Allophycocyanin Fluorescent Tandem Dye, Preparation Method Thereof and Method for Coupling with Antibody

Abstract

The present invention relates to the field of immunology technology, and more particularly to a fluorescent protein tandem dye and a method for labeling a conjugated monoclonal antibody therewith, as well as a kit. The tandem dye is composed of two fluorescent molecules, with a macromolecular protein dye APC serving as a donor and a small molecule organic dye serving as an acceptor, the two being covalently bonded. After the tandem dye is combined, the donor dye APC can be excited by a laser emitted by an exciter, while the small molecule acceptor dye cannot. The energy emitted by the donor dye APC after being excited is transferred to the acceptor dye, causing the acceptor dye to excite light of a specific wavelength. The tandem dye has the excitation spectrum of the donor dye APC and the emission spectrum of the small molecule acceptor dye, becoming a unique fluorescent dye that is completely different from the donor dye APC and the acceptor dye and has a relatively large Stokes shift.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a method for forming a tandem dye by serially connecting allophycocyanin and a fluorescent dye, and a method for coupling the tandem dye to an antibody. Background Art

[0002] Phycobiliproteins are light-harvesting pigment proteins found in algae such as cyanobacteria and red algae. They are multi-subunit protein complexes. Phycobiliproteins covalently bind to one or more phycobilipigments, resulting in strong fluorescence and can be used as fluorescent probes in immunofluorescence assays. Compared to other immunolabeling techniques such as radioimmunoassay and enzyme-linked immunosorbent assays, immunofluorescence has become a hot topic of research due to its high sensitivity and safety.

[0003] Immunofluorescence technology is also known as fluorescent antibody probe technology. Fluorescent antibodies are antibodies labeled with fluorescent dyes to make fluorescent-labeled antibody solutions. Fluorescent-labeled antibodies and specific antigens undergo antigen-antibody specific reactions. Fluorescence microscopy technology can be used to very sensitively observe the distribution of antigen-antibody complexes in tissues, and flow cytometry technology can be used to analyze the expression of specific antigens.

[0004] However, in most cases, the detection limit of fluorescence determination is often affected by the background fluorescence in serum and other biological samples. Taking serum as an example, the background fluorescence in the 400-600 nm band overlaps with the fluorescence emission spectrum of commonly used fluorescent markers (such as FITC), causing excessive interference and affecting the sensitivity of detection. Summary of the Invention

[0005] The present invention connects fluorescent protein and small molecule dye in series, so that the Stoke's shift of the fluorescent protein tandem dye is significantly increased, which makes the tandem dye have the advantages of low background interference, strong penetration into biological sample points, and high detection sensitivity in near-infrared imaging technology. Specifically, the present invention provides a tandem dye obtained by coupling allophycocyanin APC with a small molecule dye, which is composed of two fluorescent molecules, with the macromolecular protein dye APC as the donor and the small molecule organic dye as the acceptor, and the two are bound by a covalent bond. After the tandem dye is combined, the donor dye APC can be excited by the laser emitted by the exciter, while the small molecule acceptor dye cannot. The energy emitted by the donor dye APC after being excited is transferred to the acceptor dye, causing the acceptor dye to excite light of a specific wavelength. The tandem dye has the excitation spectrum of the donor dye APC and the emission spectrum of the small molecule acceptor dye, becoming a unique fluorescent dye completely different from the donor dye APC and the acceptor dye and having a relatively large Stoke's shift.

[0006] The present invention adopts the following technical solutions:

[0007] An allophycocyanin fluorescent tandem dye is prepared by reacting allophycocyanin with a cross-linking agent and then reacting with a small molecule dye to obtain the allophycocyanin fluorescent tandem dye.

[0008] A method for coupling an allophycocyanin fluorescent tandem dye to an antibody comprises the following steps: reacting allophycocyanin with a cross-linking agent, and then reacting with a small molecule dye to obtain an allophycocyanin fluorescent tandem dye; and then coupling the allophycocyanin fluorescent tandem dye to a modified antibody to complete the allophycocyanin fluorescent tandem dye-coupled antibody.

[0009] The existing technology requires first protecting the fluorescent protein, then connecting the fluorescent protein and the dye in series to prepare a tandem dye, and then cross-linking with a cross-linker before coupling with a modified antibody. The present invention adopts a different technical approach, first using a cross-linker to directly cross-link the fluorescent protein, then connecting it in series with a small molecule dye, and finally coupling it with the modified antibody. Compared with the existing technology, the present invention omits the subsequent processing steps of the tandem dye, avoiding the unstable degradation of the tandem dye itself, while simplifying the reaction conditions and greatly improving the reaction efficiency.

[0010] In the present invention, allophycocyanin is reacted with a cross-linker, amino groups are introduced, and then reacted with a small molecule dye to obtain an allophycocyanin fluorescent tandem dye. Specifically, allophycocyanin is reacted with a cross-linker, carboxyl groups are activated, amino groups are introduced, and then reacted with a small molecule dye to obtain an allophycocyanin fluorescent tandem dye. Preferably, the carboxyl groups are activated using EDC, and the amino groups are introduced using di-Gly peptide. Further preferably, the reaction concentration of allophycocyanin is 5 mg / ml, and the working concentration of di-Gly peptide is 5-50 mM. This retains the amino reactive sites, does not affect the subsequent binding efficiency of the small molecule dye to the amino groups on the NHS ester, and simultaneously stabilizes the allophycocyanin.

[0011] In the present invention, the amount of the cross-linking agent is 30 to 100 equivalents, preferably 30 to 50 equivalents, of the molar amount of allophycocyanin APC, and the cross-linking agent is a hetero-bifunctional cross-linking agent; preferably, SPDP (3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester) and its analogs or SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) and its analogs are used as hetero-bifunctional cross-linking agents to directionally couple to the amino site on the fluorescent protein APC, while the carboxyl site on the fluorescent protein APC is activated, while the maleimide group end on the coupling agent is retained, and directionally coupled to the thiol end of the reduced monoclonal antibody IgG, thereby achieving directional coupling of the monoclonal antibody and the fluorescent protein-small molecule tandem dye, and improving the specificity of the reaction between the fluorescent protein tandem dye and the monoclonal antibody.

[0012] In the present invention, antibody modification involves the use of a reducing agent. Preferably, the reducing agent is a novel bioreducing agent, a dithioaminoalkane, such as (S)-2-aminobutane-1,4-dithiol hydrochloride (DTBA), also known as dithiobutylamine, 3-aminopentane-1,5-dithiol hydrochloride, or 2,3-diaminobutane-1,4-dithiol hydrochloride. These reducing agents exhibit superior performance compared to other commonly used reducing agents. Compared to commonly used agents such as dithiothreitol (DTT) and 2-mercaptoethanol (BME), these reducing agents reduce small molecule disulfides 3-5 times faster than DTT. Furthermore, given the structural properties of the reducing agent itself, antibodies modified with dithioaminoalkane can be stored for approximately 7 days, while antibodies reduced with DTT or BME can only maintain their stability for 1-3 days.

[0013] The present invention aims to provide a method for linking allophycocyanin (APC) to a fluorescent dye to form a tandem dye. This method also involves using this tandem dye to label an antibody, resulting in a comprehensive solution for producing APC-tandem-dye-labeled antibodies. These antibodies can be used for both immunofluorescence assays and flow cytometry. The raw materials involved include antibodies (preferably monoclonal antibodies), allophycocyanin (preferably in liquid solution), small molecule fluorescent dyes, crosslinkers, reducing agents, stabilizers, buffers, and more. Specifically involved are monoclonal antibodies (IgG); fluorescent proteins (APC); small molecule dyes, such as Sulfo-cy7 SE, Sulfo-cy5.5 SE, YF750 SE and other similar small molecule fluorescent dyes; crosslinkers Sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt), SMCC, or SMCC analogs, such as GMBS (4-Maleimidobutyric acid N-hydroxysuccinimide ester, N-γ-maleimidobutyryl-oxysuccinimide ester), 5-Maleimidovaleric acid-NHS (5-maleimidovaleric acid-NHS), N-Succinimidyl 6-maleimidohexanoate (6-(maleimido)hexanoic acid succinimide ester), LC-SPDP (succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (succinimidyl 3-(2-pyridyldithio)propionate), Sulfo-LC-SPDP (sulfosuccinimidyl Bifunctional cross-linkers include (S)-2-aminobutane-1,4-dithiol hydrochloride (DTBA), also known as dithiobutylamine, 3-aminopentane-1,5-dithiol hydrochloride, 2,3-diaminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), 2-mercaptoethanol (BME), and tris-2-carboxyethylphosphine hydrochloride (TCEP); agents for enhancing stability include carbodiimide hydrochloride (EDC), diglycine (Gly-Gly), Cysteine, bovine serum albumin (BSA), EDTA, Na3N, and trehalose; and buffers include pH 7.2 PBS, pH 8.4 NaHCO3, and pH 5.4 MES.

[0014] Compared with the prior art, the positive effects of the present invention are as follows:

[0015] Existing fluorescent protein-dye usually requires multiple treatments to modify the fluorescent protein before it can be coupled to the antibody. However, the present invention first cross-links the fluorescent protein, then connects the small molecule dye to the fluorescent protein in series, and then directly couples it with the desired modified monoclonal antibody. This omits the steps of multiple treatments to modify the fluorescent protein before coupling with the antibody, reduces the degradation of the tandem dye, and the application effect of the tandem dye-labeled antibody of the present invention is excellent, even better than existing products.

[0016] Tandem dyes bring about spectral shifts: Taking the APC-cy7 conjugated dye as an example, the fluorescent protein APC alone has an Ex / Em ratio of 652 / 660 nm, while the small molecule dye cy7 alone has an Ex / Em ratio of 745 / 774 nm. When the tandem dye APC-cy7 is directional coupled to a monoclonal antibody, the fluorescence resonance energy transfer (FRET)-induced Stokes shift reaches as high as 132 nm (Ex / Em = 652 / 784 nm). A larger Stokes shift shifts the emitted fluorescence closer to the red end of the visible spectrum, facilitating the analysis of biochemical samples by significantly reducing fluorescence interference from red-region biochemical matrices and serum components, thereby improving detection sensitivity. Furthermore, due to the large Stokes shift, this tandem dye can also be used for multicolor labeling, enabling simultaneous multicomponent analysis with distinctly differentiated fluorescence colors, enabling rapid, simple, and reliable implementation in applications such as cell typing.

[0017] The present invention not only solves the problem that the existing method for preparing tandem dyes is complicated and may cause the tandem dyes to degrade, but also solves the problem that the tandem dyes prepared by the existing method are themselves easily degraded. The tandem dyes obtained by the present invention have the advantages of high specificity and good stability, omitting the subsequent processing steps of the tandem dyes, avoiding the unstable degradation of the tandem dyes themselves, and simplifying the reaction conditions, thereby greatly improving the reaction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the specific operation process of the allophycocyanin fluorescent tandem dye and coupled antibody of the present invention.

[0019] Figure 2 The fluorescence spectra of the single fluorescent protein dye APC (red, upper figure), the small molecule dye cy7 (blue, upper figure) and the fluorescence emission spectrum of the tandem dye APC-cy7 (red, lower figure) in the present invention.

[0020] Figure 3These are the absorption spectra of the single dye APC (a, blue), the tandem dye APC-CY5.5 (b, red), and the tandem dye APC-YF750 (c, yellow) of the present invention.

[0021] Figure 4 The present invention provides an operating procedure for labeling a monoclonal antibody using a tandem dye kit.

[0022] Figure 5 This is an immunofluorescence labeling detection diagram of the monoclonal antibody mouse secondary antibody labeled with the tandem dye APC-cy7 kit of the present invention.

[0023] Figure 6 This is a fluorescence labeling detection diagram of the monoclonal antibody rabbit secondary antibody labeled with the tandem dye APC-cy7 kit of the present invention.

[0024] Figure 7 This is the flow cytometry result of direct staining of suspended cells using the tandem dye APC-cy7 kit labeled with primary antibody CD45.

[0025] Figure 8 The mouse secondary antibody was labeled with the tandem dye APC-cy7 kit of the present invention, and CD45 was used as the primary antibody to indirectly stain the suspended cells K562, followed by immunofluorescence detection.

[0026] Figure 9 APC-cy7-IgG stained cells labeled with the present invention and competitors.

[0027] Figure 10 The flow cytometry results of the APC-cy7-CD45 antibodies labeled with the present invention and the competitor are shown. DETAILED DESCRIPTION

[0028] The reagents used in the present invention are all conventional products in the field, and the specific experimental operations and testing methods are conventional techniques in the field.

[0029] The absorbance (A) at different wavelengths was measured using a Shimadzu UV-1800 spectrophotometer. Taking SMCC modified with APC as an example, 488-SPDP was reacted with the modified APC to determine its modification capacity, i.e., DOL value. The DOL value was calculated according to the formula:

[0030]

[0031] aC refers to the collected APC-488 SPDP concentration;

[0032] b. Dilution factor refers to the dilution multiple during photometric measurement;

[0033] cA 652 and A 496They refer to the absorbance at 652 nm and the absorbance at 496 nm, respectively;

[0034] d. 0.1 refers to the correction factor of 488-SPDP, C f value;

[0035] e.6.7 refers to the extinction coefficient of APC (-MAL).

[0036]

[0037] a. DOL refers to the reaction efficiency of APC and SMCC;

[0038] b.A 496 Refers to the absorbance at 496nm;

[0039] c. 105000 refers to the molecular weight of APC (-MAL);

[0040] d. Dilution factor refers to the dilution multiple during photometric measurement;

[0041] e. 70000 refers to the molar absorptivity (ε value) of 488-SPDP;

[0042] C refers to the collected APC-488 SPDP concentration.

[0043] The specific operation process of the allophycocyanin fluorescent tandem dye and coupled antibody of the present invention can be found in Figure 1 , including pretreatment of allophycocyanin before tandem and blocking of amino sites; pretreatment of allophycocyanin into NHS ester with stable amino-reactive activity; tandem treatment of phycocyanin with amino-reactive NHS ester and small molecule fluorescent dye; thiol reaction of target antibody; cross-linking reaction of tandem dye and target antibody.

[0044] In order to more clearly illustrate the technical solution of the present invention in the prior art, an example of the use of the present invention will be briefly described below.

[0045] Example 1

[0046] (1) APC is commercially available in the form of salt. Place the fluorescent protein APC in a 1.5 mL EP tube, add 10 mM pH 7.2 PBS buffer, vortex and mix thoroughly, centrifuge at 9000 rpm for 5 min, and repeat this operation three times for desalting. Collect the desalted allophycocyanin APC solution and adjust the phycocyanin concentration to 2.5 mg / mL with 10 mM pH 7.2 PBS buffer.

[0047] (2) The cross-linking agent SMCC was added to the phycocyanin buffer at a ratio of 35 equivalents to the molar amount of allophycocyanin, and the coupling reaction was carried out at room temperature for 2 hours. Then, the mixture was washed by conventional ultrafiltration with PBS buffer, and then resuspended with pH 5.6 0.1M MES buffer, and then ultrafiltered. The reaction liquid in the ultrafiltration tube was collected to obtain modified allophycocyanin (APC-MAL), which was in a pH 5.6 0.1M MES buffer environment. At this time, the N-hydroxysuccinimide (NHS) ester at one end of the heterobifunctional cross-linking agent was covalently coupled to the amino group of the fluorescent protein APC, and the maleimide group remained at the other end, which could retain the covalent coupling with the sulfhydryl end of the subsequent antibody molecule.

[0048] (3) 10 equivalents of EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) were added to a 5 mg / mL modified allophycocyanin in MES buffer (0.1 M pH 5.6) and reacted at room temperature for 1 hour. The carboxyl group of APC was activated by EDC. Then 12 equivalents of N-hydroxysulfosuccinimide (sulfo-NHS) and 11 equivalents of diglycerides were added and reacted at room temperature for 1 hour. After the reaction, PBS at pH 7.2 was added to terminate the reaction. Then, conventional ultrafiltration was performed to obtain amino allophycocyanin. An amino group that can react with the subsequent Sulfo-Cy7 SE was introduced and no precipitation was produced after protein denaturation. The molar amount of modified allophycocyanin was taken as 1 equivalent.

[0049] (4) The aminophycocyanin was diluted to a concentration of 2.5 mg / ml using the buffer solution in Table 1. Then, the small molecule dye Sulfo-Cy7 SE was added according to 30 molar equivalents of aminophycocyanin and reacted at room temperature. The small molecule dye was conjugated to the large molecule fluorescent protein dye APC to form a tandem dye in the form of APC-dye. After the reaction, conventional ultrafiltration was performed to wash the supernatant to obtain the APC tandem dye APC-cy7 (APC-cy7-SMCC). The reaction efficiency in different buffer environments is shown in Table 1. After replacing the commonly used pH 7.2 PBS buffer environment with a pH 8.4 NaHCO3 buffer environment, the reaction time was greatly shortened and the coupling efficiency of the dye to the fluorescent protein was greatly improved.

[0050]

[0051] Figure 2 The fluorescence spectra of the single fluorescent protein dye APC (red, upper figure), the small molecule dye cy7 (blue, upper figure) and the fluorescence emission spectrum of the tandem dye APC-cy7 (red, lower figure) in the present invention.

[0052] Figure 3These are the absorption spectra of the single dye APC (a, blue), the tandem dye APC-CY5.5 (b, red), and the tandem dye APC-YF750 (c, yellow) of the present invention.

[0053] The small molecule dye Sulfo-Cy7 SE is replaced with Sulfo-Cy5.5 SE or YF750 SE, and the reaction is carried out at room temperature for 15 to 60 minutes. The small molecule dye is connected in series to the large molecule fluorescent protein dye APC to form more tandem dyes in the form of APC-dye.

[0054] Example 2

[0055] Monoclonal antibody thiolation: Dithioaminoalkanes (such as or one of the following: (S)-2-aminobutane-1,4-dithiol hydrochloride (DTBA), also known as dithiobutylamine, 3-aminopentane-1,5-dithiol hydrochloride, or 2,3-diaminobutane-1,4-dithiol hydrochloride) are highly effective thiol reducing agents that can specifically and efficiently reduce disulfide bonds in monoclonal antibody IgG to sulfhydryl (-HS) forms. These disulfide bonds can then be coupled to the maleimide end of the tandem dye APC-cy7-SMCC heterobifunctional bond (SMCC), forming a stable sulfhydryl bond at a pH of 6.5-7.5. Cysteine hydrochloride is a sulfur-containing amino acid that provides disulfide bonds in proteins. Cys residues are used to block excess sulfhydryl sites on the fluorescent protein APC.

[0056] Taking the modification of 1mg IgG antibody as an example, the specific modification steps are as follows:

[0057] Take 1mg of IgG antibody and put it into a 30K ultrafiltration tube. Add pH7.2 PBS to wash and desalt, and repeat the washing three times at 9000rpm for 5min. Then add 40eq DTBA (2mM) and add pH7.2 PBS buffer to the final reaction concentration of IgG to 1mg / ml. React at room temperature (25℃) and 1000rpm for 1h to modify IgG. After the reaction is completed, collect the reaction liquid into a 30K ultrafiltration tube, centrifuge at 9000rpm for 5min, concentrate the reduced IgG product, and ultrafilter to remove unreacted DTBA. After centrifugation, pour out the waste liquid in the collection tube, add pH7.2 PBS buffer to the ultrafiltration tube to resuspend the reaction liquid, centrifuge at 9000rmp for 5min to remove unreacted DTBA, wash three times and collect the reaction liquid in the ultrafiltration tube.

[0058] 30 μl of the reaction solution containing the modified and reduced antibody IgG was mixed with 0.4 μl of Flourescen-5-Maleimide (5 mM) and added to a pH 7.2 PBS buffer to bring the volume to 20 μl. The reaction was incubated at 25°C, 1000 rpm, for 2 hours to determine the ability to reduce the antibody IgG. The test results are shown in Table 2. Two conventional reducing agents were also used for comparison under different reaction times. Table 2 compares the reducing abilities of the different reducing agents used in the present invention: DTBA, BME, and DTT, under the same conditions, as well as the stability test results of the modified antibody (treated at 25°C for 60 minutes) and stored at 4°C for different periods of time.

[0059]

[0060] Example 3 Labeling method of monoclonal antibody using tandem dye kit of the present invention

[0061] The immunofluorescence detection kit of the present invention contains 4 components, namely:

[0062] Component A: APC tandem dye APC-cy7; Component B: antibody linking modifier DTBA (Linking Reagent); Component C: Tris buffer, neutral; Component D: ultrafiltration tube (MWCO = 10KDa).

[0063] Specific marking test methods such as Figure 4 , the specific steps are as follows:

[0064] The thiol-modified target monoclonal antibody (the reaction solution collected in the ultrafiltration tube in Example 2) was added to component B and reacted at 25°C for 1 hour. The excess component B was then removed by ultrafiltration using the ultrafiltration tube of component D, resulting in a final antibody concentration of 1 mg / mL. The antibody was then dissolved and mixed with component A using PBS as the solvent, and the coupling reaction was carried out at 25°C for 4 hours to ensure full coupling. After the coupling was completed, 1 / 10 volume of component C buffer was added to the reaction solution to obtain the APC tandem dye-labeled monoclonal antibody. The target antibody was then detected and analyzed by detecting the fluorescence of the tandem dye.

[0065] The same analysis was performed by replacing component A with other dyes to obtain monoclonal antibodies labeled with different dyes.

[0066] Figure 5 This is an immunofluorescence labeling detection diagram of the monoclonal antibody mouse secondary antibody labeled with the tandem dye APC-cy7 kit of the present invention. Figure 6 This is a fluorescence labeling detection diagram of the monoclonal antibody rabbit secondary antibody labeled with the tandem dye APC-cy7 kit of the present invention. The detection instrument is a NIB900 fluorescence microscope.

[0067] Figure 7This is the flow cytometry result of direct staining of suspended cells using the tandem dye APC-cy7 kit labeled with primary antibody CD45. Figure 8 The present invention's tandem dye APC-cy7 kit was used to label mouse secondary antibodies, and CD45 was used as the primary antibody to indirectly stain K562 cells in suspension, followed by immunofluorescence detection. The detection instrument was an Attune Nxt flow cytometer.

[0068] Example 4 Comparison of the Effects of Antibody Labeling with the Tandem Dye Kit of the Present Invention and Competitor's Kit

[0069] The competitive product was the commercially available product with the best current industrial application results. The present invention labeled the antibody according to the labeling method described in Example 3, and the competitive product (Abcam-ab102859) was labeled according to the instructions in the competitive company's kit instructions, respectively, for the monoclonal mouse secondary antibody and CD45 primary antibody. The present invention modified the antibody with thiol groups according to Example 2, and the competitive product processed the antibody according to the instructions in the competitive company's kit instructions. The two labeled APC-cy7-IgG secondary antibodies were used to indirectly stain suspended K562 cells using CD45 as the primary antibody, and microscopic images were taken for comparison. The two labeled APC-cy7-CD45 antibodies were then used to directly stain K562 cells for flow cytometry analysis.

[0070] The results showed that the APC-cy7-IgG stained cells labeled with the present invention and the competitor did not cause nonspecific staining (no fluorescence in the negative group), but the fluorescence brightness of the positive group of the present invention was significantly better than that of the competitor ( Figure 9 The APC-cy7-CD45 antibody labeled by the present invention also showed significantly better discrimination than competing products in flow cytometry, with more obvious differentiation between negative and positive groups ( Figure 10 ).

[0071] In existing tandem dye-labeled antibody schemes, a fluorescent protein is first coupled with a small molecule dye to obtain a coupled protein dye, such as PE-Cy5. After desalting, the coupled protein reacts with a thiol blocking agent NEM to block the thiol groups on the coupled protein. After the thiol groups are blocked, the amino groups on the coupled protein react with a crosslinker to obtain a coupled protein dye with a crosslinker. The crosslinker S-LC-SPDP is first crosslinked with a monoclonal antibody. After the monoclonal antibody and the crosslinker are crosslinked, the disulfide bonds in the monoclonal antibody are reduced using the reducing agent TCEP. The thiol-modified monoclonal antibody is then subjected to a directed coupling reaction with the crosslinked coupled protein. After the reaction is complete, the fluorescent-labeled antibody dye is obtained by treating with NEM, Cys, and PBS. In the scheme of tandem dye-labeled antibodies of the present invention, the amino group on the fluorescent protein directly reacts with the crosslinker SMCC to form a crosslinked fluorescent protein dye; the fluorescent protein is modified with NHS, EDC, etc. to form a stable functional group that can be directly coupled with small molecule dyes such as Cy7 and Cy5.5; the crosslinked fluorescent protein is coupled with the small molecule dye to form a coupled dye that can be directionally crosslinked with the sulfhydryl group; the reducing agent DTBA reduces the disulfide bonds in the monoclonal antibody to form a sulfhydryl monoclonal antibody; the sulfhydryl monoclonal antibody directly reacts with the crosslinked coupled protein in a directionally coupled manner; after the reaction is complete, Cys is added for treatment to obtain a fluorescently labeled antibody dye. Existing methods for preparing tandem dyes are complex and can cause the tandem dyes to degrade. The tandem dyes prepared by the existing methods are solved, and the problem that the tandem dyes themselves are easily degraded is solved. The tandem dyes obtained by the present invention have the advantages of high specificity and good stability, omitting the subsequent processing steps of the tandem dyes, avoiding the unstable degradation of the tandem dyes themselves, simplifying the reaction conditions, and greatly improving the reaction efficiency.

Claims

1. A method for preparing an allophycocyanin fluorescent tandem dye, characterized in that: Allophycocyanin is reacted with a heterobifunctional crosslinker, and then the carboxyl group is activated, and then the amino group is introduced, and then the allophycocyanin is reacted with a small molecule dye to obtain an allophycocyanin fluorescent tandem dye; SPDP or its analogs, or SMCC or its analogs is used as the heterobifunctional crosslinker; the amount of the heterobifunctional crosslinker is 30 to 100 equivalents of the molar amount of allophycocyanin.

2. The allophycocyanin fluorescent tandem dye prepared according to the method for preparing the allophycocyanin fluorescent tandem dye according to claim 1.

3. The method of using the allophycocyanin fluorescent tandem dye-coupled antibody according to claim 2, characterized in that: The method comprises the following steps: coupling the allophycocyanin fluorescent tandem dye with the modified antibody to complete the allophycocyanin fluorescent tandem dye-coupled antibody; the modified antibody is an antibody modified with a reducing agent.

4. An immunofluorescence detection kit comprising a dye, characterized in that: The dye is the allophycocyanin fluorescent tandem dye according to claim 2.

5. A method for preparing an antibody for immunofluorescence detection, characterized in that: The method comprises the following steps: coupling the allophycocyanin fluorescent tandem dye according to claim 2 with the modified antibody to complete the allophycocyanin fluorescent tandem dye-coupled antibody; and then performing fluorescence detection to complete the immunofluorescence detection of the antibody; The modified antibody is an antibody modified with a reducing agent.

6. Use of the allophycocyanin fluorescent tandem dye according to claim 2 as an immunofluorescence detection reagent.

7. Use of the allophycocyanin fluorescent tandem dye according to claim 2 in preparing an immunofluorescence detection reagent.

8. Use of the allophycocyanin fluorescent tandem dye according to claim 2 or the immunofluorescence detection kit according to claim 4 in the preparation of conjugated antibodies or immunofluorescence detection antibodies.

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