A pe / florescent tandem dye, its preparation method and a method for labeling antibodies

Abstract

The application discloses a PE / fluorescent tandem dye, a preparation method thereof and a method for labeling an antibody, and the free thiol group on phycoerythrin is blocked; then small-molecule organic dye tandem treatment is carried out; then protein protection treatment is carried out on the obtained tandem dye; finally, activation treatment is carried out by using a crosslinking agent; and a PE / fluorescent tandem dye is obtained. When the antibody is labeled, firstly, thiolation treatment is carried out on the target antibody by using a reducing agent; then the thiolated target antibody is crosslinked with the activated tandem dye; then a blocking agent is added into the crosslinked tandem dye-antibody system for dye blocking treatment, and the fluorescent antibody is preserved, and the antibody labeling is completed. The PE and the small-molecule organic dye are combined by a covalent bond, the donor PE is excited by laser emitted by an exciter, the energy emitted after excitation is transferred to the small-molecule organic dye, the small-molecule organic dye is excited to emit light of a specific wavelength, and the PE / fluorescent tandem dye is a fluorescent dye with a unique fluorophore.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically relating to a method for preparing a phycoerythrin tandem fluorescent dye and a method for labeling antibodies with this tandem dye. Background Technology

[0002] Phycoerythrin (PE) is a widely used protein-based fluorescent dye isolated and purified from red algae. Like other phycobiliproteins, phycoerythrin emits strong fluorescence under specific wavelength excitation, with an intensity 30-100 times that of fluorescein. It exhibits excellent absorption properties and a high quantum yield, with a wide excitation and emission range in the visible spectrum. Phycoerythrin and its tandem dyes are important fluorescent proteins widely used in flow cytometry. Current techniques often employ the method of combining phycoerythrin and its tandem dyes with antibodies, biotin, avidin, immunoproteins, etc., to create fluorescent probes. The emitted fluorescence is then detected for multicolor fluorescence analysis, cell surface antigen detection, and analysis of biomolecules such as proteins or nucleic acids. This is commonly used in immunofluorescence detection platforms such as fluorescence microscopy and flow cytometry. However, existing phycoerythrin tandem dyes for antibody labeling have low labeling efficiency, are prone to antibody denaturation, result in poor separation between negative and positive groups, and exhibit weak fluorescence intensity in the positive group. Summary of the Invention

[0003] This invention belongs to the field of immunology technology, specifically relating to a method for preparing a phycoerythrin tandem fluorescent dye and a method for labeling antibodies with this tandem dye.

[0004] The present invention adopts the following technical solution:

[0005] A PE / fluorescent tandem dye, the preparation method of which includes the following steps:

[0006] (1) The thiol-blocked phycoerythrin was reacted with a small molecule organic dye to obtain dye / PE;

[0007] (2) After activating the carboxyl groups on the dye / PE, react with a stabilizer to obtain a stable dye / PE;

[0008] (3) React the stable dye / PE with the crosslinking agent to obtain PE / fluorescent tandem dye.

[0009] This invention discloses a flow cytometry antibody labeling kit, which includes the above-mentioned PE / fluorescent tandem dye, as well as antibody modifiers, antibody preservation solutions, filter tubes, etc.

[0010] This invention discloses a method for labeling antibodies using the above-mentioned PE / fluorescent tandem dye, wherein the above-mentioned PE / fluorescent tandem dye is reacted with thiolized antibody to complete antibody labeling.

[0011] This invention discloses the application of the above-mentioned PE / fluorescent tandem dye or the above-mentioned PE / fluorescent tandem dye labeled antibody in the preparation of fluorescent probes.

[0012] This invention involves blocking the free thiol groups on phycoerythrin; then subjecting it to tandem treatment with a small molecule organic dye; followed by protein protection treatment of the obtained tandem dye; and finally activation treatment with a cross-linking agent to obtain a PE / fluorescent tandem dye. For antibody labeling, the target antibody is first thiolated with a reducing agent; then the thiolated target antibody is cross-linked with the activated tandem dye; finally, a blocking agent is added to the cross-linked tandem dye-antibody system for dye blocking treatment, and the fluorescent antibody is preserved, thus completing antibody labeling.

[0013] In this invention, the thiol groups of phycoerythrin are blocked using a blocking agent to obtain thiol-blocked phycoerythrin; the molar ratio of blocking agent to phycoerythrin is 1:(50-80); the reaction temperature during blocking is 0-35°C and the time is 1-25 hours, for example, the reaction temperature during blocking is 15-35°C and the time is 1-3 hours, or the reaction temperature during blocking is 4°C and the time is 10-20 hours. After the blocking reaction is completed, conventional ultrafiltration is performed.

[0014] In this invention, the molar ratio of thiol-blocked phycoerythrin to small molecule organic dye is 1:(10-30); the reaction temperature of thiol-blocked phycoerythrin and small molecule organic dye is 15-35℃, and the reaction time is 10-60 minutes. After the reaction, conventional ultrafiltration is performed.

[0015] In this invention, EDC and NHS are used to activate the carboxyl groups on the dye / PE; the ratio of dye / PE to stabilizer is (2.5-5) g : (0.1-1) mol; when the carboxyl-activated dye / PE reacts with the stabilizer, the reaction temperature is 0-35℃ and the time is 1-25 hours. For example, when the carboxyl-activated dye / PE reacts with the stabilizer, the reaction temperature is 15-35℃ and the time is 1-5 hours, or the reaction temperature is 4℃ and the time is 10-20 hours. After the sealing reaction is completed, conventional ultrafiltration is performed.

[0016] In this invention, the molar ratio of stable dye / PE to crosslinking agent is 1:(20-80); the reaction temperature is 15-35℃ and the reaction time is 0.5-4 hours. After the reaction is completed, conventional ultrafiltration is performed.

[0017] In this invention, a reducing agent reacts with an antibody to obtain a thiolized antibody; the reaction temperature is 15–35°C and the time is 1–5 hours. After the reaction, conventional ultrafiltration is performed.

[0018] In this invention, the molar ratio of PE / fluorescent tandem dye to thiolized antibody is (0.5–5):1; the reaction temperature is 15–35°C, and the reaction time is 2–6 hours. Preferably, the concentration of thiolized antibody is 0.5–5 mg / mL. Preferably, after the reaction of PE / fluorescent tandem dye with thiolized antibody is completed, a blocking agent is added, and the reaction is carried out at 15–35°C for 0.5–2 hours to complete antibody labeling, which can then be directly used for fluorescence detection.

[0019] The raw materials involved in this invention include antibodies, small molecule organic dyes, phycoerythrin, preservatives, stabilizers, cross-linking agents, reducing agents, blocking agents, and buffer solutions, wherein the small molecule organic dyes are YF568, YF594, and Texas. The dyes are any one of Rhodamine dyes or cyanine dyes, such as Red, YF620, YF647, YF680, YF750, Cy5, Cy5.5, and Cy7; the preservative is any one of NaN3 or Proclin series, used for antibody preservation solutions; the stabilizer is an amino compound (optionally a protein or polypeptide, such as bovine serum albumin BSA, glycine, alanine, serine, arginine, histidine, lysine, glargine peptide, glargine peptide, glargine peptide, glargine serine peptide, etc.), sugar alcohols (such as sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, etc.), trehalose, gelatin, glycerol, or polyethylene glycol polymers (such as PEG300, PEG400, PEG600, PEG4000, PEG10000). The crosslinking agent is any one or more combinations of PEG20000, NaCl, and EDTA; the crosslinking agent is SMCC (succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate), Sulfo-SMCC (sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate), LC-SMCC (4-(N-maleimide methyl)cyclohexane-1-carboxy-(6-aminohexanoic acid)), SM(PEG)2 (polyethylene glycolated SMCC crosslinking agent), SM(PEG)4 (polyethylene glycolated SMCC crosslinking agent), SM(PEG)6 (polyethylene glycolated, long-chain SMCC crosslinking agent), SM(PEG)8 (polyethylene glycolated, long-chain SMCC crosslinking agent), SM(PEG) 12 (Polyethylene glycolation, long-chain SMCC crosslinking agent), SM(PEG) 24(Polyethylene glycolation, long-chain SMCC crosslinking agent), SMPB (succinimide-4-(p-maleimide-phenyl)butyrate), AMAS (N-α-maleimide-acetyl-oxysuccinimide), GMBS (N-γ-maleimide-butyryl-oxysuccinimide), MBS (m-maleimide-benzoyl-N-hydroxysuccinimide), EMCS (N-ε-maleimide-hexanoyl-oxysuccinimide), BMPS (N-β-maleimide-propyl) 3-[2-pyridyldithio]propionate, Sulfo-LC-SPDP (sulfosuccinimide 6-(3'-(2-pyridyldithio)propionylamino)hexanoate), LC-SPDP (succinimide-6(3-[2-pyridyldithio]propionylamino)hexanoate), PEG4-SPDP (polyethylene glycolated, long-chain SPDP crosslinking agent), PEG12-SPDP (polyethylene glycolated, long-chain SPD) P crosslinking agent), carbodiimide (such as DIC (N,N'-diisopropylcarbodiimide), DCC (dicyclohexylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), EDAC (1-ethyl(3-dimethylaminopropyl)-3-carbodiimide), PC-40 (polycarbodiimide)), hydroxysuccinimide (such as NHS (N-hydroxysuccinimide), Sulfo-NHS (N-hydroxysulfosuccinimide)); and any one of the following: The stock solution is any one or more combinations of DTE (1,4-dithioerythritol), DTT (dithiothreitol), TCEP (tris(2-carbonylethyl)phosphohydrochloride), 2IT (2-iminothione hydrochloride), DTBA ((2S)-2-amino-1,4-dimercaptobutane hydrochloride), ME (mercaptoethanol), and TGA (mercaptoacetic acid), preferably 2IT and DTT are used together, and more preferably, the mass ratio of 2IT to DTT is 1:1 to 10; the blocking agent is any one or more combinations of NEM (N-ethylmaleimide), MEA (cysteamine), and cysteamine hydrochloride; the buffer solution is any one or more combinations of HEPES buffer, MES buffer, citrate buffer, carbonate buffer, and phosphate buffer.

[0020] Compared with existing technologies, this invention employs a novel method to increase protein stability, avoiding the precipitation phenomenon of tandem dyes. This invention enhances the reduction performance of small-molecule disulfides, significantly increasing reaction efficiency. By tandemly connecting phycoerythrin with small-molecule organic dyes, this invention increases the Stokes shift of the tandem dyes, thereby increasing the number of fluorescent dyes detectable by the same laser excitation, which is more beneficial for the analysis of biochemical samples. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the present invention in the prior art, some examples of the present invention will be briefly described below.

[0022] Figure 1 The labeling flowchart for antibodies labeled with PE tandem fluorescent dyes is shown.

[0023] Figure 2 The image shows the immunofluorescence staining of HeLa cells labeled with PE / Cy5-IgG (GAM) using the antibody labeling method described in this invention. In the image, blue represents the cell nuclei stained with DAPI, with an exposure time of 50ms; red represents RAB27A (1C4B8) cells stained with PE / Cy5-IgG (GAM), with an exposure time of 100ms.

[0024] Figure 3 This is a flowchart illustrating the usage of a PE tandem fluorescent dye labeled antibody kit.

[0025] Figure 4 The image shows a flow cytometry result of PE / Cy7-CD45-stained Jurkat cells labeled with the PE / Cy7 flow cytometry antibody labeling kit described in this invention. Purple represents the control group, and blue represents the PE / Cy7-CD45-stained group.

[0026] Figure 5 The absorption spectra of the tandem dyes PE / 647 (A, purple), PE / 680 (B, yellow), and PE / 750 (C, green) of this invention are shown.

[0027] Figure 6 The absorption spectra of the tandem dyes PE / Cy5 (D, gray), PE / Cy5.5 (E, blue), and PE / Cy7 (F, red) of this invention are shown.

[0028] Figure 7 The image shows the precipitation of PE / Cy5-labeled antibody (A) without protein protection treatment and PE / Cy5-labeled antibody (B) treated with protein protection method in this invention.

[0029] Figure 8 The image shows flow cytometry results of CD45 antibody-labeled k562 cells stained with the PE / Cy7 antibody labeling kit of this invention and the competing PE / Cy7 antibody labeling kit. Yellow represents the control group, green represents the group of this invention, and red represents the competing group. Detailed Implementation

[0030] This invention involves blocking the free thiol groups on phycoerythrin; then subjecting it to tandem treatment with a small molecule organic dye; followed by protein protection treatment of the obtained tandem dye; and finally activation treatment with a cross-linking agent to obtain a PE / fluorescent tandem dye. For antibody labeling, the target antibody is first thiolated with a reducing agent; then the thiolated target antibody is cross-linked with the activated tandem dye; finally, a blocking agent is added to the cross-linked tandem dye-antibody system for dye blocking treatment, and the fluorescent antibody is preserved, completing antibody labeling. The preparation process is described in [reference needed]. Figure 1 This invention increases the stability of phycoerythrin and avoids precipitation.

[0031] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only. The raw materials involved in the present invention are all conventional commercially available raw materials, and the methods for preparing buffer solutions, reagents, and testing methods are conventional techniques.

[0032] Example 1: Preparation of PE / Cy5-IgG

[0033] 1. Preprocessing before PE marking

[0034] (1) Vortex the 20 mg / mL PE suspension (commercially available) in the bottle.

[0035] (2) Pipette 50 μL of PE suspension into an EP tube, add 1 mL of PBS buffer, vortex to mix, and centrifuge at 14000 x g for 1 min.

[0036] (3) Transfer the supernatant to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3min, remove the filtrate, add 500μL PBS buffer to the filter, mix by blowing and aspiration, centrifuge at 14000xg for 3min, and repeat the washing 3 times.

[0037] (4) Collect the desalted PE solution and adjust the solution concentration to 2.5 mg / mL with PBS buffer.

[0038] 2. PE thiol-blocking treatment

[0039] (1) Add NEM solution with a molar ratio of 50 to the PE solution and mix well.

[0040] (2) Shake the reaction at room temperature in the dark for 2 hours to allow the maleimide groups of NEM to fully react with the free thiol groups of PE; then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3 minutes, remove the filtrate, add 500μL of PBS buffer to the filter device, mix by blowing and aspiration, centrifuge at 14000xg for 3 minutes, and repeat the washing 3 times.

[0041] (3) Collect the ultrafiltration PE solution and adjust the solution concentration to 2.5 mg / mL with PBS buffer.

[0042] 3. PE / Cy5 processing

[0043] (1) Add Cy5 SE stock solution (commercially available, Cy5 SE is 20 times the molar amount of PE) to the PE solution and mix well.

[0044] (2) Shake the reaction at room temperature in the dark for 30 min to allow the succinimide ester on Cy5 SE to fully react with the primary amine group on PE to form PE tandem dye; then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3 min, remove the filtrate, add 500μL PBS buffer to the filter device, mix by blowing and aspiration, centrifuge at 14000xg for 3 min, and repeat washing until the filtrate is colorless.

[0045] (3) Collect the ultrafiltration PE / Cy5 solution and adjust the solution concentration to 2.5 mg / mL with MES buffer.

[0046] 4. PE / Cy5 stability modification treatment

[0047] (1) Add 20 times the molar concentration of EDC solution to the PE / Cy5 solution and mix well; then add high concentration NHS solution (2M) until the final NHS concentration is 2mM and mix well; then shake and react at room temperature in the dark for 20min to fully activate the carboxyl groups on PE; then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3min, remove the filtrate, add 500μL PBS buffer to the filter device, mix by blowing and aspiration, centrifuge at 14000xg for 3min, and repeat the washing 3 times.

[0048] (2) Collect the ultrafiltration PE / Cy5-1 solution, adjust the solution concentration to 2.5 mg / mL with PBS buffer; then add histidine to the final histidine concentration of 0.5 M, and mix well.

[0049] (3) Shake the reaction at room temperature in the dark for 2 hours to allow the primary amine group of histidine to fully react with the activated carboxyl group on PE; then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3 min, remove the filtrate, add 500μL of PBS buffer to the filter device, mix by blowing and aspiration, centrifuge at 14000xg for 3 min, and repeat the washing 3 times; collect the ultrafiltration PE / Cy5-2 solution, and adjust the solution concentration to 2.5mg / mL with PBS buffer.

[0050] 5. PE / Cy5 activation reaction

[0051] (1) Add 50 times the molar concentration of SMCC solution to the PE / Cy5-2 solution and mix well.

[0052] (2) Shake the reaction at room temperature in the dark for 2 hours to allow the N-hydroxysuccinimide (NHS) active ester on the SMCC to fully react with the primary amine group on the PE; then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3 min, remove the filtrate, add 500μL PBS buffer to the filter device, mix by blowing and aspiration, centrifuge at 14000xg for 3 min, and repeat the washing 3 times.

[0053] (3) Collect the activated PE / Cy5 solution after ultrafiltration and adjust the solution concentration to 2.5 mg / mL with PBS buffer.

[0054] 6. IgG (GAM) thiolation reaction

[0055] (1) In this reaction, 2IT and DTT are used together. First, 2IT and DTT are mixed in a mass ratio of 1:5, and diH2O is added to prepare a 2IT / DTT mixed solution of 15 mg / mL.

[0056] (2) Add 1 / 10 volume of 2IT / DTT mixed solution to the 2mg / mL IgG (GAM) solution, mix well, and shake at room temperature in the dark for 2h to allow it to fully react with IgG (GAM); then transfer the reaction solution to a 30KD ultrafiltration tube, centrifuge at 14000xg for 3min, remove the filtrate, add 500μL PBS buffer to the filter device, mix well by blowing and aspiration, centrifuge at 14000xg for 3min, and repeat the washing 3 times.

[0057] (3) Collect the ultrafiltration thiolized IgG (GAM) solution and adjust the solution concentration to 2 mg / mL with PBS buffer.

[0058] 7. Crosslinking reaction of PE / Cy5 with IgG (GAM)

[0059] (1) Mix the activated PE / Cy5 solution with the thiolized IgG (GAM) solution at a dye-to-antibody molar ratio of 2:1, and adjust the thiolized IgG (GAM) reaction concentration to 2 mg / mL with PBS buffer.

[0060] (2) Shake the reaction at room temperature in the dark for 3 hours to allow the phycoerythrin tandem dye to fully couple with IgG (GAM) and complete antibody labeling.

[0061] (3) Further, add antibody preservation solution, mix well, and increase preservation time; the antibody preservation solution consists of 1M Tris, 10% BSA, 0.05% PC300, 0.5mM NEM, and water.

[0062] 8. When conducting the test, the indirect IF test method is used, 2×10 4 HeLa cells were blocked with immunofluorescence blocking solution for 10 min and washed three times with 1×PBS. The positive group was added with 100 μL of RAB27A (1C4B8) monoclonal antibody diluted 1:100 with primary antibody dilution buffer, while the control group was added with 100 μL of 1×PBS. Cells were incubated at 37°C for 1 h and washed three times with 1×PBS. Both groups were then added with 100 μL of PE / Cy5-IgG (GAM) diluted 1:500 with secondary antibody dilution buffer, incubated at 37°C in the dark for 1 h, and washed three times with 1×PBS. Both groups were then added with 100 μL of ready-to-use DAPI staining solution (5 μg / mL), incubated at room temperature in the dark for 5 min, washed three times with 1×PBS, and covered with 50 μL of 1×PBS. Cells were then analyzed using a fluorescence microscope (Ex: 470 / 40, Em: 695 / 40). The results are shown below. Figure 2 As shown.

[0063] Depend on Figure 2 It is known that the PE / Cy5-IgG (GAM) labeled by the labeling scheme of the present invention has good staining effect and fluorescence intensity, and no non-specific staining.

[0064] Example 2: PE / Cy7 flow cytometry antibody labeling kit and its labeled CD45

[0065] The PE / Cy7 flow cytometry antibody labeling kit consists of component A (activated PE / Cy5), component B (antibody modifier), component C (antibody preservation solution), and component D (ultrafiltration tube).

[0066] Figure 3 This is a schematic diagram illustrating the usage of the PE / Cy7 flow cytometry antibody labeling kit of the present invention. Taking its CD45 labeling as an example:

[0067] 1. B component (antibody modifier, lyophilized powder of 2IT and DTT in a mass ratio of 1:5) was brought to room temperature; then 50 μg of CD45 antibody (1 mg / mL) was added, dissolved and mixed, and the mixture was shaken at room temperature for 1 h.

[0068] 2. Transfer the entire antibody reaction solution from the previous step to an ultrafiltration tube, add 200 μL of 1×PBS (pH 7.2), and centrifuge at 14000×g for 2 min; discard the liquid in the collection tube, as the antibody remains in the ultrafiltration tube. Repeat the washing process once.

[0069] 3. Collect the antibody and, based on the amount of antibody added to the reaction, resuspend the antibody in 1×PBS (pH 7.2) to a final concentration of 1 mg / mL.

[0070] 4. Transfer the collected antibody to a tube containing 100 μg of component A (pre-activated PE / Cy7 lyophilized powder) and dissolve and mix well. Incubate at room temperature in the dark with shaking for 4 hours, then perform antibody labeling.

[0071] 5. Add 1 / 10 volume of antibody preservation solution to the reaction solution from the previous step, vortex to mix, and react at room temperature in the dark with shaking for 1 hour to obtain the PE / Cy7-CD45 antibody solution. The antibody preservation solution consists of 1M Tris, 10% BSA, 0.05% PC300, 0.5mM NEM, and water.

[0072] 6. When conducting the test, the FC test method is used, 1×10 6 Jurkat cells were divided into two groups. The positive control group received 100 μL of fluid antibody dilution buffer diluted 1:100 with PE / Cy7-CD45 antibody, while the control group received 100 μL of fluid antibody dilution buffer. Cells were incubated at 4°C in the dark for 30 min, washed three times with fluid antibody washing buffer, and resuspended in 500 μL of fluid antibody washing buffer. Cells were then analyzed by flow cytometry. Results are shown below. Figure 4 As shown. The flow resistant washing solution was 1×PBS containing 2% BSA, and the flow resistant dilution solution was 1×PBS containing 1% BSA.

[0073] 7. By Figure 4 It can be seen that, compared with the control group, the CD45 monoclonal antibody labeled by the PE / Cy7 flow cytometry antibody labeling kit of the present invention has good staining effect and fluorescence intensity signal.

[0074] Example 3

[0075] By changing the small molecule dye according to the method in Example 1, a variety of tandem dyes were obtained. Figure 5 The absorption spectra of the tandem dyes PE / 647 (A, purple), PE / 680 (B, yellow), and PE / 750 (C, green) are shown. Figure 6 The absorption spectra of the tandem dyes PE / Cy5 (D, gray), PE / Cy5.5 (E, blue), and PE / Cy7 (F, red) are shown.

[0076] Example 4

[0077] Based on step 6 of Example 1, DTT was used instead of 2IT / DTT as the reducing agent for the IgG (GAM) thiolation reaction, and the rest were the same.

[0078] The absorbance of the labeled antibody at 280 nm and 492 nm was measured using a UV spectrophotometer, and the labeling efficiency (DOL) was calculated.

[0079] Table 1 compares the efficiency of DTT reduction of IgG in Example 4 alone with that in Example 1 using a combination of 2IT and DTT (DOL: a parameter for characterizing and optimizing biological conjugates (e.g., fluorophore-labeled proteins), expressed as a molar ratio of label to protein).

[0080]

[0081] Comparative Example 1

[0082] Based on Example 1, step 4 was omitted, meaning the PE / Cy5 solution obtained in step 3 of Example 1 was directly subjected to the activation reaction in step 5. The resulting tandem dye was then used for antibody labeling experiments in subsequent steps 6 and 7. The resulting labeled antibody showed significant denaturation (precipitation). See [link to relevant documentation]. Figure 7 A, this is also a problem with similar products currently on the market. In contrast, the labeled antibody obtained in Example 1 did not show denaturation (precipitation), see [link to example]. Figure 7 B.

[0083] In addition, when histidine in step 4 of Example 1 was replaced with glargine peptide, xylitol or trehalose, the labeled antibody did not denature.

[0084] Example 5: Comparison of Antibody Labeling Effects between the Invention's Tandem Dye Kit and Competitor Kits. The best-performing commercially available product in industrial applications was used as the competitor. The present invention's kit was used for antibody labeling according to the method described in Example 2, while the competitor's kit (AAT-1342) was labeled using the monoclonal antibody CD45 according to the competitor's instructions. The two labeled PE / Cy7-CD45 antibodies were then used to stain K562 cells using the FC assay method described in Example 2, followed by flow cytometry analysis.

[0085] The results showed that the PE / Cy7-CD45 antibody labeled in this invention exhibited significantly better fluorescence intensity signal intensity than competing products in flow cytometry detection, such as... Figure 8 As shown.

[0086] This invention provides a method for tandemly binding phycoerythrin with a fluorescent dye and a method for conjugating this tandem dye with an antibody. The phycoerythrin tandem dye consists of a macromolecular fluorescent protein dye (PE) and another small molecule organic dye. PE acts as an energy donor, and the small molecule organic dye acts as an energy acceptor. The two are covalently bonded. The donor PE is excited by a laser emitted from an exciter, and the emitted energy is transferred to the adjacent acceptor small molecule organic dye, causing the acceptor small molecule organic dye to emit light of a specific wavelength. This tandem dye possesses the excitation characteristics of the donor PE and the emission characteristics of the acceptor, making it a fluorescent dye with a unique fluorophore. Importantly, this invention effectively solves the problem of antibody denaturation in existing tandem dye-labeled antibodies, ensuring accurate antibody concentration and reducing deviations in fluorescence detection ratios caused by differences in antibody concentration.

Claims

1. A method for preparing a PE / fluorescent tandem dye, characterized in that, Includes the following steps: (1) The thiol-blocked phycoerythrin was reacted with a small molecule organic dye to obtain dye / PE; (2) After activating the carboxyl groups on the dye / PE, react with a stabilizer to obtain a stable dye / PE; (3) React the stable dye / PE with the crosslinking agent to obtain PE / fluorescent tandem dye; The thiol groups of phycoerythrin are blocked using a blocking agent to obtain thiol-blocked phycoerythrin; the molar ratio of blocking agent to phycoerythrin is 1:(50-80); the reaction temperature during blocking is 15-35℃ and the time is 1-3 hours, or the reaction temperature during blocking is 4℃ and the time is 10-20 hours; the molar ratio of thiol-blocked phycoerythrin to small molecule organic dye is 1:(10-30); the reaction temperature of thiol-blocked phycoerythrin with small molecule organic dye is 15-35℃ and the time is 10-60 minutes; the small molecule organic dye is any one of rhodamine dye or cyanine dye; the blocking agent is any one or a combination of NEM, MEA, and cysteine ​​hydrochloride. The carboxyl groups on the dye / PE are activated using EDC and NHS. When the carboxyl-activated dye / PE reacts with the stabilizer, the ratio of dye / PE to stabilizer is (2.5-5) g:(0.1-1) mol, the reaction temperature is 15-35℃ and the time is 1-5 hours, or the reaction temperature is 4℃ and the time is 10-20 hours. The stabilizer is histidine, glargine peptide, xylitol or trehalose. The molar ratio of stable dye / PE to crosslinking agent is 1:(20-80); the reaction temperature is 15-35℃ and the reaction time is 0.5-4 hours; the crosslinking agent is any one of SMCC, SM(PEG)2, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)12, SM(PEG)24, SMPB, AMAS, GMBS, MBS, EMCS, BMPS, SPDP, LC-SPDP, PEG4-SPDP, PEG12-SPDP, and carbodiimide.

2. The PE / fluorescent tandem dye prepared by the method described in claim 1.

3. A flow cytometry antibody labeling kit, characterized in that, Includes the PE / fluorescent tandem dye as described in claim 2.

4. The application of the PE / fluorescent tandem dye according to claim 2 in the preparation of fluorescent probes.

5. The application of the PE / fluorescent tandem dye according to claim 2 in fluorescence detection or in the preparation of fluorescence detection reagents.

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