CN116444662B - Monoclonal antibody pair of anti-canine distemper virus N protein, nucleic acid molecule, cell, preparation method and application - Google Patents

Monoclonal antibody pair of anti-canine distemper virus N protein, nucleic acid molecule, cell, preparation method and application Download PDF

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CN116444662B
CN116444662B CN202310244290.3A CN202310244290A CN116444662B CN 116444662 B CN116444662 B CN 116444662B CN 202310244290 A CN202310244290 A CN 202310244290A CN 116444662 B CN116444662 B CN 116444662B
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amino acid
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CN116444662A (en
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张涛
邹柳
林奕婷
周国栋
盛布恩
张松
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Territory Animal Diagnosis And Treatment Technology Xiamen Co ltd
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Abstract

The invention belongs to the technical fields of immunology and molecular virology, and discloses a monoclonal antibody pair of anti-canine distemper virus N protein, a nucleic acid molecule, a cell, a preparation method and application. A monoclonal antibody pair or antigen binding fragment thereof comprises a first antibody or antigen binding fragment thereof, the amino acid sequence of which is shown as SEQ ID NO. 1-3, the amino acid sequence of which is shown as SEQ ID NO. 4-6, of which the amino acid sequence of which is shown as SEQ ID NO. 1-3; and, the amino acid sequence of the heavy chain variable region complementarity determining regions 1-3 is shown in SEQ ID NO. 7-9, and the amino acid sequence of the light chain variable region complementarity determining regions 1-3 is shown in SEQ ID NO. 10-12. The monoclonal antibody provided by the invention has high titer, has strong binding capacity with the N protein of the canine distemper virus, can not be interfered by other viruses and medicines, and realizes high-specificity and high-sensitivity detection of the canine distemper virus.

Description

Monoclonal antibody pair of anti-canine distemper virus N protein, nucleic acid molecule, cell, preparation method and application
Technical Field
The invention belongs to the technical fields of immunology and molecular virology, and particularly relates to a monoclonal antibody pair of anti-canine distemper virus N protein, a nucleic acid molecule, a cell, a preparation method and application.
Background
Canine Distemper (CD) is a severe systemic disease caused by Canine Distemper Virus (CDV) infection in dogs, with major clinical manifestations mainly fever, catarrhal respiratory symptoms, gastrointestinal disease, rash, and central nervous system disease. CD was originally thought to be a canine susceptible infectious disease, but in recent years there has been an increasing report of the onset of animals such as weasels, raccoons, hyridae, xiong Maoke, primates, artiodactyles, finpodales, seal, and whales dolphins, and there is evidence that CDV can spread across host species with an ever-expanding trend in the spectrum of infection, and the resulting hazard is also increasing. Therefore, developing specific, rapid and sensitive detection techniques for early detection of CDV is of great significance.
CDV belongs to measles virus genus of Paramyxoviridae, is a single-stranded, non-segmented negative-strand RNA virus with envelope, has a total length of 15-16 kb, and has a genome mainly encoding nucleocapsid protein (N protein), hemagglutinin (H protein), fusion protein (F protein), phosphoprotein (P protein), large protein (L protein and matrix protein (M protein), wherein the encoding gene variability of the N protein is small compared with other proteins, plays an important role in the virus replication process, can cause body humoral immunity and cellular immune response, and can be used as target antigen in CDV immunological detection.
Currently, CDV immunological detection techniques mainly include methods such as an agar diffusion method, an enzyme-linked immunosorbent method, a serum neutralization method, an immunofluorescence method, an immunohistochemical method, a colloidal gold immunochromatography method, and the like. The method needs an antibody with good binding capacity with N protein as a detection substance to detect whether the N protein of the canine distemper virus exists in a sample; however, the antibodies used in the prior art have problems of low specificity or low sensitivity, which may lead to the occurrence of false positive or false negative detection results.
Disclosure of Invention
The invention aims to obtain a canine distemper virus N protein with excellent specificity and sensitivity, realize high-specificity and high-sensitivity detection of canine distemper virus, and provide a monoclonal antibody pair, nucleic acid molecules, cells, a preparation method and application of the anti-canine distemper virus N protein.
In a first aspect, the monoclonal antibody pair or antigen binding fragment thereof provided by the present invention adopts the following technical scheme:
a monoclonal antibody pair or antigen-binding fragment thereof comprising: the amino acid sequences of the complementarity determining regions 1-3 of the heavy chain are shown as SEQ ID NO. 1-3, respectively, and the amino acid sequences of the complementarity determining regions 1-3 of the light chain are shown as SEQ ID NO. 4-6, respectively; and, the amino acid sequences of the heavy chain variable region complementarity determining regions 1-3 are shown in SEQ ID NO. 7-9, respectively, and the amino acid sequences of the light chain variable region complementarity determining regions 1-3 are shown in SEQ ID NO. 10-12, respectively.
In some embodiments, the amino acid sequence of the heavy chain variable region of the first antibody or antigen binding fragment thereof is shown in SEQ ID NO. 13 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the second antibody or antigen binding fragment thereof is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
In some specific embodiments, the first antibody or antigen binding fragment thereof is selected from the group consisting of Fab, fab ', F (ab') 2 Fd, fv, dAb and one or more of complementarity determining region fragments, single chain antibodies, chimeric antibodies and multispecific antibodies.
In some embodiments, the second antibody or antigen binding fragment thereof is selected from the group consisting of Fab, fab ', F (ab') 2 Fd, fv, dAb and one or more of complementarity determining region fragments, single chain antibodies, chimeric antibodies and multispecific antibodies.
In a second aspect, the invention provides a nucleic acid molecule encoding the monoclonal antibody pair described above or an antigen binding fragment thereof.
In a third aspect, the present invention provides a cell capable of secreting a monoclonal antibody pair as described above or an antigen binding fragment thereof, the cell comprising a nucleic acid molecule as described above.
In a fourth aspect, the present invention provides a method of preparing a monoclonal antibody pair as described above, or an antigen binding fragment thereof, comprising: the above-described cells are cultured under appropriate conditions, and the monoclonal antibody pair or antigen-binding fragment thereof is recovered from the cell culture.
In a fourth aspect, the present invention provides a kit according to the following technical solution:
a kit comprises a first antibody or antigen binding fragment thereof with the amino acid sequences of the complementarity determining regions 1-3 of the heavy chain variable region shown in SEQ ID NO. 1-3 and the amino acid sequences of the complementarity determining regions 1-3 of the light chain variable region shown in SEQ ID NO. 4-6;
and, a second antibody or antigen-binding fragment thereof having the amino acid sequences of the complementarity determining regions 1 to 3 of the heavy chain variable region shown in SEQ ID NOS.7 to 9 and the amino acid sequences of the complementarity determining regions 1 to 3 of the light chain variable region shown in SEQ ID NOS.10 to 12.
In some embodiments, the first antibody or antigen-binding fragment thereof is a capture antibody and the second antibody or antigen-binding fragment thereof is a labeled antibody.
In some specific embodiments, the first antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.
In some embodiments, the second antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.
In a fifth aspect, the invention provides the use of a monoclonal antibody pair as described above, or an antigen binding fragment thereof, a nucleic acid molecule and/or a kit for the detection of canine distemper virus for non-diagnostic purposes.
The beneficial effects are that:
the primary antibody or the antigen binding fragment thereof, the secondary antibody or the antigen binding fragment thereof provided by the invention have high affinity with the N protein of the canine distemper virus; when the first antibody or the antigen binding fragment thereof is used as a capture antibody, the second antibody or the antigen binding fragment thereof is used as a labeled antibody, the spatial structure of the canine distemper virus N protein is changed after the first antibody or the antigen binding fragment thereof is combined with the second antibody or the antigen binding fragment thereof, so that the first antibody or the antigen binding fragment thereof has better combination capability with the canine distemper virus N protein, and the first antibody or the antigen binding fragment thereof can not be interfered by other disease virus strains and drugs, thereby realizing high-specificity and high-sensitivity detection on the canine distemper virus N protein.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold chromatographic strip according to embodiment 2 of the present invention;
FIG. 2 is a graph showing the experimental results of the colloidal gold chromatographic strip provided in example 2 of the present invention for testing the inactivated canine distemper virus liquid with different dilution factors;
FIG. 3 is a graph showing the experimental results of the colloidal gold chromatographic strip provided in example 3 of test example 2 for testing the inactivated canine distemper virus liquid with different dilution factors;
fig. 4 is a graph showing the experimental results of detecting the inactivated canine distemper virus liquid with different dilution factors by using the colloidal gold chromatographic strip provided in comparative example 1 in test example 2.
Detailed Description
The monoclonal antibody pair or the antigen binding fragment thereof provided by the invention comprises a first antibody and the antigen binding fragment thereof, and a second antibody and the antigen binding fragment thereof.
In the first antibody and antigen binding fragment thereof, the adjacent heavy chain variable region with the amino acid sequence shown as SEQ ID NO. 13 and the light chain variable region with the amino acid sequence shown as SEQ ID NO. 14 are folded to form a canine distemper virus N protein binding site with a specific three-dimensional space structure, and antigen determinants on canine distemper virus N proteins are specifically identified and targeted through the heavy chain variable region complementarity determining regions 1-3 with the amino acid sequences shown as SEQ ID NO. 1-3 and the light chain variable region complementarity determining regions 1-3 with the amino acid sequences shown as SEQ ID NO. 4-6.
In the invention, the amino acid sequence of the heavy chain constant region of the primary antibody and the antigen binding fragment thereof is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 18.
In the second antibody and antigen binding fragment thereof, the adjacent heavy chain variable region with the amino acid sequence shown as SEQ ID NO. 15 and the light chain variable region with the amino acid sequence shown as SEQ ID NO. 16 are folded to form a canine distemper virus N protein binding site with a specific three-dimensional space structure, and the other antigenic determinant on the canine distemper virus N protein is specifically identified and targeted through the heavy chain variable region complementarity determining regions 1-3 with the amino acid sequences shown as SEQ ID NO. 7-9 and the light chain variable region complementarity determining regions 1-3 with the amino acid sequences shown as SEQ ID NO. 10-12.
In the invention, the amino acid sequence of the heavy chain constant region of the second antibody and the antigen binding fragment thereof is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 20.
Among the first antibody and antigen-binding fragment thereof and the second antibody and antigen-binding fragment thereof, the antigen-binding fragment referred to is a polypeptide having a specific binding ability to the N protein of canine distemper virus, which can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of the whole first antibody or second antibody.
In some embodiments, the primary antibody and antigen binding fragments thereof may be, but are not limited to, fab ', F (ab') 2 Fd, fv, dAb and one or more of complementarity determining region fragments, single chain antibodies, chimeric antibodies and multispecific antibodies.
In some embodiments, the secondary antibody and antigen binding fragments thereof may be, but are not limited to, fab ', F (ab') 2 Fd, fv, dAb and one or more of complementarity determining region fragments, single chain antibodies, chimeric antibodies and multispecific antibodies.
In the invention, fv refers to a small molecular genetic engineering antibody, and is specifically formed by connecting a heavy chain variable region and a light chain variable region through a short peptide; fab refers to a fragment having antigen binding ability obtained after papain hydrolysis of an antibody molecule, specifically a fragment obtained by S-S linkage of a heavy chain variable region and a light chain variable region; f (ab') 2 Refers to a macromolecular fragment obtained by pepsin hydrolysis of antibody molecules, in particular to a fragment obtained by S-S linkage between heavy chain variable regions of two Fab, which has bivalent anti-tumor effectAnd the somatic activity can be combined with the antigenic determinants of two corresponding recombinant canine distemper virus N proteins.
The invention also provides a nucleic acid molecule which encodes the monoclonal antibody pair or antigen binding fragment thereof.
In some embodiments, the nucleic acid molecule comprises a fragment encoding the heavy chain variable region of the first antibody and antigen-binding fragments thereof, the nucleotide sequence of which is shown in SEQ ID NO. 21; also included are fragments encoding the light chain variable region of the primary antibody and antigen binding fragments thereof, the nucleotide sequences of which are shown in SEQ ID NO. 22.
In some embodiments, the nucleic acid molecule comprises a fragment encoding the heavy chain variable region of the second antibody and antigen-binding fragments thereof, the nucleotide sequence of which is shown in SEQ ID NO. 23; also included are fragments encoding the light chain variable region of the secondary antibody and antigen binding fragments thereof, the nucleotide sequences of which are shown in SEQ ID NO. 24.
The method of producing the nucleic acid molecule is not limited in the present invention, and may be specifically, but not limited to, extraction from hybridoma cells by a DNA extraction technique or synthesis by an engineering recombinant technique and a chemical synthesis method.
The invention provides a cell comprising the nucleic acid molecule, which can synthesize and secrete a first antibody or an antigen binding fragment thereof, or synthesize and secrete a second antibody or an antigen binding fragment thereof, or secrete the above substances simultaneously.
In the present invention, the cells may be plasma cells obtained by an immune reaction and capable of secreting the first antibody or the second antibody, hybridoma cells obtained by a hybridoma technique and capable of secreting the first antibody or the second antibody, or cells obtained by a genetic engineering technique and capable of secreting the first antibody or an antigen-binding fragment thereof, or the second antibody or an antigen-binding fragment thereof.
The preparation method of the monoclonal antibody pair or the antigen binding fragment thereof provided by the invention specifically comprises the following steps: culturing the cells under suitable conditions, and recovering the monoclonal antibody pair or antigen-binding fragment thereof from the cell culture.
In some specific embodiments, the monoclonal antibody pair or antigen binding fragment thereof may be prepared by recombinant DNA technology to obtain recombinant canine distemper virus N protein with the amino acid sequence shown as SEQ ID NO. 25, immunizing mice with the recombinant canine distemper virus N protein to obtain immune spleen cells, preparing hybridoma cells by hybridoma technology, and secreting and expressing the first antigen or the second antigen.
In some specific embodiments, the monoclonal antibody pair or antigen binding fragment thereof may be prepared by obtaining recombinant canine distemper virus N protein with an amino acid sequence shown as SEQ ID NO. 25 by recombinant DNA technology, immunizing mice with the recombinant canine distemper virus N protein to obtain immune spleen cells, extracting nucleic acid molecules encoding the synthetic monoclonal antibody pair or antigen binding fragment thereof from the immune spleen cells by DNA extraction technology, transforming receptor cells by genetic engineering technology, and secreting and expressing the first antigen or antigen binding fragment thereof, or the second antigen or antigen binding fragment thereof from the receptor cells.
In the present invention, cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures and the like used in the above preparation methods are conventional procedures widely used in the corresponding fields.
The kit provided by the invention comprises a first antibody or antigen binding fragment thereof, wherein the amino acid sequences of the complementarity determining regions 1-3 of the heavy chain variable region are shown as SEQ ID NO. 1-3, and the amino acid sequences of the complementarity determining regions 1-3 of the light chain variable region are shown as SEQ ID NO. 4-6;
and, a second antibody or antigen-binding fragment thereof having the amino acid sequences of the complementarity determining regions 1 to 3 of the heavy chain variable region shown in SEQ ID NOS.7 to 9 and the amino acid sequences of the complementarity determining regions 1 to 3 of the light chain variable region shown in SEQ ID NOS.10 to 12.
The kit provided by the invention takes a first antibody or antigen binding fragment thereof and a second antibody or antigen binding fragment thereof as detection reagents, and detects the presence or the level of the canine distemper virus or the N protein thereof or the RDB of the N protein thereof in a sample by using methods such as an enzyme-linked immunosorbent assay, an enzyme immunoassay, a chemiluminescent immunoassay, a radioimmunoassay, a fluorescent immunoassay, an immunochromatography or a competition method.
In particular, the first antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a luminescent substance, a colored substance, and an enzyme.
In particular, the second antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.
In the present invention, a radioisotope may be used 125 And I, labeling the primary antibody or the antigen binding fragment thereof, the secondary antibody or the antigen binding fragment thereof by using a chloramine T (N-chlorobenzenesulfonamide) method or an Indogen coating test tube method.
In the present invention, the fluorescent dye is one or more selected from fluorescein fluorescent dye, rhodamine fluorescent dye, cyanine fluorescent dye and coumarin fluorescent dye, and may be one or more selected from fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate and phycoerythrin.
In the present invention, the enzyme used for labeling the primary antibody or the antigen-binding fragment thereof, the secondary antibody or the antigen-binding fragment thereof may be, but is not limited to, horseradish peroxidase and/or alkaline phosphatase.
In some specific embodiments, provided are kits, wherein the first antibody or antigen-binding fragment thereof is used as a capture antibody, the second antibody or antigen-binding fragment thereof is used as a labeled antibody, and the canine distemper virus or the N protein or the RDB of the N protein thereof is detected by a double antibody sandwich method; specifically, the second antibody or antigen binding fragment thereof is firstly combined with the RDB of the canine distemper virus or the N protein thereof to obtain a complex, the complex is combined with the first antibody or the antigen binding fragment thereof, and the qualitative and/or quantitative detection of the RDB of the canine distemper virus or the N protein thereof is realized by detecting the detectable label.
When the second antibody or the antigen binding fragment thereof is combined with the RDB of the canine distemper virus or the N protein thereof, the spatial conformation of the RDB of the canine distemper virus or the N protein thereof is found to be changed, so that the first antibody or the antigen binding fragment thereof has more excellent combination capability with the RDB of the canine distemper virus or the N protein thereof, and finally, the high-specificity and high-sensitivity detection of the RDB of the canine distemper virus or the N protein thereof is realized.
In the application provided by the invention, the monoclonal antibody is used for detecting the canine distemper virus or antigen binding fragments, nucleic acid molecules and/or kits thereof, and has higher sensitivity and specificity compared with the monoclonal antibodies of other canine distemper virus N proteins.
The amino acid and nucleotide sequences involved in the invention are shown in Table 1:
table 1.
The following detailed description of the preparation examples and examples of the present invention is intended to be illustrative of the invention and is not to be construed as limiting the invention. The preparation examples and examples do not identify specific techniques or conditions, which may be followed by those described in the literature in the field or by the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The reagents and sources involved in the preparation examples and examples are as follows:
freund's complete adjuvant (sigma, cat# F5881);
freund's incomplete adjuvant (sigma company, cat# F5506);
myeloma cells (ATCC, cat No. BNCC 100908);
1640 medium (Vivacell, cat. No. C3010-0500);
50% PEG1500 (Roche, cat. No. 10783641001);
fetal bovine serum FBS (herba Siraitiae Grosvenorii, product number 11011-8611);
penicillin-streptomycin diabodies (100×, shanghai, cat# E607011-0100);
HAT medium additive (50×, sigma company, cat# H0262);
HRP-labeled goat anti-mouse secondary antibody (Xiamen taijing, cat No. TJ-211229 CN);
HT Medium additive (sigma, cat# H0137);
an adjuvant special for ascites (Beijing boolone, product number is KX 0210048);
protein a column (boilon biosome, cat No. AA 301307);
horseradish peroxidase (risc reagent, cat No. RS 20220118);
DNP antibody (Merck company, cat. Number D9781-2 ML);
0.01% colloidal gold solution (micllin, cat No. C805628);
Anti-Canine Distemper Virus I (sea peptide biotechnology (Shanghai) Co., ltd.,. Trade mark 3CD 10-5-4);
Anti-Canine Distemper Virus II (sea peptide biotechnology (Shanghai) Inc., cat# 3CD 10-8-1).
Preparation example 1.
The preparation example provides a preparation method of hybridoma cells, which comprises animal immunity, cell immunity, screening of positive hybridoma cells and cloning of positive hybridoma cells, and comprises the following specific steps:
1. immunization of animals
(1) Preparation of the immunogen:
mixing the recombinant canine distemper virus N protein with the amino acid sequence shown as SEQ ID NO. 25 with Freund's complete adjuvant in an equal volume to obtain a first immunogen;
mixing the recombinant canine distemper virus N protein with the amino acid sequence shown as SEQ ID NO. 25 with Freund's incomplete adjuvant in an equal volume to prepare a second immunogen;
mixing the recombinant canine distemper virus N protein with the amino acid sequence shown as SEQ ID NO. 25 with normal saline in an equal volume to prepare the third immunogen.
(2) Immunization process:
primary immunization: injecting 50 mug of a first immunogen into the back of a BALB/C female mouse at 3-4 points subcutaneously;
two weeks later, a second immunization is carried out, and 50 mug of a second immunogen is injected into the back of the BALB/C female mouse at about 4 points subcutaneously;
after two weeks, performing third immunization, and injecting 50 mug of a second immunogen into the back of the BALB/C female mouse subcutaneously at 3-4 points;
two weeks later, a fourth immunization-impact immunization was performed, and 50 μg of the third immunogen was intraperitoneally injected into BALB/C female mice;
a fifth immunization was performed 24h later and 50 μg of the third immunogen was intravenously injected into the BALB/C female rat tail.
2. Cellular immunity
(1) Preparation of spleen cell suspension: on the third day after the fifth immunization of the BALB/C female mice, the eyeballs of the BALB/C female mice are removed for blood sampling, and the serum of the BALB/C female mice is separated as a positive control in antibody detection; meanwhile, the cervical dislocation kills the BALB/c female mice, and spleen is taken to prepare spleen cell suspension;
preparation of myeloma cell suspension: resuscitates myeloma cells two weeks in advance (ensuring that myeloma cells are in logarithmic growth phase when in use), and prepares myeloma cell suspension;
preparation of feeder cells: the day before cell fusion, blank BALB/c female mouse peritoneal macrophages and spleen cells were taken and cultured in 96-well plates to obtain feeder layer cell-containing plates (cell concentration 1X 10) 4 Well), feeder cells are prepared;
(2) Cell fusion process:
adopting PEG (polyethylene glycol) to mediate cell fusion, taking spleen cell suspension and myeloma cell suspension, uniformly mixing in a serum-free 1640 culture medium according to the ratio of cell number to cell number of 5:1, centrifuging for 5min under the condition of 1200rpm, and removing supernatant;
flick the bottom of the centrifuge tube with finger to mix the two kinds of cells, put into a beaker filled with 37 ℃ water for heat preservation, add 1mL 50% peg1500 fused cells in 1min, shake while adding, and stand for 30s after adding; adding serum-free 1640 culture medium to terminate fusion, centrifuging at 800rpm for 5min, suspending the precipitate with HAT culture medium, packaging into cell plate containing feeder cells to obtain cell plate containing fused cell-feeder cells, and placing at 37deg.C and 5% CO 2 Is cultured in a cell culture box;
wherein, the preparation of 500ml HAT medium requires the following reagents: 100mL of fetal bovine serum FBS, 5mL of penicillin-streptomycin diabody for cell culture, 10mL of HAT medium additive and 385mL of 1640 medium.
3. Screening of positive hybridoma cells
And culturing the cell plate containing the fusion cells and the feeder layer cells until the 4 th day, performing half liquid exchange, continuously culturing until the 7 th day, performing full liquid exchange, and screening positive holes by adopting a conventional indirect ELISA method when the fusion cells cover 10-50% of the bottoms of the holes.
The indirect ELISA method specifically comprises the following steps:
(1) And (3) wrapping the plate: recombinant canine distemper virus N protein with the amino acid sequence shown as SEQ ID NO. 25 is used as a coating antigen, and 0.05mol/L CB buffer (Na) with the pH value of 9.6 is used 2 CO 3 31.8g，NaHCO 3 58.8g, 2L of ultrapure water) to 2. Mu.g/mL, adding an ELISA plate at 100. Mu.L/hole, coating at 4 ℃ overnight, then beating to dry, sealing with gelatin-PBS buffer solution with the mass-volume ratio of 1%, sealing at 300. Mu.L/hole, sealing at 4 ℃ overnight, and beating to dry for later use.
(2) And (3) detection: cell culture supernatant in cell plates containing fused cell-feeder cells was added to an ELISA plate at 100. Mu.L/well, incubated at 37℃for 60min, washed with 0.01mol/LPBST buffer containing Tween-20 and then patted dry, 100. Mu.L/well of HRP-labeled goat anti-mouse secondary antibody was added, incubated at 37℃for 60min, washed and patted dry, 100. Mu.L/well of TMB chromogenic solution was added, incubated at 37℃for 10min in the absence of light, and 50. Mu.L/well of 1mol/L HCl was added to terminate the reaction.
Meanwhile, BALB/c female mouse serum obtained by eyeball blood sampling and separation in the step of cell immunization is used as a positive control, and fusion cells with higher antibody titer are screened out, namely positive hybridoma cells.
4. Cloning of positive hybridoma cells
Positive hybridoma cells obtained by screening from the cell plate containing the fusion cells-feeder cells are derived from more than two hybridoma cells, so that antibodies secreted by the hybridoma cells obtained by screening are of different qualities. In order to obtain fully homogeneous monoclonal antibodies, positive hybridoma cells need to be cloned.
The day before cloning, preparing feeder cells according to the method of the step (1) in cellular immunity and plating to obtain a cell plate containing the feeder cells; suspending positive hybridoma cells obtained by screening by using HT culture medium, blowing and mixing by using a liquid transfer device, inoculating to cell plates containing feeder cells, diluting cells in cell plate holes to 1 cell in each hole by using HT culture medium, placing at 37deg.C, 5% CO 2 The antibody can be detected after the wet culture for 7 to 10 days and the appearance of macroscopic cloned cells.
Wherein, the following reagents are required for preparing 500mL of HT medium: 100mL of fetal bovine serum FBS, 5mL of penicillin-streptomycin diabody for cell culture, 10mL of HT medium additive, 385mL of 1640 medium.
Observing under an inverted microscope, marking out a hole in which only a single clone grows, finally primarily screening to obtain 10 monoclonal antibody hybridoma cell strains, and taking the supernatant for the next functional screening. The above 10 hybridoma cell lines were designated and are specifically shown in table 2.
TABLE 2.10 monoclonal antibody hybridoma cell strains and antibodies
Cell strain Monoclonal antibodies HRP-antibody conjugated complex protein
3A7 cell lines 3A7 antibodies HRP-3A7 antibodies
5G11 cell line 5G11 antibodies HRP-5G11 antibodies
8G8 cell line 8G8 antibodies HRP-8G8 antibodies
8B5 cell line 8B5 antibodies HRP-8B5 antibodies
2H6 cell line 2H6 antibodies HRP-2H6 antibodies
6E9 cell lines 6E9 antibodies HRP-6E9 antibodies
12F11 cell line 12F11 antibodies HRP-12F11 antibodies
6A2 cell lines 6A2 antibodies HRP-6A2 antibodies
9C12 cell line 9C12 antibodies HRP-9C12 antibodies
3C3 cell lines 3C3 antibodies HRP-3C3 antibodies
Preparation example 2.
The preparation example provides a method for preparing monoclonal antibodies against canine distemper virus N protein, wherein the monoclonal antibodies are prepared by respectively preparing ascites fluid and purifying monoclonal antibodies by using monoclonal antibody hybridoma cell strains prepared in preparation example 1, and specifically comprises the following steps:
s1, preparation of ascites: ab/c mice of 8-10 weeks old were intraperitoneally injected with an adjuvant specific for ascites, and the monoclonal antibody hybridoma cell line (1X 10d after injection 6 Individual cells/individual) were injected into the abdominal cavity of Balb/c mice, and the ascites of Balb/c mice was collected by a medical syringe over 12 d.
S2, purifying the monoclonal antibody: (1) The Balb/c mouse ascites collected in S1 was poured into a centrifuge tube, centrifuged at 12500rpm for 20min, and the supernatant was collected and sequentially filtered with a 0.22 μm filter membrane, and purified by Protein A column affinity chromatography, and the monoclonal antibodies prepared by each monoclonal antibody hybridoma cell line were named as shown in Table 2.
Preparation example 3.
The preparation example provides a method for preparing HRP-antibody coupled complex protein, and the 10 monoclonal antibodies prepared in the preparation example 2 are subjected to HRP labeling, which specifically comprises the following steps:
s1, diluting the monoclonal antibody prepared in the preparation example 2 to 2mg/mL by using 0.05mol/L CB buffer with pH=9.6, stirring and dialyzing at 4 ℃, changing the liquid once every 1h, and dialyzing for 5 times.
S2 Horseradish peroxidase (HRP) with concentration of 20mg/mL) Solution and NaIO with concentration of 20mg/mL 4 After the solutions are mixed according to the volume ratio of 1:1, a centrifuge tube is immediately wrapped by tin foil, and HRP is activated for 30min at 4 ℃ in the dark;
the ethylene glycol was then slowly added dropwise to the HRP-activated centrifuge tube while gently shaking (1. Mu.L of ethylene glycol per 1mg of HRP), continued to block light at 4deg.C for 30min, and the activated HRP was terminated.
S3, adding the activated HRP solution into an antibody dialysis membrane (1 mg of antibody is added with 1mg of HRP), coupling overnight at 4 ℃ in a CB buffer with the pH value of 9.6 and 0.05mol/L,
and (3) continuing to dialyze for 2 hours after changing the CB buffer solution the next day, and transferring the coupled dialysate into a centrifuge tube after dialysis to obtain the antibody-HRP coupling solution.
S4, according to HRP and NaBH 4 The mass ratio of NaBH to NaBH is 25:1, and NaBH with the concentration of 20mg/mL is taken 4 Mixing the solution with an antibody-HRP coupling solution, reacting for 2 hours at 4 ℃, and uniformly mixing every 0.5 hour in an upside down manner;
the reaction solution was precipitated with 50% ammonium sulfate solution at 4℃for 15min and centrifuged at 10000rpm for 10min to obtain HRP-antibody conjugated complex proteins, as shown in Table 2.
S5, blowing dissolution or vortex dissolution precipitation of HRP-antibody coupled composite protein with preservation solution, and preserving at-20 ℃; the stock solution was 50% glycerol, 10% calf serum and 20mM PBS pH 7.4.
The final HRP-antibody conjugated complex protein designations are shown in Table 2.
Test example 1.
Pairing 10 monoclonal antibodies prepared in preparation example 2 and 10 HRP-antibody coupled composite proteins prepared in preparation example 3 in pairs, taking the monoclonal antibodies as capture antibodies and the HRP-antibody coupled composite proteins as labeled antibodies, and adopting a double antibody sandwich method, wherein the specific steps are as follows: coating 10 monoclonal antibodies prepared in preparation example 2 serving as capture antibodies on an ELISA plate respectively, adding canine distemper virus N protein into holes of the ELISA plate, and washing out unbound canine distemper virus N protein after incubation; respectively adding HRP-antibody conjugated composite protein as a labeled antibody, and washing off unbound HRP-antibody conjugated composite protein after incubation; finally, the color development liquid was added to develop color, and the absorbance was measured at 280nm using a spectrophotometer, and the results are shown in Table 3.
TABLE 3 detection results of pairwise pairing of 10 monoclonal antibodies and 10 HRP-antibody-coupled composite proteins
The test result shows that the pair of monoclonal antibodies 12F11 and 8B5 has high titer and can be applied to qualitative and/or quantitative detection of canine distemper virus and N protein or RDB of N protein thereof.
Example 1.
The results of cell sequencing of the 12F11 cell line and the 8B5 cell line are provided in this example and are shown in Table 4.
Table 4.
Example 2.
The present embodiment provides a kit comprising a colloidal gold chromatographic strip prepared by using the 12F11 antibody and the 8B5 antibody in embodiment 1, wherein the colloidal gold chromatographic strip uses the 12F11 antibody as a capture antibody and the 8B5 antibody as a labeled antibody, and the specific preparation method comprises the following steps:
s1, preparing a detection pad: 2.901g of Na is weighed 2 HPO 4 ·12H 2 O, 0.2914g of NaH 2 PO 4 ·2H 2 O, 8.5g of NaCl and 25g of sucrose are dissolved in 1000mL of ultrapure water to prepare a coating buffer solution; taking 1mg/mL of 12F11 antibody, and spraying the nitrocellulose membrane according to the spraying amount of 1.0 mu L/cm to obtain a detection line with the length of 30 cm; taking 1mg/mL DNP antibody, spraying the nitrocellulose membrane according to the spraying amount of 1.0 mu L/cm to obtain a quality control line with the length of 30cm, and controlling the qualityThe distance between the line and the detection line is 5mm; and (3) the nitrocellulose membrane coated with the detection line and the quality control line is the detection pad, the detection pad is stuck on a PVC lining plate, and the PVC lining plate is placed in a 50 ℃ oven for drying for 24+/-2 hours.
S2, labeling of gold label conjugate: weighing 0.36g of Tris, 1.0g of BSA, 0.2g of PEG20000, 100 mu L of Tween-20, 2g of sucrose and 100 mu L of proclin300, and dissolving in 100mL of ultrapure water to obtain a compound solution; 100mL of 0.01% colloidal gold solution is taken and placed in a clean container, and 1-3 mL of K with the concentration of 0.2M is added 2 CO 3 Stirring the solution uniformly, adding 500-1000 mug of 8B5 antibody, stirring and reacting for 15min, adding 500 mug of 20% BSA solution, stirring, sealing for 10min, centrifuging for 20min at 8000-10000 r/min, discarding the supernatant, re-dissolving and precipitating by adopting a re-solution to obtain the marked antibody, and storing at 4 ℃ for later use.
S3, preparing a bonding pad: 0.362g of Tris, 0.3g of sodium caseinate, 200. Mu.L of Triton X-405, 100. Mu.L of Tween-20, 3g of sucrose, 100. Mu.L of proclin300 were weighed and dissolved in 100mL of ultrapure water to obtain a gold-labeled conjugate solution; adding a certain amount of labeled antibody into the gold-labeled conjugate solution, uniformly mixing, wherein the concentration of the labeled antibody in the solution is 5-15 wt%, uniformly coating the solution on a glass fiber membrane (25 cm multiplied by 30 cm) according to the coating amount of 35 mL/sheet, and drying in an oven at 50 ℃ for 24 hours to obtain the conjugate pad.
S4, preparing a sample pad: 2.901g of Na is weighed 2 HPO 4 ·12H 2 O, 0.2914g of NaH 2 PO 4 ·2H 2 O, 8.5g of NaCl, 25g of sucrose, 0.5g of BSA and 1mL of proclin300 are dissolved in 1000mL of ultrapure water to obtain a sample pad treatment solution; uniformly coating the sample pad treatment liquid in 32mL of each plate on a glass fiber membrane (25 cm multiplied by 30 cm), and drying in an oven at 50 ℃ for 24+/-2 hours to obtain a sample pad;
s5, assembling the gold-labeled paper strips: according to the structure shown in FIG. 1, a sample pad and a bonding pad are sequentially stuck to one end of a PVC board, which is close to a detection line, under the conditions that the temperature is 18-28 ℃ and the humidity is 10-30%, and the sample pad is partially overlapped on the bonding pad and the bonding pad is partially overlapped on the detection pad; the water absorbing paper is stuck to one end of the PVC plate, which is far away from the detection line, and the water absorbing paper is partially overlapped on the detection pad to form a large plate, and then the large plate is cut into strips according to the width of 3mm, so that the colloidal gold chromatographic strip is obtained, and the assembled colloidal gold chromatographic strip is arranged in the shell of the test paper card for facilitating the subsequent detection.
Example 3.
The present example provides a kit, and a colloidal gold chromatographic strip was prepared according to the method provided in example 2, except that the 8B5 antibody was used as the capture antibody, and the 12F11 antibody was used as the labeled antibody in the colloidal gold chromatographic strip, with the same other conditions.
Comparative example 1.
The comparative example provides a kit, and a colloidal gold chromatographic strip was prepared according to the method provided in example 2, except that Anti-Canine Distemper Virus I was used as the capture antibody and Anti-Canine Distemper Virus II was used as the labeled antibody in the colloidal gold chromatographic strip, with the same other conditions.
Test example 2.
The colloidal gold chromatographic strips provided in examples 2 and 3 and comparative example 1 were evaluated for sensitivity and specificity.
(1) Sensitivity detection: the inactivated canine distemper virus of different concentrations was detected by using a diluent (diluent component: 0.85% NaCl), the sensitivity was calculated, the test results are shown in FIGS. 2 to 4, and the test results were collated according to FIGS. 2 to 4 to obtain Table 5.
TABLE 5 sensitivity test results
Dilution factor Example 2 Example 3 Comparative example 1
10 times of Strengthen yang Strengthen yang Middle yang
100 times of Strengthen yang Middle yang Weak yang
1000 times Middle yang Weak yang Weak yang
2000 times Weak yang Weak yang Weak yang
5000 times Weak yang Weak yang Negative of
10000 times Weak yang Negative of Negative of
Dilution liquid Negative of Negative of Negative of
As can be seen from fig. 2 to 4 and table 5, the monoclonal antibody pairs used in examples 2 and 3 have higher detection sensitivity for canine distemper virus N protein than the monoclonal antibody pair provided in comparative example 1, and have higher detection sensitivity when the 12F11 antibody is a capture antibody and the 8B5 antibody is a labeled antibody.
(2) And (3) specificity detection: the test results of the inactivated canine distemper virus liquid, recombinant N protein, measles virus liquid, respiratory syncytial virus liquid, newcastle disease virus liquid, peste des petits ruminants virus liquid, canine parainfluenza virus liquid, infectious hepatitis virus liquid, canine parvovirus liquid, herpes simplex virus liquid, pseudomonas aeruginosa liquid, escherichia coli liquid, vero cell lysate, newborn bovine serum, MEM medium, 1% BSA and 1% skimmed milk powder are shown in Table 5.
TABLE 5 specificity detection results
Detecting substance Example 2 Example 3 Comparative example 1
Inactivated canine distemper virus liquid Middle yang Middle yang Middle yang
Recombinant N protein Middle yang Middle yang Middle yang
Measles virus liquid Negative of Negative of Negative of
Respiratory syncytial virus liquid Negative of Negative of Weak yang
New castle disease virus liquid Negative of Negative of Negative of
Peste des petits ruminants epidemic venom Negative of Negative of Negative of
Canine parainfluenza virus liquid Negative of Negative of Negative of
Infectious hepatitis virus liquid Negative of Negative of Negative of
Canine parvovirus liquid Negative of Negative of Negative of
Herpes simplex virus liquid Negative of Negative of Weak yang
Pseudomonas aeruginosa liquid Negative of Negative of Negative of
Escherichia coli liquid Negative of Negative of Negative of
Vero cell lysate Negative of Negative of Negative of
New born calf serum Negative of Negative of Weak yang
MEM medium Negative of Negative of Negative of
1％BSA Negative of Negative of Negative of
1% skimmed milk powder Negative of Negative of Negative of
As can be seen from the detection results, the monoclonal antibodies provided in examples 2 and 3 are positive for the detection results of the inactivated canine distemper virus liquid and the recombinant N protein, and are negative for the detection results of other substances, and compared with the monoclonal antibodies provided in comparative examples which are weak positive for the detection results of the respiratory syncytial virus liquid and the neonatal bovine serum, the monoclonal antibodies provided in examples 2 and 3 have higher detection specificity, and have higher detection precision and efficiency when applied to the detection of the canine distemper virus for practical non-diagnostic purposes.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims (12)

1. A monoclonal antibody pair against a canine distemper virus N protein, or an antigen binding fragment thereof, wherein the monoclonal antibody pair, or antigen binding fragment thereof, comprises: the amino acid sequences of the complementarity determining regions 1-3 of the heavy chain variable region are respectively shown as SEQ ID NO. 1-3, and the amino acid sequences of the complementarity determining regions 1-3 of the light chain variable region are respectively shown as SEQ ID NO. 4-6;
and a second antibody or antigen binding fragment thereof, wherein the amino acid sequences of the complementarity determining regions 1-3 of the heavy chain variable region are shown as SEQ ID NO. 7-9, and the amino acid sequences of the complementarity determining regions 1-3 of the light chain variable region are shown as SEQ ID NO. 10-12.
2. The monoclonal antibody pair or antigen-binding fragment thereof against canine distemper virus N protein of claim 1, wherein the amino acid sequence of the heavy chain variable region of the first antibody or antigen-binding fragment thereof is shown in SEQ ID No. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 14;
the amino acid sequence of the heavy chain variable region of the second antibody or antigen binding fragment thereof is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
3. The pair of monoclonal antibodies against canine distemper virus N protein, or antigen binding fragments thereof, of claim 1, wherein the first antibody or antigen binding fragment thereof is selected from Fab, fab ', F (ab') 2 One or more of Fv, dAb, single chain antibody, chimeric antibody and multispecific antibody.
4. The pair of monoclonal antibodies against canine distemper virus N protein, or antigen binding fragments thereof, of claim 1, wherein the second antibody or antigen binding fragment thereof is selected from Fab, fab ', F (ab') 2 One or more of Fv, dAb, single chain antibody, chimeric antibody and multispecific antibody.
5. A nucleic acid molecule encoding the first antibody or antigen-binding fragment thereof of any one of claims 1-4;
or, the nucleic acid molecule encodes the second antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.
6. A cell capable of secreting a first antibody or antigen binding fragment thereof and/or a second antibody or antigen binding fragment thereof according to any one of claims 1 to 4, comprising a nucleic acid molecule according to claim 5.
7. A method of preparing the first antibody or antigen-binding fragment thereof and/or the second antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising: culturing the cell of claim 6 under suitable conditions, recovering the primary antibody or antigen-binding fragment thereof and/or the secondary antibody or antigen-binding fragment thereof from the cell culture.
8. A kit is characterized by comprising a first antibody or antigen binding fragment thereof, wherein the first antibody has the amino acid sequence of a heavy chain variable region complementarity determining region 1-3 shown in SEQ ID NO. 1-3 and the amino acid sequence of a light chain variable region complementarity determining region 1-3 shown in SEQ ID NO. 4-6;
and a second antibody or antigen binding fragment thereof having the amino acid sequence of the heavy chain variable region complementarity determining region 1-3 shown in SEQ ID NO. 7-9 and the amino acid sequence of the light chain variable region complementarity determining region 1-3 shown in SEQ ID NO. 10-12.
9. The kit of claim 8, wherein the first antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.
10. The kit of claim 8, wherein the second antibody or antigen binding fragment thereof further comprises a detectable label selected from one or more of a radioisotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.
11. The kit of claim 8, wherein the first antibody or antigen-binding fragment thereof is a capture antibody and the second antibody or antigen-binding fragment thereof is a labeled antibody.
12. Use of the monoclonal antibody pair or antigen binding fragment thereof according to any one of claims 1-4, the nucleic acid molecule according to claim 5 and/or the kit according to any one of claims 8-11 for the detection of canine distemper virus for non-diagnostic purposes.
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