of phycoerythrin, mix well, and react at room temperature above 25°C for 1.5 h;
[0042] In this step, Sulfo-S-4FB molecules bind to free amino groups in phycoerythrin molecules to prepare Sulfo-S-HyNic-PE;
[0043] (2.2) Transfer the reaction solution obtained in step (2.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5 min, remove the filtrate, add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of PE to 30ul;
[0044] In this step, desalting is performed to remove the unreacted Sulfo-S-4FB residue in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-4FB residue in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0045] (3) cross-linking the Sulfo-S-HyNic-CD4 obtained in step (1) with the Sulfo-S-HyNic-PE obtained in step (2) to obtain CD4-PE of this embodiment;
[0046] Specifically include:
[0047] (3.1) Mix 46ul of Sulfo-S-HyNic-CD4 antibody obtained in step (1) and 30ul of Sulfo-S-HyNic-PE obtained in step (2) (n(Ab):n(PE)=1:3), and then place the mixture in a dark place above 25°C for 3 hours, then dilute the antibody concentration to 0.25mg / ml and store at 4°C to obtain the prepared CD4-PE.
[0048] (3.2) CD4-PE labeled with the same concentration was co-stained with CD8-FITC on 1 million mouse spleen cells (0.25 μg of iFluor labeled antibody and 0.125 μg of PE labeled antibody were added to each reaction system), and the fluorescence signal was detected by flow cytometry after staining for 15 minutes.
[0049] Figure 1-Figure 3 The flow cytometry scatter plot of the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC co-stained mouse spleen cells prepared in Example 1 and the histogram of the PE fluorescence signal intensity detection results.
[0050] Example 2
[0051] This example uses anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody) as the target protein to introduce a method for preparing a phycoerythrin immunofluorescent probe (blocking is performed on the basis of Example 1). The preparation method includes the following steps:
[0052] (1) Reaction of the amino groups on the target protein with Sulfo-S-HyNic;
[0053] Specifically include:
[0054] (1.1) Add 0.7ul 60mM Sulfo-S-HyNic solution to 200ug CD4 antibody, mix well, and react at room temperature above 25°C for 1.5h;
[0055] In this step, Sulfo-S-HyNic molecules bind to free amino groups in CD4 antibody molecules to prepare Sulfo-S-HyNic-CD4;
[0056] (1.2) Transfer the reaction solution obtained in step (1.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5 min, remove the filtrate, add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of antibody to 46ul;
[0057] In this step, desalting is performed to remove the unreacted Sulfo-S-HyNic residues in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-HyNic residues in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0058] (2) reacting the amino group on the fluorescent protein (phycoerythrin) with Sulfo-S-4FB;
[0059] Specifically include:
[0060] (2.1) Add 2.3 μl of 60 mM Sulfo-S-4FB solution to 600 μg of phycoerythrin, mix well, and react at room temperature above 25°C for 1.5 h;
[0061] In this step, Sulfo-S-4FB molecules bind to free amino groups in phycoerythrin molecules to prepare Sulfo-S-HyNic-PE;
[0062] (2.2) Transfer the reaction solution obtained in step (2.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5 min, remove the filtrate, add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of PE to 30ul;
[0063] In this step, desalting is performed to remove the unreacted Sulfo-S-4FB residue in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-4FB residue in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0064] (3) cross-linking the Sulfo-S-HyNic-CD4 obtained in step (1) with the Sulfo-S-HyNic-PE obtained in step (2) to obtain CD4-PE of this embodiment;
[0065] Specifically include:
[0066] (3.1) Take 46ul Sulfo-S-HyNic-CD4 antibody obtained in step (1) and mix with 30ul Sulfo-S-HyNic-PE obtained in step (2) (n(Ab):n(PE)=1:3). At the same time, add 10mM catalyst 2,4-dimethoxyaniline (2,4-DMA) to the reaction and place it at room temperature above 25°C in the dark for 3 hours. Then dilute the antibody concentration to 0.25mg / ml and store it at 4 degrees to prepare CD4-PE.
[0067] (3.2) CD4-PE labeled with the same concentration was co-stained with CD8-FITC on 1 million mouse spleen cells (0.25 μg of iFluor labeled antibody and 0.125 μg of PE labeled antibody were added to each reaction system), and the fluorescence signal was detected by flow cytometry after staining for 15 minutes.
[0068] Figure 4-Figure 6 The flow cytometry scatter plot of the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC co-stained mouse spleen cells prepared in Example 1 and the histogram of the PE fluorescence signal intensity detection results.
[0069] Example 3
[0070] This example uses anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody) as the target protein to introduce a method for preparing a phycoerythrin immunofluorescent probe (blocking is performed on the basis of Example 1). The preparation method includes the following steps:
[0071] (1) Reaction of the amino groups on the target protein with Sulfo-S-HyNic;
[0072] Specifically include:
[0073] (1.1) Add 0.7ul 60mM Sulfo-S-HyNic solution to 200ug CD4 antibody, mix well, and react at room temperature above 25°C for 1.5h;
[0074] In this step, Sulfo-S-HyNic molecules bind to free amino groups in CD4 antibody molecules to prepare Sulfo-S-HyNic-CD4;
[0075] (1.2) Transfer the reaction solution obtained in step (1.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5 min, remove the filtrate, add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of antibody to 46ul;
[0076] In this step, desalting is performed to remove the unreacted Sulfo-S-HyNic residues in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-HyNic residues in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0077] (2) reacting the amino group on the fluorescent protein (phycoerythrin) with Sulfo-S-4FB;
[0078] Specifically include:
[0079] (2.1) Add 2.3 μl of 60 mM Sulfo-S-4FB solution to 600 μg of phycoerythrin, mix well, and react at room temperature above 25°C for 1.5 h;
[0080] In this step, Sulfo-S-4FB molecules bind to free amino groups in phycoerythrin molecules to prepare Sulfo-S-HyNic-PE;
[0081] (2.2) Transfer the reaction solution obtained in step (2.1) to an ultrafiltration centrifuge tube, add 500ul sodium phosphate (PH 6.0) buffer, centrifuge at 12000g for 5 min, remove the filtrate, add 500ul sodium phosphate (PH 6.0) buffer, mix and centrifuge, repeat this operation 5 times and adjust the volume of PE to 30ul;
[0082] In this step, desalting is performed to remove the unreacted Sulfo-S-4FB residue in the reaction solution as much as possible, ensuring that the amount of Sulfo-S-4FB residue in the reaction solution obtained after desalting is at least 10% of the total amount of antibodies. -3 times or more;
[0083] (3) cross-linking the Sulfo-S-HyNic-CD4 obtained in step (1) with the Sulfo-S-HyNic-PE obtained in step (2) to obtain CD4-PE of this embodiment;
[0084] Specifically include:
[0085] (3.1) Take 46ul Sulfo-S-HyNic-CD4 antibody obtained in step (1) and 30ul Sulfo-S-HyNic-PE obtained in step (2) and mix them evenly (n(Ab):n(PE)=1:3). At the same time, add 10mM catalyst 2,4-dimethoxyaniline (2,4-DMA) to the reaction and place it at room temperature above 25°C in the dark for 3 hours. Then add 2.3ul Sulfo-S-HyNic to block the unreacted Sulfo-S-HyNic-PE. After leaving it at room temperature for 1.5 hours, add 10ul 1M cysteine to block the groups on the excess Sulfo-S-HyNic that react with cysteine for 1.5 hours. After the reaction, the mixed solution is ultrafiltered through a 200kDa ultrafiltration tube to remove uncrosslinked PE, and the antibody solution is diluted to a concentration of 0.25mg / ml and stored at 4 degrees. This is the prepared CD4-PE.
[0086] Figure 7-Figure 9 The flow cytometry scatter plot of mouse spleen cells co-stained with the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC prepared in Example 1 and the histogram of the PE fluorescence signal intensity detection results;
[0087] Comparative Example 1
[0088] The phycoerythrin immunofluorescent probe CD4-PE was prepared using the same method as the phycoerythrin immunofluorescent probe labeling method (patent number 202010087828.0).
[0089] The labeled CD4-PE was co-stained with CD8-FITC at the same concentration on 1 million mouse spleen cells (0.25 μg of labeled iFluor labeled antibody and 0.125 μg of PE labeled antibody were added to each reaction system), and the fluorescence signal was detected by flow cytometry after staining for 15 minutes.
[0090] Figure 10-12 The flow cytometry scatter plot of the phycoerythrin immunofluorescence probe CD4-PE and CD8-FITC co-stained mouse spleen cells prepared in Example 1 and the histogram of the PE fluorescence signal intensity detection results.
[0091] Result analysis:
[0092] CD4 flow cytometry positive signal: According to the PE fluorescence signal intensity detection result histogram, Example 1 positive signal: 19200 ( Figure 3 ), Example 2 positive signal: 27319 ( Figure 6 ), Experimental Example 3 Positive signal: 26486 ( Fig. 9 ), greater than the positive signal of comparative example 1 ( Fig.12 ):17846
[0093] Compared with the traditional SMCC labeling method (Comparative Example 1), Example 1 has a closer PE positive signal. After adding a catalyst for the cross-linking reaction, the positive signal of PE labeling is significantly improved, see the positive signals of Examples 2 and 3.
[0094] Negative flow cytometry signal:
[0095] Example 3 Negative signal: 167 ( Fig. 9 ), Comparative Example 1 Negative signal: 216 ( Fig.12 ), Example 1 negative signal: 259 ( Figure 3 ) Example 2 Negative signal: 429 ( Figure 6 )
[0096] Compared with the traditional SMCC labeling method (Comparative Example 1), Example 1 and Example 3 have higher PE negative signals, indicating that the new cross-linking method produces more PE non-specific signals. After adding a cross-linking blocking agent, cysteine and ultrafiltration treatment, the PE negative signal of Example 3 has significantly decreased, which is lower than that of Comparative Example 1.
[0097] In summary, Example 3 has a higher PE positive signal and the lowest PE negative signal, so Example 3 has the highest signal-to-noise ratio and is superior to the traditional labeling method.
Claims
1. A method for improving the signal-to-noise ratio of antibody-fluorescent protein directional coupling labeling, characterized in that: The method comprises the following steps: step 1) reacting phycoerythrin with a first cross-linking agent; step 2) reacting an antibody with a second cross-linking agent; step 3) cross-linking the cross-linked phycoerythrin in step 1) with the cross-linked antibody in step 2); Wherein, the first cross-linking agent is Sulfo-S-HyNic or S-HyNic, and the second cross-linking agent is Sulfo-S-4FB or S-4FB; or the first cross-linking agent is Sulfo-S-4FB or S-4FB, and the second cross-linking agent is Sulfo-S-HyNic or S-HyNic; In the step 1) and the step 2), the first cross-linking agent and the second cross-linking agent that have not participated in the reaction are removed by desalting; the molar ratio of the antibody to phycoerythrin is 1:1-5; in the step 3), an excess of the second cross-linking agent is added to the reaction mixture to block the excess phycoerythrin that has reacted with the first cross-linking agent, and after blocking, excess cysteine is added and the unreacted phycoerythrin is removed by an ultrafiltration tube.
2. The method according to claim 1, characterized in that In the step 2), the second cross-linking agent is combined with the amino group in the antibody molecule; and in the step 1), the amino group on the phycoerythrin reacts with the first cross-linking agent.
3. The method according to claim 1, characterized in that In the step 3), a nucleophilic catalyst is added to the cross-linked phycoerythrin in step 1) and the cross-linked antibody in step 2) to carry out a cross-linking reaction.
4. The method according to claim 3, characterized in that The nucleophilic catalyst is a hydrazone-based catalyst.
5. The method according to claim 4, characterized in that The hydrazone-based catalyst is selected from 2, 4-dimethoxyaniline, aniline, aniline derivatives, 5-aminoindole, phenylenediamine derivatives, 3,5-diaminobenzoic acid, and o-aminobenzoic acid derivatives.
6. The method according to claim 1, characterized in that The specific method of step 2) is: add the second cross-linking agent solution to the antibody, mix and react. In this step, the second cross-linking agent molecules bind to the free amino groups in the antibody molecules to prepare the second cross-linking agent-antibody; transfer the obtained reaction solution to an ultrafiltration centrifuge tube, add sodium phosphate buffer, centrifuge and remove the filtrate, then add sodium phosphate buffer, mix and centrifuge, repeat this operation several times and fix the antibody to a fixed volume.
7. The method according to claim 1, characterized in that The specific method of step 1) is: add the first cross-linking agent solution to phycoerythrin, mix and react. In this step, the first cross-linking agent molecules combine with the free amino groups in the phycoerythrin molecules to prepare the first cross-linking agent-phycoerythrin; transfer the obtained reaction solution to an ultrafiltration centrifuge tube, add sodium phosphate buffer, centrifuge and remove the filtrate, then add sodium phosphate buffer, mix and centrifuge, repeat this operation several times and make the phycoerythrin volume constant.
8. The method according to claim 1, characterized in that The molar ratio of the antibody to phycoerythrin is 1:3.