An iRFP monoclonal antibody and a preparation method and application thereof

Abstract

This invention discloses an iRFP monoclonal antibody, its preparation method, and its applications, relating to the fields of biotechnology and protein antibody technology. The antigen-determining region of the monoclonal antibody described in this invention is (Ac-EPPQRDVAEPQAFFRRC-NH2). The synthetic antigen of the iRFP monoclonal antibody binds to sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC). The same batch of antibodies from this invention can be seamlessly applied to three core biological technologies: cell immunofluorescence, flow cytometry, and Western blotting, ensuring wide applicability and data consistency and comparability across different experimental platforms. This invention exhibits high dilution ratios (1:50 to 1:1000) in all three applications, meaning low cost per use, high cost-effectiveness, and significant economic benefits. This invention demonstrates excellent performance in all applications, exhibiting high specificity, low background, and high signal-to-noise ratio.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and protein antibody technology, specifically an iRFP monoclonal antibody, its preparation method, and its application. Background Technology

[0002] In recent years, fluorescent proteins (FPs) have become important tools for studying biomolecules or cellular functions through dynamic imaging in live cells or in vivo. Fluorescent proteins are a series of luminescent proteins that produce fluorescence upon ultraviolet light excitation, possessing advantages such as good fluorescence stability, high sensitivity, and non-toxicity. The excitation and emission light of green fluorescent protein (GFP) and red fluorescent protein (RFP) are located in the visible light region (390 nm–650 nm). However, light below 650 nm is largely absorbed by hemoglobin in biological tissues and melanin in skin, thus interfering with the fluorescence detection and imaging of labeled substances, significantly impacting the sensitivity and accuracy of visible light fluorescence analysis.

[0003] Near-infrared fluorescent proteins are novel fluorescent proteins based on phytochrome protein. iRFP (Near-infrared fluorescent protein) is a newly developed stable near-infrared fluorescent protein. This near-infrared fluorescent protein shows superior characteristics compared to commonly used GFP and RFP. iRFP is a class of fluorescent proteins homologous to GFP. It can catalyze the formation of chromophores using its special structure and has the following excellent characteristics: (1) Easy to detect, high sensitivity, and stable fluorescence properties; the maturation of the fluorescent protein chromophore does not require the addition of substrates or other auxiliary factors. It can emit fluorescence only under excitation light. Single-cell labeling is clear and easy to observe under a fluorescence microscope; (2) The coding sequence of the fluorescent protein gene is short and easy to construct vectors; (3) Low cytotoxicity and can directly label living cells or living tissues; (4) Compared with GFP, its excitation and emission wavelengths are longer, located in the near-infrared region (650nm~900nm). It has lower light absorption and light scattering in animal tissues, higher penetration, and is more suitable for deep imaging of animal living tissues. It is a more ideal fluorescent labeling molecule for live imaging.

[0004] Currently, fluorescent antibody technology is used in clinical testing for the detection of bacteria, viruses, and parasites, as well as for the diagnosis of autoimmune diseases. Commercially available antibodies for related substances such as GFP and RFP are widely used, but there are currently no commercially available antibodies for iRFP, which has lower light absorption and scattering and higher penetrability.

[0005] Therefore, we propose an iRFP monoclonal antibody, its preparation method, and its application to address the problems mentioned above.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide an iRFP monoclonal antibody, its preparation method, and its application, in order to solve the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: an iRFP monoclonal antibody, wherein the antigen-determining region of the monoclonal antibody is (Ac-EPPQRDVAEPQAFFRRC-NH2).

[0009] Preferably, the synthetic antigen of the iRFP monoclonal antibody is bound to sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester (Sulfo-SMCC).

[0010] This invention also provides a method for preparing an iRFP monoclonal antibody, the method comprising:

[0011] Step 1: Select the antigen-determining region (Ac-EPPQRDVAEPQAFFRRC-NH2).

[0012] Step 2: React excess Sulfo-SMCC with iRFP monoclonal antibody protein containing amino groups and antigen-determining regions to obtain modified amino-iRFP monoclonal antibody protein.

[0013] Step 3: Remove unreacted Sulfo-SMCC;

[0014] Step 4: The protein containing thiol groups is linked to the modified amino-iRFP monoclonal antibody protein to enhance immunogenicity;

[0015] Step 5: Screening and preparation of iRFP monoclonal antibodies.

[0016] Preferably, step 5 specifically includes the following steps:

[0017] a. Collect pre-immune serum;

[0018] b. Animal immunization;

[0019] c. Blood collection and titer testing;

[0020] d. Fusion and filtering;

[0021] e. Small-scale culture of hybridoma cells;

[0022] f. Purification.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The antibodies of this invention from the same batch can be seamlessly applied to the three core biological technologies of cell immunofluorescence, flow cytometry and Western blotting, with wide application, ensuring the consistency and comparability of data between different experimental platforms.

[0025] 2. The present invention exhibits a high dilution ratio (1:50 to 1:1000) in all three applications, which means low cost per use, high cost performance and significant economic benefits; the present invention demonstrates excellent performance with high specificity, low background and high signal-to-noise ratio in all applications.

[0026] 3. This invention provides a standard reagent with stable performance and controllable quality for the field, which strongly promotes the standardization process of near-infrared fluorescent protein research. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0028] Figure 1 The graph shows the serum titer results of mice after three immunizations, as determined by ELISA.

[0029] Figure 2 The image shows the WB detection results of the master clone cells with the highest ELISA binding value.

[0030] Figure 3 The image shows the Western blot (WB) results of secondary subclonal cells that were positive for ELISA binding.

[0031] Figure 4 Figure 1 shows the experimental results of applying iRFP monoclonal antibody to cellular immunofluorescence detection.

[0032] Figure 5 Figure showing the experimental results of applying iRFP monoclonal antibody in flow cytometry detection;

[0033] in:

[0034] Red: HEK293T cells transfected with T-iRFP-his6;

[0035] Orange: HEK293T cells transfected with F-iRFP-his6;

[0036] Blue: Blank;

[0037] Channels: A: iRFP antibody / FITC; B: His antibody / PE-CF594; C: APC;

[0038] Figure 6 The figure shows the experimental results of the application of iRFP monoclonal antibody in Western blot detection. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] This invention provides an iRFP monoclonal antibody, wherein the antigen-determining region of the monoclonal antibody is (Ac-EPPQRDVAEPQAFFRRC-NH2).

[0041] It should be noted that the full-length iRFP protein sequence is 315 amino acids:

[0042] AEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEV RKITGFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE;

[0043] For example, related iRFP proteins containing the above iRFP protein sequence include:

[0044] 1. (GenBank ID: JN247409 / protein ID: AEL88490.1 ) pNLS-iRFP713

[0045] 316 amino acids (34.6 kDa)

[0046] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0047] 2. (GenBank ID: KC991145 / protein ID: AGN32866.1) pNLS-iRFP720

[0048] 316 amino acids (34.6 kDa)

[0049] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0050] ]>3. (GenBank ID: JN247409 / protein ID: AEL88490.1 ) pMito-iRFP713

[0051] 316 amino acids (34.6 kDa)

[0052] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0053] 4. (GenBank ID: KC991145 / protein ID: AGN32866.1) pMito-iRFP720

[0054] 316 amino acids (34.6 kDa)

[0055] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0056] 5. (GenBank ID: KC991145 / protein ID: AGN32866.1) piRFP720-N1

[0057] 316 amino acids (34.6 kDa)

[0058] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0059] 6. (GenBank ID: KC991145 / protein ID : AGN32866.1) pBAD / HisB-iRFP720

[0060] 316 amino acids (34.6 kDa)

[0061] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0062] 7. (GenBank ID: KC991143 / protein ID : AGN32864.1) piRFP682-N1

[0063] 316 amino acids (34.6 kDa)

[0064] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQE VRKITGFDRVMIYRFASDFSGVVIAEDRCAEVESKLGLHYPASAVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0065] 8. (GenBank ID: KC991143 / protein ID: AGN32864.1) pBAD / HisB-iRFP682

[0066] 316 amino acids (34.6 kDa)

[0067] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQE VRKITGFDRVMIYRFASDFSGVVIAEDRCAEVESKLGLHYPASAVPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE

[0068] A comprehensive comparison of the above-mentioned related iRFP proteins;

[0069] The antigenic determinant region (Ac-EPPQRDVAEPQAFFRRC-NH2) of the iRFP antibody was selected, and the antigen was prepared.

[0070] This invention also provides a method for preparing an iRFP monoclonal antibody, the method comprising:

[0071] Step 1: Select the antigen-determining region (Ac-EPPQRDVAEPQAFFRRC-NH2).

[0072] Step 2: The synthesized antigen is bound to an excess of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC).

[0073] Step 3: Remove unreacted Sulfo-SMCC;

[0074] Step 4: The protein containing thiol groups is linked to the modified amino-iRFP monoclonal antibody protein to enhance immunogenicity;

[0075] Step 5: Screening and preparation of iRFP monoclonal antibodies.

[0076] The preparation of iRFP monoclonal antibodies for screening includes the following steps:

[0077] a. Collect pre-immune serum;

[0078] b. Animal immunization;

[0079] c. Blood collection and titer testing;

[0080] d. Fusion and filtering;

[0081] e. Small-scale culture of hybridoma cells;

[0082] f. Purification.

[0083] For example, step 5 specifically involves:

[0084] Pre-immunization serum collection: 50-60 μl of blood was collected from the orbital venous plexus of mice, incubated overnight at 4°C, and the supernatant serum was separated by centrifugation and stored.

[0085] Animal immunization: Five Balb / c mice aged 6-8 weeks were immunized with two polypeptides (an unrelated polypeptide chain and an iRFP antigen-determining polypeptide chain). The immunogen was an HPV-conjugated polypeptide. Blood was collected one week after the third immunization to determine serum titers. Mice were selected for booster immunization, and spleens were harvested three days after the booster immunization for hybridoma fusion.

[0086] Blood collection and titer assay: One week after the last immunization, 50-60 μl of blood was collected from the orbital venous plexus of mice. After standing overnight at 4°C, the supernatant serum was separated by centrifugation for testing. Appropriate amounts of free peptide and OVA-conjugated peptide were taken and diluted with coating buffer to 5 μg / ml and 1 μg / ml, respectively. Then, 100 μl was added to each well of a 96-well plate using a single-channel pipette. The plate was gently tapped to mix the sample, sealed tightly with plastic wrap, and coated overnight at 4°C. The plate was washed once with 200 μl / well of washing buffer and the plate was dried. The plate was then blocked with 300 μl / well of blocking buffer at room temperature for 1 hour. After washing twice with 400 μl / well of washing buffer, the plate was added (100 μl of serially diluted sample and sample diluent were added to each well), along with the detection antibody, at 100 μl / well. The plate was incubated at room temperature for 2 hours. The plate was then washed twice with 400 μl / well of washing buffer. Wash the plate 5 times with μl / well, add 200 μl / well of chromogenic solution and incubate at room temperature for 12 minutes; stop the reaction by adding 50 μl / well of stop solution; detect the reaction using an ELISA reader at a wavelength of 450 nm.

[0087] It should be noted that the blood collection and titer test results can be represented by ELISA results.

[0088] The above ELISA process is as follows:

[0089] Sample loading: Add sample loading buffer to cell lysis buffer, and load at a rate of 10 ug / lane.

[0090] Electrophoresis: constant voltage 100 V until the bottom of the bromophenol blue gel plate.

[0091] Transfer: constant voltage 110 V, 90 minutes.

[0092] Sealing: Sealed overnight at 4℃.

[0093] Primary antibody incubation: Add hybridoma supernatant and control antibody to a multichannel hybridization apparatus and incubate at room temperature for 2 hours.

[0094] Washing: Wash the membrane three times using a decolorizing shaker, five minutes each time.

[0095] Secondary antibody incubation: Goat anti-mouse IgG (H+L) / HRP final concentration 1:10000, room temperature for 2 hours.

[0096] Washing: Wash the membrane three times using a decolorizing shaker, five minutes each time.

[0097] Color development: A chemiluminescence imaging system is used for color development.

[0098] Fusion and filtering:

[0099] All spleen cells from immunized mice were collected and mixed with mouse myeloma cells at a 1:1 ratio. The mixture was then fused using an electrofusion method to obtain hybridoma cells.

[0100] A fusion protein containing the target gene and epitope marker was constructed using recombinant DNA technology, and then identified using a specific tag antibody. The fusion protein containing CD63 and iRFP peptide chains and a His tag was constructed into the eukaryotic vector pcDNA3.1, and HEK293 cells were transfected with the empty vector pcDNA3.1. Two cell lysates, pcDNA3.1-CD63-iRFP-his transfected HEK293 and pcDNA3.1 vehicle transfected HEK293, were used as antigen and negative controls, respectively. The cell supernatant was coated with the antigen protein, and ELISA was used to detect all positive clones binding to the antigen protein. The supernatant of positive clones was then analyzed by Western blot (WB). Three rounds of specific affinity screening of the clones were performed using Western blot.

[0101] Positive clones were selected and subjected to two rounds of limiting dilution. The subclonal supernatants obtained after limiting dilution were screened by ELISA and WB to select the final monoclonal cells for production (10C5E1G7 cells).

[0102] Small-scale culture of hybridoma cells:

[0103] Transfer 1 ml of hybridoma cells into a 100 ml culture flask, add a certain amount of culture medium periodically to expand the cells, and culture for 10-12 days.

[0104] purification:

[0105] The Protein A affinity chromatography column was rinsed with ultrapure water and then equilibrated with equilibration buffer. The treated hybridoma cell supernatant was loaded onto the affinity chromatography column, and after loading, it was rinsed with equilibration buffer. Elution was performed with elution buffer, and the elution peak was collected and neutralized with Tris buffer. The solution was desalted to PBS pH 7.4.

[0106] During the aforementioned blood collection and titer testing process:

[0107] Mouse serological test results are shown in Figure 1 Table 1 shows the results, indicating that the serum titers of all 10 mice reached the fusion standard (5 μg / ml coated free polypeptide, 1000-fold diluted serum, OD-B>0.5) after three immunizations, making them suitable for hybridoma fusion. SBI200429A# was selected for fusion. Table 1 shows the serum detection results one week after the third immunization of SBI200428; Table 2 shows the serum detection results 14 weeks after the third immunization of SBI200428.

[0108] Table 1. Serum test results one week after the SBI 200428 triple immunization.

[0109]

[0110] Table 2 shows the serum test results 14 weeks after the SBI200428 triple immunization.

[0111]

[0112] After one fusion, a second round of fusion was restarted. Due to the long interval between the last immunization and the initial immunization, blood samples were collected again from the remaining 9 animals for ELISA testing. The results of the mouse serological measurements are shown in Tables 3 and 4. The results showed that the serum titers of the remaining 9 mice still met the fusion criteria 14 weeks after the third immunization, and could be used for hybridoma fusion. SBI200428D# was ultimately selected for fusion.

[0113] Among them, the ELISA result, which measures the serum titer of mice after three immunizations, is referred to as the ELISA result. Figure 1 As shown.

[0114] The following are the screening results during the above fusion and screening process:

[0115] First fusion:

[0116] SBI200429A# mice were used for fusion, and 83 ELISA-positive master clone cells were obtained. Ten of these cells were selected for subcloning and screening. All of them were positive by ELISA. All 10 cells were then subjected to a second limiting dilution and ELISA screening.

[0117] The above 10 secondary subclones were screened, and all were positive by ELISA. Two of them were selected for further limiting dilution, and the supernatant of the remaining 8 were collected.

[0118] SBI200428D# mice were used for fusion, yielding 126 ELISA-positive master clones. The supernatants of the master clones with the highest ELISA binding values ​​were selected for Western blotting (WB). WB analysis showed that the supernatants of four master clones (10C5, 14B4, 14A6, and 20A1) were positive. All four master clones were then subjected to a first limiting dilution.

[0119] The WB results are shown below. Figure 2 , and by Figure 2 It can be seen that, according to the requirement that positive samples have a target band and control samples have no impurities, qualified positive strains were screened out as follows: 9A7, 10C5, 12G11, 14A6, 14B4, and 16F7.

[0120] All four ELISA-positive subclones were subjected to Western blotting (WB), and WB-positive clones were found in all of them. Five of these clones (10C5E1, 14B4A12, 14A6H11, 20A1G5, and 14B4A7) were selected for a second limiting dilution.

[0121] All five ELISA-positive secondary subclones were subjected to Western blotting (WB), and WB-positive clones were screened out. Five of these clones (14B4A12G11, 14A6H11B4, 14B4A7G8, 20A1G5A2, and 14B4A7D10) were selected for a third limiting dilution, and one clone (10C5E1G7) was used for amplification and production.

[0122] WB results are available Figure 3 ; and by Figure 3 It can be seen that, according to the requirement that positive samples have the target band and control samples have no impurities, qualified positive strains were screened, and 10C5E1G7 was selected for amplification and production.

[0123] All five ELISA-positive subclones were subjected to Western blot (WB) testing, and WB-positive clones were screened out. All of them were then amplified and produced.

[0124] During the above purification process:

[0125] The purified monoclonal antibody was subjected to ELISA detection with standard anti-BALB / c mouse IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies. The results showed that the monoclonal antibody type was mIgG1.

[0126] To verify the practicality of the antibody of this invention, two eukaryotic expression plasmids for near-infrared fluorescent proteins were constructed: pcDNA3.1-T-iRFP-his6 and pcDNA3.1-F-iRFP-his6. These plasmids were transfected into HEK293T cells using liposome transfection (Lipo2000), with a blank transfection group as a control. A cell model expressing the target protein was successfully established for subsequent antibody performance testing.

[0127]

[0128] 1. Application of the antibody of this invention in cellular immunofluorescence detection: The experiment was conducted using HEK293T cells expressing iRFP. After fixation with 4% paraformaldehyde, permeabilization with 0.1% Triton X-100, and blocking with 5% BSA, the cells were incubated overnight at 4°C with the primary antibody of this invention at a dilution of 1:150. Subsequently, they were incubated at room temperature for 1 hour with the fluorescent secondary antibody diluted 1:1000. The nuclei were counterstained with DAPI and observed.

[0129] The results are as follows Figure 4 As shown, the experimental group exhibited clear, specific near-red fluorescence signals with accurate localization and low background, confirming that the antibody of this invention is suitable for cell immunofluorescence imaging and sub-localization studies.

[0130] 2. Application of the invented antibody in flow cytometry: Cells expressing iRFP were collected, fixed, permeabilized, and blocked. The primary antibody of this invention was incubated at room temperature for 2 hours at a 1:50 dilution, followed by labeling with a 1:1000 dilution of fluorescent secondary antibody. Results are as follows... Figure 5 As shown, flow cytometry analysis indicates that the antibody of this invention can effectively distinguish between iRFP-positive and iRFP-negative cell populations, exhibiting high signal intensity and low non-specific binding, making it suitable for precise quantitative analysis and sorting of cells expressing near-infrared fluorescent proteins.

[0131] 3. Application of the antibody of this invention in Western blot detection: Total protein was extracted from transfected cells, quantified and denatured using the BCA method, then subjected to SDS-PAGE electrophoresis and transferred to a membrane. The primary antibody of this invention, diluted 1:1000, was incubated overnight at 4°C, followed by incubation at room temperature for 1 hour with a near-infrared fluorescent secondary antibody (M800) diluted 1:10000. Results are as follows: Figure 6 As shown, a single, clear, specific band is displayed at the expected molecular weight, with no visible non-specific bands, demonstrating that the antibody of the present invention has high specificity and high sensitivity in protein level detection.

[0132] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An iRFP monoclonal antibody, characterized in that: The antigen-determining region of the monoclonal antibody is (Ac-EPPQRDVAEPQAFFRRC-NH2).

2. The iRFP monoclonal antibody according to claim 1, characterized in that: The synthetic antigen of the iRFP monoclonal antibody is bound to sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester (Sulfo-SMCC).

3. A method for preparing an iRFP monoclonal antibody, characterized in that, The method includes: Step 1: Select the antigen-determining region (Ac-EPPQRDVAEPQAFFRRC-NH2). Step 2: React excess Sulfo-SMCC with iRFP monoclonal antibody protein containing amino groups and antigen-determining regions to obtain modified amino-iRFP monoclonal antibody protein. Step 3: Remove unreacted Sulfo-SMCC; Step 4: The protein containing thiol groups is linked to the modified amino-iRFP monoclonal antibody protein to enhance immunogenicity; Step 5: Screening and preparation of iRFP monoclonal antibodies.

4. The method for preparing an iRFP monoclonal antibody according to claim 3, characterized in that: Step 5 specifically includes the following steps: a. Collect pre-immune serum; b. Animal immunization; c. Blood collection and titer testing; d. Fusion and filtering; e. Small-scale culture of hybridoma cells; f. Purification.

5. The application of an iRFP monoclonal antibody as described in any one of claims 1-2 in cellular immunofluorescence detection.

6. The application of an iRFP monoclonal antibody as described in any one of claims 1-2 in flow cytometry detection.

7. The application of an iRFP monoclonal antibody as described in any one of claims 1-2 in Western blot detection.

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