Hybridoma cell preparation method based on electrofusion technology and application thereof

Abstract

The invention relates to a preparation method and application of hybridoma cells based on an electrofusion technology, the preparation method of the hybridoma cells based on the electrofusion technology comprises the following steps: taking splenocytes and myeloma cells of an antigen-immunized mouse for electrofusion, screening to obtain the hybridoma cells, and the antigen is serum amyloid protein A. The invention also relates to a preparation method of the hybridoma cells based on the electrofusion technology. The hybridoma cell capable of generating the serum amyloid protein A antibody is obtained in an electrofusion mode, the fusion efficiency of the hybridoma cell is improved, and the problem that the yield of the antibody is reduced due to the fact that the fusion efficiency of the hybridoma cell is low is solved.

Description

[0001] This application is a divisional application of the patent application with application number 202410555506.2 (the filing date of the original application is May 7, 2024, and the name of the invention is A method for preparing hybridoma cells based on electrofusion technology and its application). Technical Field

[0002] The present invention relates to the field of biomedical engineering, and in particular to a method for preparing hybridoma cells based on electric fusion technology and its application. Background Art

[0003] Serum amyloid A (SAA) is a non-specific acute phase protein mainly derived from liver cells. The blood concentration of SAA is extremely low when the body is healthy, but when the body is infected or damaged, its concentration in the blood will increase rapidly, and can increase rapidly by about 1,000 times within 4-6 hours. After the pathogen is cleared, it can quickly decrease to normal levels. Therefore, SAA level determination is often used clinically for the diagnosis and disease monitoring of infectious diseases, such as pneumonia and mumps in children.

[0004] In the fields of biotechnology, immunology and recombinant protein expression technology, fluorescent immunochromatography is a widely used detection technology that uses fluorescently labeled antibodies to specifically recognize and bind to target antigens, thereby achieving qualitative or quantitative detection of target antigens. Measuring the SAA protein content in serum using fluorescent immunochromatography is an effective method for quickly diagnosing whether the body has viral or bacterial infections.

[0005] One of the key steps to achieve this method is to obtain fluorescent microsphere-labeled antibodies and coated antibodies with high affinity and high sensitivity. The traditional method is mainly to obtain polyclonal antibodies through animal immunization, but this method has problems such as large batch differences and poor stability. Monoclonal antibodies have gradually become the first choice to replace polyclonal antibodies due to their strong specificity and good stability. However, the preparation process of monoclonal antibodies is complicated and costly, and hybridoma cell lines are prone to problems such as antibody sequence loss and degradation.

[0006] CN101906159A discloses a method for preparing OTA monoclonal antibodies, which comprises: immunizing mice with a coupling protein OTA-BSA as an immune antigen, taking spleen cells of the immunized mice, adding PEG to fuse with myeloma cells, and screening and obtaining hybridoma cells that secrete anti-OTA antibodies. The invention uses the PEG method to fuse mouse spleen cells and myeloma cells to obtain hybridoma cells, but the method has complicated operation steps and low fusion efficiency.

[0007] CN117106728A discloses a method for preparing hybridoma cells, the method comprising using recombinant S1 401-769The mouse was immunized with protein, and the immune spleen cells were fused with hybridoma cells. The positive hybridoma cell lines were screened by indirect ELISA method. After subcloning, only the stable secretion of recombinant S1 was screened. 401-769 The invention only uses the ELISA method for screening, and the obtained antibodies cannot be applied to the preparation of the colloidal gold test strips mentioned in the invention.

[0008] In summary, how to efficiently and quickly prepare hybridoma cells and apply the antibodies secreted by them to fluorescent immunochromatography technology has become a problem that needs to be solved urgently in this field. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a hybridoma cell preparation method based on electrofusion technology and its application, wherein hybridoma cells capable of producing SAA antibodies are obtained by electrofusion, and the fusion efficiency of hybridoma cells is improved by parameter design related to the electrofusion technology, thereby solving the problem of reduced antibody production caused by low hybridoma cell fusion efficiency.

[0010] To achieve this object, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for preparing hybridoma cells based on electrofusion technology, the method for preparing hybridoma cells based on electrofusion technology comprising: taking spleen cells and myeloma cells from mice immunized with an antigen for electrofusion, and obtaining the hybridoma cells after screening, wherein the antigen is serum amyloid A.

[0012] Preferably, the fusion voltage of the electric fusion is 700-1000V (for example, 700V, 750V, 800V, 850V, 900V, 950V or 1000V, etc.), preferably 800-900V (for example, 800V, 820V, 840V, 860V, 880V or 900V, etc.).

[0013] Preferably, the pulse time of the electrofusion is 20-50 μs (for example, 20 μs, 25 μs, 30 μs, 35 μs, 40 μs, 45 μs or 50 μs, etc.), preferably 30-40 μs (for example, 30 μs, 32 μs, 34 μs, 36 μs, 38 μs or 40 μs, etc.).

[0014] Preferably, the electrofusion is repeated 2-4 times (eg, 2, 3 or 4 times).

[0015] Preferably, the screening includes screening using HAT medium, ELISA screening and fluorescent immunochromatographic test strip screening.

[0016] Preferably, the ELISA screening further includes a step of antibody purification.

[0017] Preferably, the antibody purification method comprises: using magnetic beads to purify the supernatant of ELISA positive wells to obtain coated antibodies to be screened and labeled antibodies to be screened.

[0018] Preferably, the fluorescent immunochromatographic test strip comprises a blank nitrocellulose membrane, a sample pad and absorbent paper, and the step of screening the fluorescent immunochromatographic test strip comprises: dripping the coated antibody to be screened onto the blank nitrocellulose membrane, and after drying, dripping a mixture of the labeled antibody to be screened and the antigen labeled with fluorescent microspheres onto the sample pad, and screening for positive results.

[0019] Preferably, the fluorescent immunochromatographic test strip further comprises a step of monoclonal hybridoma cells after screening.

[0020] Preferably, the method for monocloning hybridoma cells comprises: sorting hybridoma cells by flow cytometry and screening again using the ELISA screening and fluorescent immunochromatography test strip screening.

[0021] In a second aspect, the present invention provides the use of the hybridoma cell preparation method based on the electrofusion technology as described in the first aspect in the preparation of coated antibodies and labeled antibodies for fluorescent immunochromatography technology.

[0022] In a third aspect, the present invention provides a coated antibody, wherein the coated antibody is purified from hybridoma cells prepared by the hybridoma cell preparation method based on the electrofusion technology described in the first aspect.

[0023] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the coated antibody include the sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0024] SEQ ID NO.5: DTYMH.

[0025] SEQ ID NO.6: RIDPANGNTKYDPKFQG.

[0026] SEQ ID NO. 7: HDGYYWFAY.

[0027] Preferably, the amino acid sequences of the light chain variable regions CDR1, CDR2 and CDR3 of the coated antibody include the sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0028] SEQ ID NO. 8: RASQSVSTSSYSYMH.

[0029] SEQ ID NO.9: YASNLES.

[0030] SEQ ID NO.10: QHSWEIPPT.

[0031] Preferably, the amino acid sequence of the heavy chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.2.

[0032] SEQ ID NO. 1: EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLE WIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARHDGYYWFAY WGQGTLVTVSA.

[0033] SEQ ID NO. 2: DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPP KLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPPTFGGGTKLEIK.

[0034] In a fourth aspect, the present invention provides a labeled antibody, wherein the labeled antibody is purified from hybridoma cells prepared by the hybridoma cell preparation method based on the electrofusion technology described in the first aspect.

[0035] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the labeled antibody include the sequences shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0036] SEQ ID NO.11: GYGVN.

[0037] SEQ ID NO. 12: MIWGDGSTDYNSALKS.

[0038] SEQ ID NO.13: GLGRRYFDY.

[0039] Preferably, the amino acid sequences of the light chain variable regions CDR1, CDR2 and CDR3 of the labeled antibody include the sequences shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0040] SEQ ID NO. 14: RASSSVSSSYLH.

[0041] SEQ ID NO. 15: STSNLAS.

[0042] SEQ ID NO. 16: QQYSGYPLT.

[0043] Preferably, the amino acid sequence of the heavy chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.4.

[0044] SEQ ID NO.3: QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWL GMIWGDGSTDYNSALKSRLSISKDNKSSQVFLKMNSLQTDDTARYYCARGLGRRYFDYW GQGTTLTVSS.

[0045] SEQ ID NO.4: ENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKL WLYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELK.

[0046] In the fifth aspect, the present invention provides the use of the hybridoma cell preparation method based on electrofusion technology as described in the first aspect, the coated antibody as described in the third aspect, or any one or a combination of at least two of the labeled antibodies as described in the fourth aspect in the preparation of a fluorescent immunochromatographic test strip for detecting serum amyloid A.

[0047] In a sixth aspect, the present invention provides a fluorescent immunochromatographic test strip for detecting serum amyloid A, wherein the fluorescent immunochromatographic test strip for detecting serum amyloid A comprises a plastic substrate, a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper, wherein the conjugate pad contains the labeled antibody described in the fourth aspect, and a detection line and a quality control line are drawn on the nitrocellulose membrane, wherein the detection line contains the coated antibody described in the third aspect.

[0048] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The present invention obtains hybridoma cells capable of producing SAA antibodies by electrofusion, and improves the fusion efficiency of hybridoma cells by designing parameters related to the electrofusion technology, thereby solving the problem of reduced antibody production caused by low hybridoma cell fusion efficiency;

[0051] (2) The present invention obtains monoclonal hybridoma cells through multiple screening, which solves the problem of large differences between batches of polyclonal antibodies and poor stability, significantly improves the specificity and stability of antibodies, uses fluorescent immunochromatography technology to screen coated antibodies and labeled antibodies, simplifies the operation steps, reduces costs and saves time;

[0052] (3) The present invention screened and obtained SAA antibodies that can be used for fluorescent immunochromatography technology and obtained their sequences. Through gene synthesis and the construction of expression vectors, a stable antibody production system was obtained, and SAA antibodies that can be used for fluorescent immunochromatography technology were successfully prepared, providing strong support for the subsequent development of immunofluorescence diagnostic kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the structure of the fluorescent immunochromatographic test strip;

[0054] Figure 2 This is a flow cytometric sorting diagram of hybridoma cells;

[0055] Figure 3 This is the linear fitting result of SAA fluorescent immunochromatography. DETAILED DESCRIPTION

[0056] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0057] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0058] Reagents and instruments used in the following examples:

[0059] Electrofusion instrument: purchased from BTX Company, USA, product model ECM2001;

[0060] Electrofusion solution: purchased from BTX, USA, product number 47-0001;

[0061] High-throughput magnetic bead purification system: purchased from Shanghai Berger Medical Technology Co., Ltd., product number BG-Abot-96-CH;

[0062] High-throughput antibody purification kit: purchased from Shanghai Bio-Tech Medical Technology Co., Ltd., product number BG-CH-001;

[0063] Flow cytometer: purchased from Sony, product number SH800S.

[0064] Example 1

[0065] This embodiment provides a method for preparing hybridoma cells, the method comprising the following steps:

[0066] (1) Antigen Immunization:

[0067] SAA was injected into mice at a dose of 100 μg / mouse, and booster immunization was performed every two weeks for a total of four immunizations. For the first immunization, the immunogen was mixed with an equal amount of Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points. For the second and third immunizations, the antigen was mixed with an equal amount of Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points. Serum samples were collected after three immunizations, and the serum titer was determined by the ELISA method. Mice with high serum titers were selected and immunized once with SAA at a dose of 100 μg by intraperitoneal injection. SAA protein is purchased from the market. In order to increase the titer, four brands of antigens were purchased for this immunization.

[0068] (2) Preparation of SP / 20 cells:

[0069] SP / 20 cells were revived two weeks before antigen immunization. SP / 20 cells are a commonly used myeloma cell line. SP / 20 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at a dose of 3×10 per mouse. 7 SP / 20 cells were expanded and cultured in T75 square flasks for later use. Cell viability was maintained at more than 95% before fusion, and cells were washed 2-3 times with serum-free RPMI-1640 medium to remove serum.

[0070] (3) Preparation of mouse spleen cells:

[0071] After the mice were killed, they were soaked in 75% alcohol for 5 minutes, and then the spleen tissue was removed by aseptic operation. 10mL of serum-free medium (RPMI 1640) was drawn with a syringe, and the spleen cells were blown out. The color of the spleen was visibly lightened, and the obtained cell suspension was collected into a 50mL centrifuge tube. The spleen was cut into pieces with scissors and placed on a 200-mesh sieve. Serum-free medium was added dropwise, and the grinding was repeated 2-3 times. The cell suspension was collected into a 50mL centrifuge tube. Washed twice with serum-free DMEM medium, counted, and resuspended with 20mL serum-free medium, placed in a 50mL polypropylene plastic centrifuge tube, and placed on ice.

[0072] (4) Electrofusion:

[0073] The prepared SP / 20 cells were mixed with mouse spleen cells, washed twice with electrofusion solution, and fused using an electrofusion instrument with a voltage of 800V and a pulse time of 40μs, repeated 3 times. After the fusion was completed, the fused cells were plated onto a 96-well plate, and HAT was added for screening 24 hours later. The growth of the fused cells was observed for 5-7 days, and the fusion efficiency in the wells of different groups was counted.

[0074] Example 2

[0075] This embodiment provides a method for preparing hybridoma cells, which is different from that of embodiment 1 only in that the fusion voltage of the electrofusion in step (4) is 700 V, the pulse time is 50 μs, and it is repeated twice.

[0076] Example 3

[0077] This embodiment provides a method for preparing hybridoma cells, which is different from that of embodiment 1 only in that the fusion voltage of the electrofusion in step (4) is 1000 V, the pulse time is 20 μs, and it is repeated 4 times.

[0078] Example 4

[0079] This embodiment provides a method for preparing hybridoma cells, which is different from that of Embodiment 1 only in that the pulse time of electrofusion in step (4) is 30 μs.

[0080] Example 5

[0081] This embodiment provides a method for preparing hybridoma cells, which is different from that of Embodiment 1 only in that the pulse time of electrofusion in step (4) is 35 μs.

[0082] Example 6

[0083] This embodiment provides a method for preparing hybridoma cells, which is different from that of Embodiment 1 only in that the fusion voltage of the electrofusion in step (4) is 850V.

[0084] Example 7

[0085] This embodiment provides a method for preparing hybridoma cells, which is different from that of Embodiment 1 only in that the fusion voltage of the electrofusion in step (4) is 900V.

[0086] Example 8

[0087] In this example, hybridoma cells were obtained using the method provided in Example 1, and SAA antigen was coated on a 96-well plate at a dose of 1 μg / well at 4°C overnight. Then, 2% BSA was used to block for 2 hours at 37°C, and then 70 μL of cell serum was taken from the culture wells of the hybridoma cells and added to the coated 96-well plate, and incubated at 37°C for 1 hour. Subsequently, HRP-labeled goat anti-mouse IgG secondary antibody was added to the well and incubated at 37°C for 1 hour. Finally, TMB substrate solution was added to the well, reacted at room temperature in the dark for 15 minutes, and then the stop solution was added to terminate the reaction, and the OD450 value was read on the microplate reader. Wells with OD450 values ​​greater than 2 times the negative control were considered positive wells.

[0088] Example 9

[0089] In this example, the cell supernatant of the positive wells obtained in Example 8 was collected and added to a 96-deep-well plate. The purification program was pre-set using a high-throughput magnetic bead purification system and a matching high-throughput antibody purification kit to automatically complete the steps of equilibration, binding, washing, and elution. A maximum of 96 wells could be purified at a time.

[0090] Example 10

[0091] In this example, the purified antibodies obtained in Example 9 were screened using fluorescent immunochromatography, and a blank NC membrane, a sample pad, and absorbent paper were assembled into a chromatography test strip, and the coated antibody to be screened was diluted to a concentration of 1 μg / mL, 1.5 μL was dripped onto the center of the blank NC membrane, and dried at 37°C for 30 min. An antibody with high affinity was selected as the labeled antibody, and labeled with fluorescent microspheres. After labeling, it was diluted 20 times, and the diluted labeled antibody and antigen were mixed. The mixed labeled antibody and antigen were dripped onto the center of the sample pad for detection. If fluorescence was generated, the coated antibody and labeled antibody could be used for fluorescent immunochromatography.

[0092] Embodiment 11

[0093] In this example, the hybridoma cells expressing the coating antibody and the labeled antibody screened in Example 10 were monocloned, and the hybridoma cells were grouped according to the cell volume by flow cytometry sorter, and sorted into 96-well plates (RPMI 1640 medium with 20% serum added in advance) at a rate of 1 cell / well. After 5-7 days, ELISA and fluorescent immunochromatography screening were performed for identification.

[0094] Example 12

[0095] In this example, the antibodies produced by the monoclonal hybridoma cells obtained by the method of Example 11 were sequenced. The total RNA of the hybridoma cells was extracted using a kit, and then reverse transcription was performed using a reverse transcription kit to synthesize cDNA. Primers were designed for the nucleic acid sequence encoding the variable region of the antibody for PCR amplification. After the amplification was completed, the product was gel-recovered and purified, cloned into a pEASY vector, and sequenced for the first generation. The primers encoding the nucleic acid sequence of the heavy chain variable region of the antibody are shown in SEQ ID NO.17-18, and the primers encoding the nucleic acid sequence of the light chain variable region of the antibody are shown in SEQ ID NO.19-20.

[0096] SEQ ID NO. 17: CAGCCGGCCATGGCGCAGGTSCAGCTGCAGSAG.

[0097] SEQ ID NO. 18: CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT.

[0098] SEQ ID NO. 19: AATTTTCAGAAGCACGCGTAGATATCKTGMTSACCCAAWCTCCA.

[0099] SEQ ID NO. 20: GGGAAGATGGATCCAGTTGGTGCAGCATCAGC.

[0100] Example 13

[0101] In this example, the antibody amino acid sequence obtained in Example 12 was codon-optimized to adapt it to the expression of Expi293F cells. The sequence was synthesized into the pcDNA3.4 expression vector by gene synthesis, and transformed into Escherichia coli DH5α competent cells. After that, positive clones were picked for plasmid extraction, and then the light chain and heavy chain plasmids were transferred to Expi293F cells by transient transfection for antibody expression.

[0102] After transfection, the cells were cultured for 5-7 days, the cell supernatant was centrifuged at 4000 rpm for 2 min, filtered using a 0.22 μm pore size polypropylene membrane, and then purified using an AKTAPure purifier and a protein A affinity purification column to obtain coated antibodies and labeled antibodies.

[0103] Embodiment 14

[0104] In this example, the antibody obtained in Example 13 is used to prepare a fluorescent immunochromatographic test strip, and the preparation method comprises the following steps:

[0105] (1) Fluorescent microsphere labeled antibodies:

[0106] Mix 50 μL of ultrasensitive time-resolved fluorescent microspheres (hereinafter referred to as "microspheres") and 1 mL of activation buffer MES on a vortex mixer, centrifuge at 15,000 g at room temperature, remove the supernatant, add 500 μL of activation buffer MES to the precipitated microspheres, sonicate, add 5 μL of EDC solution to activate for 15 minutes, centrifuge at 15,000 g, and remove the supernatant. Add 500 μL of HEPES solution to the precipitated microspheres and sonicate. Add 90 μg of labeled antibody, mix on a vortex mixer, and place on a standard rotary mixer for 2 hours. Add 50 μL of blocking agent to a 1.5 mL centrifuge tube and place on a standard rotary mixer for blocking for 30 minutes. Centrifuge at 15,000 g at room temperature, remove the supernatant, and sonicate. At this time, the microspheres coupled with antibodies can be stored at 4°C for less than 2 weeks.

[0107] (2) Preparation of test strips

[0108] Place the plastic substrate, sample pad, conjugate pad, nitrocellulose membrane and absorbent paper as Figure 1 Assemble as shown, spray the labeled antibody of the coupled microspheres onto the conjugate pad using a film sprayer, and vacuum dry for 1 hour. Dilute the coated antibody with antibody coating solution, and use a film sprayer to draw a film on the detection line, and use goat anti-mouse IgG as a control antibody to draw a film on the quality control line.

[0109] Test Example 1

[0110] In this test example, hybridoma cells were prepared using the method for preparing hybridoma cells provided in Examples 1-7, and the fusion efficiency of cells in different groups was calculated. Fusion efficiency = total number of fused cell clusters in a 96-well plate / 96. The calculation results are shown in Table 1.

[0111] Table 1

[0112] Group Fusion voltage(V) Fusion time (μs) Fusion efficiency (cells / well) Example 1 800 40 9.3 Example 2 700 50 2.0 Example 3 1000 20 2.5 Example 4 800 30 3.4 Example 5 800 35 5.1 Example 6 850 40 6.4 Example 7 900 40 4.8

[0113] It can be concluded from Table 1 that the fusion efficiency of hybridoma cells can be effectively improved by adjusting the fusion voltage and fusion time of the electrofusion step.

[0114] Test Example 2

[0115] This test example used the method provided in Example 8 to obtain the ELISA screening results, and some of the results are excerpted as shown in Table 2.

[0116] Table 2

[0117] Hole No. Absorbance Time(s) Hole No. Absorbance Time(s) A05 2.0053 0.07 B01 1.6154 0.82 H06 3.3912 4.5 E06 1.2538 2.60 A12 3.4483 0.2 B03 0.9408 0.79 F05 3.5049 3.26 C09 0.7164 1.40 A01 3.54 0 G06 0.7336 3.85 F01 3.7246 3.33 G05 0.1110 3.83 A03 3.7516 0.03 H07 0.1115 4.48 F03 3.822 3.3 G04 0.1392 3.82 E01 3.9016 2.51 G08 0.1416 3.89 D06 4 1.99 Negative control 0.0541 4.44

[0118] Based on the ELISA results, hybridoma cells in wells with absorbance values ​​≥ 1.0 were selected as the antibody source.

[0119] Test Example 3

[0120] This test example uses the methods provided in Examples 9 and 10 to screen the candidate wells obtained in Test Example 2, and uses the method provided in Example 11 to monoclonalize the hybridoma cells by flow cytometry. The results are as follows: Figure 2 As shown, the hybridoma cells of group B were selected for sorting. After ELISA and fluorescent immunochromatography screening again, monoclonal hybridoma cells capable of secreting highly specific antibodies were obtained.

[0121] Test Example 4

[0122] In this test example, the variable regions of the secreted antibodies in the hybridoma cells obtained in Test Example 3 were sequenced. The amino acid sequence of the heavy chain variable region of the coated antibody was shown in SEQ ID NO.1, the amino acid sequence of the light chain variable region of the coated antibody was shown in SEQ ID NO.2, the amino acid sequence of the heavy chain variable region of the labeled antibody was shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the labeled antibody was shown in SEQ ID NO.4.

[0123] Test Example 5

[0124] In this test example, based on the results of test example 4, the coated antibody and the labeled antibody were obtained using the method provided in example 13, and the fluorescent immunochromatographic test strip was prepared using the method provided in example 14. 70 μL of each concentration of antigen was added to the sample pad, and the test was performed by a fluorescence detector after 3 minutes. The test results are shown in Table 3. The results in Table 3 were linearly fitted, and the results are shown in Table 3. Figure 3 shown.

[0125] Table 3

[0126] serial number Concentration (mg / L) T-line fluorescence value C line fluorescence value T / C 1 200 54332 33380 1.7641 2 100 43426 39660 1.1734 3 50 26999 43876 0.6664 4 10 6761 55124 0.1315 5 5 3391 53961 0.0635 6 0 0 52097 0.0000

[0127] like Figure 3 As shown, R 2 The value is 0.9999, that is, when the screened SAA antibody pair is used in a fluorescent immunochromatography detection experiment, the detection result has a good linearity, and the coated antibody and antibody can be used for the preparation of downstream kits.

[0128] In summary, the present invention obtains hybridoma cells capable of producing SAA antibodies by electrofusion, obtains monoclonal hybridoma cells by multiple screenings, successfully prepares coated antibodies and labeled antibodies, and applies them to prepare fluorescent immunochromatographic test strips. The results show good linearity and have broad application prospects.

[0129] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of coated antibodies and / or labeled antibodies in the preparation of fluorescent immunochromatographic test strips for detecting serum amyloid A, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the coated antibody include the sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the coated antibody include the sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the labeled antibody include the sequences shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the labeled antibody include the sequences shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

2. The use according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.

4.

4. The use according to any one of claims 1 to 3, characterized in that: The coated antibody is purified from hybridoma cells prepared by a hybridoma cell preparation method based on electrofusion technology; Preferably, the labeled antibody is purified from hybridoma cells prepared by a hybridoma cell preparation method based on electrofusion technology.

5. The use according to claim 4, characterized in that: The method for preparing hybridoma cells based on the electrofusion technology comprises: taking spleen cells of mice immunized with antigens and myeloma cells for electrofusion, and obtaining the hybridoma cells after screening, wherein the antigen is serum amyloid protein A.

6. The use according to claim 5, characterized in that: The fusion voltage of the electric fusion is 700-1000V, preferably 800-900V; Preferably, the pulse time of the electrofusion is 20 to 50 μs, preferably 30 to 40 μs; Preferably, the electrofusion is repeated 2 to 4 times.

7. The use according to claim 5, characterized in that: The screening includes screening using HAT culture medium, ELISA screening and fluorescent immunochromatographic test strip screening; Preferably, the ELISA screening further includes a step of antibody purification; Preferably, the antibody purification method comprises: using magnetic beads to purify the supernatant of ELISA positive wells to obtain coated antibodies to be screened and labeled antibodies to be screened; Preferably, the fluorescent immunochromatographic test strip comprises a blank nitrocellulose membrane, a sample pad and absorbent paper, and the step of screening the fluorescent immunochromatographic test strip comprises: dripping the coated antibody to be screened onto the blank nitrocellulose membrane, and after drying, dripping a mixture of the labeled antibody to be screened and the antigen labeled with fluorescent microspheres onto the sample pad, and screening for positive results; Preferably, the fluorescent immunochromatographic test strip further comprises a step of monoclonal hybridoma cells after screening; Preferably, the method for monocloning hybridoma cells comprises: sorting hybridoma cells by flow cytometry and screening again using the ELISA screening and fluorescent immunochromatography test strip screening.

8. A fluorescent immunochromatographic test strip for detecting serum amyloid A, characterized in that: The fluorescent immunochromatographic test strip for detecting serum amyloid A comprises a plastic substrate, a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper, wherein the conjugation pad contains a labeled antibody, a detection line and a quality control line are drawn on the nitrocellulose membrane, and the detection line contains a coated antibody; The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the coated antibody include the sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the coated antibody include the sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the labeled antibody include the sequences shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the labeled antibody include the sequences shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

9. The fluorescent immunochromatographic test strip according to claim 8, characterized in that: The amino acid sequence of the heavy chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the coated antibody includes the sequence shown in SEQ ID NO.

2.

10. The fluorescent immunochromatographic test strip according to claim 8 or 9, characterized in that: The amino acid sequence of the heavy chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the labeled antibody includes the sequence shown in SEQ ID NO.4.

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