Antibody fluorescent labeling kit and labeling method thereof

Abstract

The present invention provides an antibody fluorescent labeling kit and a labeling method thereof, the kit comprising the following raw materials: 0.4-0.6mg fluorescein isothiocyanate, 500-700μL dimethyl sulfoxide, 2.0-3.5mL labeling buffer, 1.5-2.5mL labeling preservation solution, 0.1-0.3mL sodium bicarbonate solution, 1 centrifugal column, 3 EP tubes. The labeling method of the present invention completes the fluorescent labeling of the antibody by the following steps: S1, preparing antibody solution, S2, adjusting the pH of the antibody solution, S3, preparing fluorescein isothiocyanate solution, S4, preparing markers, S5, centrifugal column balancing treatment, S6, preparing fluorescently labeled antibodies, S7, fluorescently labeled antibody dilution and preservation. The kit of the present invention can quickly and conveniently label antibodies, and there is no need to go through a dialysis step during the entire labeling process, thereby simplifying the labeling process, saving labeling time, and being both convenient and efficient. The fluorescently labeled antibody prepared by the present invention has high fluorescence intensity and strong stability, and can still maintain strong fluorescence intensity even after being stored for 12 months.

Description

Technical Field

[0001] The invention relates to the technical field of immunology, and in particular to an antibody fluorescent labeling kit and a labeling method thereof. Background Art

[0002] Fluorescent labeling of antibodies is an important technology in biomedical research, which gives antibodies the ability to be visualized and tracked. Fluorescently labeled antibodies specifically bind to target molecules, allowing researchers to directly observe the distribution, location, and dynamic changes of target molecules under a microscope. This technology greatly enhances the sensitivity and resolution of biomolecule detection, allowing researchers to gain a deeper understanding of complex processes in organisms, such as protein interactions, cell signaling, and the mechanisms of disease occurrence and development. In addition, fluorescently labeled antibodies are also widely used in disease diagnosis, drug screening, and treatment effect evaluation, providing a powerful tool for biomedical research and clinical applications. Among them, fluorescein thiocyanate is a commonly used fluorescent dye that is widely used in antibody labeling technology. Its working principle is based on the fact that fluorescein thiocyanate molecules can covalently bind to amino groups in antibody proteins to form a stable fluorescently labeled antibody complex. When excited by light of a specific wavelength, this complex can emit bright green fluorescence, thereby achieving tracking and detection of antibodies. However, the current preparation of fluorescein thiocyanate labeled antibodies is time-consuming and cumbersome, and dialysis is required during the labeling process to remove unbound fluorescein thiocyanate molecules. Currently, there is a lack of kits specifically for fluorescein thiocyanate-labeled antibodies on the market, which limits the convenient application and development of this technology. Summary of the invention

[0003] In view of this, the present invention provides an antibody fluorescent labeling kit and a labeling method thereof to solve the above problems.

[0004] The technical solution of the present invention is implemented as follows: an antibody fluorescent labeling kit comprises the following raw materials: 0.4-0.6 mg fluorescein isothiocyanate, 500-700 μL dimethyl sulfoxide, 2.0-3.5 mL labeling buffer, 1.5-2.5 mL labeling substance preservation solution, 0.1-0.3 mL sodium bicarbonate solution, 1 centrifugal column, and 3 EP tubes.

[0005] Furthermore, the labeling buffer consists of 15-25 mM sodium dihydrogen phosphate, 70-90 mM sodium dihydrogen phosphate, and 1-3 mM ethylenediaminetetraacetic acid.

[0006] Furthermore, the marker preservation solution is composed of dimethyl sulfoxide, glutathione and vitamin E in a mass ratio of (8-10):(1-2):(1-2).

[0007] Furthermore, the sodium bicarbonate solution is prepared by the following method: 0.672-1.008 g of sodium bicarbonate is dissolved in 9 mL of deionized water, the pH is adjusted to 9 with sodium hydroxide, and the volume of the solution is supplemented to 10 mL with deionized water.

[0008] Furthermore, a labeling method of an antibody fluorescent labeling kit comprises the following steps:

[0009] S1. Prepare antibody solution: take 0.1 mg of antibody, add 0.8-1.2 mL of labeling buffer to adjust the antibody concentration, and obtain antibody solution;

[0010] S2. Adjust the pH of the antibody solution: add the antibody solution to EP tube 1, add 10-20% of the volume of the antibody solution sodium bicarbonate solution, and shake to mix;

[0011] S3. Prepare fluorescein isothiocyanate solution: Take 0.4-0.6 mg of fluorescein isothiocyanate and add it to EP tube 2 under light-proof conditions, add 500-700 μL of dimethyl sulfoxide to adjust the concentration of fluorescein isothiocyanate, and vortex to mix to obtain a fluorescein isothiocyanate solution;

[0012] S4. Prepare a marker: add 0.1-0.4 μL of fluorescein isothiocyanate solution to the solution prepared in S2 under light-proof conditions, shake to mix, and incubate to obtain a marker;

[0013] S5. Centrifugal column equilibration: Place the centrifugal column in EP tube 3, add 350-450 μL of labeling buffer to swell for 2.5-3.5 min, then centrifuge, discard the supernatant, add 150-250 μL of labeling buffer, centrifuge, and discard the supernatant; repeat the above operation 1-2 times;

[0014] S6. Preparation of fluorescent labeled antibody: adding the marker to EP tube 3 equipped with a centrifugal column, standing at room temperature for 2.5-3.5 min, adding an indicator, centrifuging, discarding the supernatant, and obtaining the fluorescent labeled antibody;

[0015] S7. Dilution and storage of fluorescent labeled antibodies: dilute the antibody labeled product 5-10 times with labeling buffer, add labeling preservation solution, and store at 2-6℃ in the dark.

[0016] Furthermore, the antibody in S1 is an IgG antibody or an IgM antibody.

[0017] Furthermore, the antibody concentration in S1 was adjusted to 2-4 mg / mL.

[0018] Furthermore, the concentration of fluorescein thiocyanate in S3 was adjusted to 10-20 mg / mL.

[0019] Furthermore, in S4, the incubation temperature is 35-40° C. and the incubation time is 30-40 min.

[0020] Furthermore, in S5 and S6, the centrifugal treatment speed is 800-1200 r / min and the centrifugal treatment time is 1.5-2.5 min.

[0021] Compared with the prior art, the present invention has the beneficial effects that the kit of the present invention can quickly and conveniently label the antibody, and the dialysis step is not required during the labeling process, which simplifies the labeling step, saves the labeling time, and is convenient and efficient. The present invention adopts a labeling buffer prepared by a specific ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate, and ethylenediaminetetraacetic acid, wherein sodium dihydrogen phosphate and disodium hydrogen phosphate are mutually converted to maintain the pH value of the buffer in a stable range, and ethylenediaminetetraacetic acid forms a stable complex with metal ions, thereby preventing metal ions from interacting with markers or biomolecules, reducing the interference of metal ions with the experiment, thereby improving the stability of fluorescent labeled antibodies, improving their fluorescence intensity and reducing the risk of fluorescence quenching. The present invention adopts a marker preservation solution made of a specific ratio of dimethyl sulfoxide, glutathione, and vitamin E, wherein dimethyl sulfoxide prevents aggregation and precipitation by maintaining the dissolved state of the marker. Glutathione can scavenge free radicals and prevent the fluorescent labeled antibody from being damaged by oxidation reactions, thereby maintaining the stability of the fluorescent signal. Glutathione can also maintain the redox balance in fluorescently labeled antibody cells and protect fluorescently labeled antibody cells from damage by oxidative stress. Vitamin E can protect the fluorescently labeled antibody cell membrane from damage by lipid peroxidation, and protect the cell membrane integrity of cells containing fluorescent markers through its membrane antioxidant effect, preventing leakage or degradation of fluorescent markers caused by membrane damage. After scavenging lipid peroxidation free radicals, vitamin E can be regenerated by glutathione through reaction, thus forming a circulating antioxidant system. This synergistic effect enhances the stability and antioxidant capacity of the preservation solution, and further reduces the risk of fluorescence quenching. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0024] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0025] Example 1

[0026] An antibody fluorescent labeling kit includes the following raw materials: 0.4 mg fluorescein isothiocyanate, 500 μL dimethyl sulfoxide, 2.2 mL labeling buffer, 1.5 mL labeling preservation solution, 0.1 mL sodium bicarbonate solution, 1 centrifugal column, and 3 EP tubes. The labeling buffer is composed of 15 mM sodium dihydrogen phosphate, 70 mM disodium hydrogen phosphate, and 1 mM ethylenediaminetetraacetic acid. The labeling preservation solution is composed of dimethyl sulfoxide, glutathione, and vitamin E in a mass ratio of 8:1:1.

[0027] The sodium bicarbonate solution is prepared by the following method: 0.672-1.008 g of sodium bicarbonate is dissolved in 9 mL of deionized water, the pH is adjusted to 9 with sodium hydroxide, and the volume of the solution is supplemented to 10 mL with deionized water.

[0028] The above-mentioned labeling method of an antibody fluorescent labeling kit comprises the following steps:

[0029] S1. Prepare antibody solution: take 0.1 mg IgG antibody, then add 1.2 mL labeling buffer to adjust the antibody concentration to 2 mg / mL to obtain an antibody solution;

[0030] S2. Adjust the pH of the antibody solution: add the antibody solution to EP tube 1, add 10% sodium bicarbonate solution by volume of the antibody solution, and shake to mix;

[0031] S3. Prepare fluorescein isothiocyanate solution: Take 0.4 mg of fluorescein isothiocyanate and add it to EP tube 2 under light-proof conditions, add 500 μL of dimethyl sulfoxide to adjust the concentration of fluorescein isothiocyanate to 10 mg / mL, and vortex mix to obtain a fluorescein isothiocyanate solution;

[0032] S4. Prepare the marker: add 0.1 μL of fluorescein isothiocyanate solution to the solution prepared in S2 under light-proof conditions, shake and mix, and incubate at 35°C for 40 min to obtain the marker;

[0033] S5, centrifugal column equilibration treatment: place the centrifugal column into EP tube 3, add 350 μL labeling buffer to swell for 2.5 min, then centrifuge at 800 r / min for 2.5 min, discard the supernatant, add 150 μL labeling buffer, centrifuge at 800 r / min for 2.5 min, discard the supernatant; repeat the above operation once;

[0034] S6. Preparation of fluorescent labeled antibody: adding the marker to EP tube 3 equipped with a centrifugal column, standing at room temperature for 2.5 min, centrifuging at 800 r / min for 2.5 min, discarding the supernatant, and obtaining the fluorescent labeled antibody;

[0035] S7. Dilution and storage of fluorescent labeled antibodies: dilute the antibody labeled product 5 times with labeling buffer, add labeling storage solution, and store at 2°C in the dark.

[0036] Example 2

[0037] An antibody fluorescent labeling kit includes the following raw materials: 0.5 mg fluorescein isothiocyanate, 600 μL dimethyl sulfoxide, 2.8 mL labeling buffer, 2.0 mL labeling preservation solution, 0.2 mL sodium bicarbonate solution, 1 centrifugal column, and 3 EP tubes. The labeling buffer is composed of 20 mM sodium dihydrogen phosphate, 80 mM disodium hydrogen phosphate, and 2 mM ethylenediaminetetraacetic acid. The labeling preservation solution is composed of dimethyl sulfoxide, glutathione, and vitamin E in a mass ratio of 9:1.5:1.5.

[0038] The sodium bicarbonate solution was prepared by the following method: 0.840 g of sodium bicarbonate was dissolved in 9 mL of deionized water, the pH was adjusted to 9 with sodium hydroxide, and the volume of the solution was supplemented to 10 mL with deionized water.

[0039] The above-mentioned labeling method of an antibody fluorescent labeling kit comprises the following steps:

[0040] S1. Prepare antibody solution: take 0.1 mg IgG antibody, then add 1.0 mL labeling buffer to adjust the antibody concentration to 3 mg / mL to obtain an antibody solution;

[0041] S2. Adjust the pH of the antibody solution: add the antibody solution to EP tube 1, add 15% sodium bicarbonate solution by volume of the antibody solution, and shake to mix;

[0042] S3. Prepare fluorescein isothiocyanate solution: Take 0.5 mg of fluorescein isothiocyanate and add it to EP tube 2 under light-proof conditions, add 600 μL of dimethyl sulfoxide to adjust the concentration of fluorescein isothiocyanate to 15 mg / mL, and vortex mix to obtain a fluorescein isothiocyanate solution;

[0043] S4. Prepare the marker: add 0.25 μL of fluorescein isothiocyanate solution to the solution prepared in S2 under light-proof conditions, shake and mix, and incubate at 37.5°C for 35 min to obtain the marker;

[0044] S5, centrifugal column equilibration treatment: place the centrifugal column into EP tube 3, add 400 μL of labeling buffer to swell for 3.0 min, then centrifuge at 1000 r / min for 2.0 min, discard the supernatant, add 200 μL of labeling buffer, centrifuge at 1000 r / min for 2.0 min, discard the supernatant; repeat the above operation twice;

[0045] S6. Preparation of fluorescent labeled antibody: adding the marker to EP tube 3 equipped with a centrifugal column, standing at room temperature for 3.0 min, centrifuging at 1000 r / min for 2.0 min, discarding the supernatant, and obtaining the fluorescent labeled antibody;

[0046] S7. Dilution and storage of fluorescent labeled antibodies: dilute the antibody labeled product 7 times with labeling buffer, add labeling preservation solution, and store at 4°C in the dark.

[0047] Example 3

[0048] An antibody fluorescent labeling kit includes the following raw materials: 0.6 mg fluorescein isothiocyanate, 700 μL dimethyl sulfoxide, 3.0 mL labeling buffer, 2.5 mL labeling preservation solution, 0.3 mL sodium bicarbonate solution, 1 centrifugal column, and 3 EP tubes. The labeling buffer is composed of 25 mM sodium dihydrogen phosphate, 90 mM disodium hydrogen phosphate, and 3 mM ethylenediaminetetraacetic acid. The labeling preservation solution is composed of dimethyl sulfoxide, glutathione, and vitamin E in a mass ratio of 10:2:2.

[0049] The sodium bicarbonate solution was prepared by the following method: 1.008 g of sodium bicarbonate was dissolved in 9 mL of deionized water, the pH was adjusted to 9 with sodium hydroxide, and the volume of the solution was supplemented to 10 mL with deionized water.

[0050] The above-mentioned labeling method of an antibody fluorescent labeling kit comprises the following steps:

[0051] S1. Prepare antibody solution: take 0.1 mg IgM antibody, then add 0.8 mL labeling buffer to adjust the antibody concentration to 4 mg / mL to obtain an antibody solution;

[0052] S2. Adjust the pH of the antibody solution: add the antibody solution to EP tube 1, add 20% sodium bicarbonate solution by volume of the antibody solution, and shake to mix;

[0053] S3. Prepare fluorescein isothiocyanate solution: Take 0.6 mg of fluorescein isothiocyanate and add it to EP tube 2 under light-proof conditions, add 700 μL of dimethyl sulfoxide to adjust the concentration of fluorescein isothiocyanate to 20 mg / mL, and vortex mix to obtain a fluorescein isothiocyanate solution;

[0054] S4. Prepare the marker: add 0.4 μL of fluorescein isothiocyanate solution to the solution prepared in S2 under light-proof conditions, shake and mix, and incubate at 40° C. for 30 min to obtain the marker;

[0055] S5, centrifugal column equilibration treatment: place the centrifugal column into EP tube 3, add 450 μL labeling buffer to swell for 3.5 min, then centrifuge at 1200 r / min for 1.5 min, discard the supernatant, add 250 μL labeling buffer, centrifuge at 1200 r / min for 1.5 min, discard the supernatant; repeat the above operation twice;

[0056] S6. Preparation of fluorescent labeled antibody: adding the marker to EP tube 3 equipped with a centrifugal column, standing at room temperature for 3.5 min, centrifuging at 1200 r / min for 1.5 min, discarding the supernatant, and obtaining the fluorescent labeled antibody;

[0057] S7. Dilution and storage of fluorescent labeled antibodies: dilute the antibody labeled product 10 times with labeling buffer, add labeling storage solution, and store at 6°C in the dark.

[0058] Comparative Example 1

[0059] Compared with Example 2, the difference between this comparative example and Example 2 is that an antibody fluorescent labeling kit includes the following raw materials: 0.8 mg fluorescein isothiocyanate, 800 μL dimethyl sulfoxide, 5.0 mL labeling buffer, 4.5 mL labeling substance preservation solution, 0.6 mL sodium bicarbonate solution, 1 centrifugal column, and 3 EP tubes.

[0060] Comparative Example 2

[0061] The difference between this comparative example and Example 2 is that the labeling buffer consists of 15-25 mM sodium dihydrogen phosphate and 70-90 mM disodium hydrogen phosphate.

[0062] Comparative Example 3

[0063] The difference between this comparative example and Example 2 is that the marker storage solution is composed of dimethyl sulfoxide.

[0064] Comparative Example 4

[0065] The difference between this comparative example and Example 2 is that step S5 is not performed, that is, the centrifugal column is not swollen and centrifuged.

[0066] Fluorescence intensity detection

[0067] The fluorescence signal intensity of the fluorescent labeled antibodies prepared by the kits of Examples 1-3 and Comparative Examples 1-4 at 0 h and after storage for 12 months was respectively detected by a fluorescence spectrophotometer. Each Example and Comparative Example was detected 5 times, and the average value was recorded in Table 1.

[0068] Table 1

[0069] 0h fluorescence intensity Fluorescence intensity after 12 months Example 1 65370 63721 Example 2 66013 65028 Example 3 65284 63125 Comparative Example 1 45319 38790 Comparative Example 2 43512 37895 Comparative Example 3 65290 36152 Comparative Example 4 52437 50476

[0070] It can be seen from Table 1 that the fluorescent antibody prepared by the kit of the present invention has high fluorescence intensity and can still maintain strong fluorescence intensity after long-term storage, especially Example 2 has the best effect.

[0071] By comparing Example 2 with Comparative Example 1, it can be seen that the components in the kit need to cooperate with each other in a specific ratio to obtain a kit with excellent performance. Specific ratios of fluorescein isothiocyanate can efficiently bind to antibodies to form stable fluorescently labeled antibodies. If fluorescein isothiocyanate is excessive, it will cause self-quenching and reduce the fluorescence intensity; if it is insufficient, the fluorescence intensity will also decrease. Excessive dimethyl sulfoxide will affect the biological activity and stability of the fluorescently labeled antibody, thereby damaging the antibody. Therefore, each component must be within a specific ratio range to better exert its effect.

[0072] By comparing Example 2 with Comparative Example 2, the ethylenediaminetetraacetic acid in the buffer solution of Example 2 can effectively chelate metal ions to prevent metal ions from binding to fluorescein or antibodies, thereby protecting the stability and intensity of the fluorescent signal. In addition, the oxidation reaction catalyzed by metal ions will accelerate the degradation of fluorescein, resulting in the attenuation of the fluorescent signal, thereby accelerating the fluorescence quenching. By chelating metal ions, ethylenediaminetetraacetic acid helps maintain the pH and ionic strength of the reaction system, preventing the adverse effects of changes in pH or fluctuations in ionic strength caused by metal ions on the reaction.

[0073] By comparing Example 2 with Comparative Example 3, in Example 2, dimethyl sulfoxide prevents the aggregation and precipitation of the marker by maintaining the dissolved state. Glutathione can remove free radicals and prevent the fluorescent-labeled antibody from being damaged by oxidation reaction, thereby maintaining the stability of the fluorescent signal. Glutathione can also maintain the redox balance in the fluorescent-labeled antibody cell and protect the fluorescent-labeled antibody cell from oxidative stress. Vitamin E can protect the fluorescent-labeled antibody cell membrane from damage by lipid peroxidation, and protect the cell membrane integrity of cells containing fluorescent markers through its membrane antioxidant effect, preventing leakage or degradation of fluorescent markers caused by membrane damage. After removing lipid peroxidation free radicals, vitamin E can be regenerated by glutathione through reaction, thereby forming a circulating antioxidant system. This synergistic effect enhances the stability and antioxidant capacity of the preservation solution, and further reduces the risk of fluorescence quenching. Therefore, dimethyl sulfoxide, glutathione, and vitamin E can better improve the stability and antioxidant capacity of the fluorescent-labeled antibody through joint action and synergistic effect, thereby reducing the risk of fluorescence quenching, and therefore can maintain the high fluorescence intensity of the fluorescent-labeled antibody for a long time, which is particularly important for samples that need long-term storage and subsequent analysis.

[0074] By comparing Example 2 with Comparative Example 4, Example 2 uses a labeling buffer solution to swell the centrifugal column and centrifuge it twice, which can effectively remove impurities, residual proteins, DNA fragments and other substances that may interfere with the labeling reaction in the centrifugal column, reduce the possibility of binding with fluorescent antibodies in subsequent steps, thereby reducing the generation of nonspecific fluorescent signals, and helping to improve the authenticity and accuracy of fluorescence intensity. Reducing the presence of nonspecific binding substances also helps to reduce the risk of fluorescence quenching. Swelling and two centrifugation treatments help to make the filler in the centrifugal column more uniform and loose, improve its adsorption and release ability for the target substance, and can more effectively capture and purify fluorescently labeled antibodies, reduce the interference of nonspecific binding, and thus improve labeling efficiency.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody fluorescent labeling kit, characterized in that: Including the following ingredients: 0.5 mg fluorescein isothiocyanate, 600 μL dimethyl sulfoxide, 2.8 mL labeling buffer, 2.0 mL labeling preservation solution, 0.2 mL sodium bicarbonate solution, 1 centrifugal column, 3 EP tubes; wherein the labeling buffer is composed of 20 mM sodium dihydrogen phosphate, 80 mM disodium hydrogen phosphate, and 2 mM ethylenediaminetetraacetic acid, and the labeling preservation solution is composed of dimethyl sulfoxide, glutathione, and vitamin E in a mass ratio of 9:1.5:1.

5. The labeling method of the antibody fluorescent labeling kit comprises the following steps: S1. Prepare antibody solution: take 0.1 mg of antibody, add 0.8-1.2 mL of labeling buffer to adjust the antibody concentration, and obtain antibody solution; S2. Adjust the pH of the antibody solution: add the antibody solution to EP tube 1, add 10-20% of the volume of the antibody solution sodium bicarbonate solution, and shake to mix; S3. Prepare fluorescein isothiocyanate solution: Take 0.5 mg of fluorescein isothiocyanate and add it to EP tube 2 under light-proof conditions, add 500-700 μL of dimethyl sulfoxide to adjust the concentration of fluorescein isothiocyanate, and vortex to mix to obtain a fluorescein isothiocyanate solution; S4. Prepare a marker: add 0.1-0.4 μL of fluorescein isothiocyanate solution to the solution prepared in S2 under light-proof conditions, shake to mix, and incubate to obtain a marker; S5, centrifugal column equilibration treatment: place the centrifugal column into EP tube 3, add 350-450 μL labeling buffer to swell for 2.5-3.5 min, then centrifuge, discard the supernatant, add 150-250 μL labeling buffer, centrifuge, discard the supernatant; repeat S5 operation 1-2 times; S6. Preparation of fluorescent labeled antibody: adding the marker to EP tube 3 equipped with a centrifugal column, standing at room temperature for 2.5-3.5 min, centrifuging, discarding the supernatant, and obtaining the fluorescent labeled antibody; S7. Dilution and storage of fluorescent labeled antibodies: dilute the antibody labeled product 5-10 times with labeling buffer, add labeling preservation solution, and store at 2-6℃ in the dark.

2. An antibody fluorescent labeling kit according to claim 1, characterized in that: The sodium bicarbonate solution is prepared by the following method: 0.672-1.008 g of sodium bicarbonate is dissolved in 9 mL of deionized water, the pH is adjusted to 9 with sodium hydroxide, and the volume of the solution is supplemented to 10 mL with deionized water.

3. The antibody fluorescent labeling kit according to claim 1, wherein the labeling method is characterized in that: The antibody in S1 is an IgG antibody or an IgM antibody.

4. The antibody fluorescent labeling kit according to claim 1, wherein the labeling method is characterized in that: The antibody concentration in the S1 was adjusted to 2-4 mg / mL.

5. The antibody fluorescent labeling kit according to claim 1, wherein the labeling method is characterized in that: The concentration of fluorescein thiocyanate in the S3 was adjusted to 15 mg / mL.

6. The antibody fluorescent labeling kit according to claim 1, wherein the labeling method is characterized in that: In the S4, the incubation temperature is 35-40° C. and the incubation time is 30-40 min.

7. The antibody fluorescent labeling kit according to claim 1, wherein the labeling method is characterized in that: In S5 and S6, the centrifugal treatment speed is 800-1200 r / min and the centrifugal treatment time is 1.5-2.5 min.