CN117447602B - Antibodies to porcine IgM and uses thereof - Google Patents
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Abstract
The invention relates to the technical field of antibodies, in particular to an antibody of pig IgM and application thereof. The antibody of the pig IgM provided by the invention can specifically bind the pig IgM, has no cross reaction with other similar proteins, has higher affinity with the pig IgM, and has higher stability; the detection of the pig IgM by using the double-antibody sandwich ELISA method based on the antibody has higher sensitivity, specificity and accuracy, can realize accurate and high-flux detection of the IgM content in pig serum or other serum analogues or products containing the pig IgM, and has better application prospect in pig IgM detection.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody of pig IgM and application thereof.
Background
IgM is the earliest synthesized and secreted antibody during the development of an animal's individual, and in antigen-stimulated induced humoral immune responses, igM is also the first antibody produced. IgM is used as a detection index to help early diagnosis of bacterial and viral disease infection of pigs. In blood products and clinical diagnosis, the method has important significance for rapidly and accurately detecting the content of IgM in pig serum.
Due to the complexity of the impact of various diseases on the immune system of the organism, early diagnosis is an important approach to avoid serious harm to the herd, and despite the numerous methods of early diagnosis, there is still a lack of timely and comprehensive diagnostic methods for live swine infectious diseases that are in-and-out or regional trade. Antibody detection is of great importance to pig infectious disease epidemic situation monitoring, vaccine immune effect evaluation and immune program optimization. The detection technology of the pig IgM can be used for detecting the response effect of the population to the vaccine, and has strong pertinence to the detection of early infection, especially the establishment of an early rapid detection method. In the detection technology of pig IgM, the performance of the antibody is a key factor influencing the detection effect. Therefore, developing antibodies with high affinity and specificity for porcine IgM is of great importance for sensitive and accurate detection of porcine IgM.
Disclosure of Invention
The invention provides antibodies to porcine IgM and uses thereof.
According to the invention, the pig IgM protein is taken as an antigen to immunize a mouse, and an antibody with high affinity and high specificity for the pig IgM protein is obtained through cell fusion and hybridoma cell screening, and a kit and a method for detecting the pig IgM are developed based on the antibody.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides an antibody or antigen binding fragment thereof of porcine IgM, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of said antibody or antigen binding fragment thereof are shown in SEQ ID nos. 7, 8, 9, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region are shown in SEQ ID nos. 10, 11, 12, respectively.
The antibody can specifically bind with pig IgM, has higher affinity and higher stability, and the double-antibody sandwich ELISA method detection kit developed based on the antibody can be used for detecting the content of pig IgM, has a wider detection concentration range and higher linear correlation, and has higher sensitivity, specificity and accuracy.
Preferably, the heavy chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID NO.15 or has at least 80% similarity to the sequence shown in SEQ ID NO.15, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.16 or has at least 80% similarity to the sequence shown in SEQ ID NO. 16.
The above sequence similarity is preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%.
In some embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.15 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16.
Preferably, the antibody or antigen binding fragment thereof is a monoclonal antibody, fab ', F (ab') 2, fd, fv or single chain antibody.
In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.19 and the nucleotide sequence of a nucleic acid molecule encoding the light chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 20.
In a third aspect, the invention provides a biological material comprising a nucleic acid molecule as described above, said biological material being an expression cassette, a vector or a host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling an antibody or antigen binding fragment thereof as described above with a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radiolabel.
In a fifth aspect, the invention provides an antibody composition for porcine IgM comprising the antibodies in (1) and (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4, 5 and 6 respectively;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.7, 8 and 9 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO.10, 11 and 12 respectively.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is as shown in SEQ ID NO.13 or has at least 80% similarity to the sequence as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14 or has at least 80% similarity to the sequence as shown in SEQ ID NO. 14.
The heavy chain variable region of the antibody described in (2) above has an amino acid sequence shown in SEQ ID NO.15 or at least 80% similarity to the sequence shown in SEQ ID NO.15, and the light chain variable region has an amino acid sequence shown in SEQ ID NO.16 or at least 80% similarity to the sequence shown in SEQ ID NO. 16.
The antibody composition can be used as a pairing antibody for detecting pig IgM by a double-antibody sandwich ELISA method, and two antibodies in the antibody composition are respectively used as a coating antibody and a labeling antibody. The antibody composition is used for detecting the pig IgM by adopting a double-antibody sandwich ELISA method, and has higher specificity, sensitivity and accuracy.
In some embodiments of the present invention, the antibody of (1) above is used as a coating antibody, and the antibody of (2) above is used as a labeling antibody.
In a sixth aspect, the invention provides the use of an antibody or antigen binding fragment thereof or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition as described above in the manufacture of a product for detecting the presence or level of porcine IgM in a sample.
The above-described samples include biological samples derived from pigs (e.g., blood, serum analogs, etc.), and also include products containing pig IgM prepared in vitro (e.g., drugs, feeds, feed additives, etc. containing pig IgM).
The above-described products can be used for disease diagnosis purposes, for example, for early diagnosis of bacterial and viral diseases in pigs by detecting IgM content in biological samples derived from pigs; it can also be used for non-disease diagnosis purposes, for example, to detect the content of pig IgM in the in vitro prepared pig IgM-containing products for product production, quality control, etc.
Such products include detection reagents or kits.
In a seventh aspect, the invention provides any one of the following uses of an antibody or antigen binding fragment thereof or the antibody conjugate or antibody composition as described above:
(1) Use of a detection of the presence or level of porcine IgM in a sample for non-disease diagnostic purposes;
(2) The application in vaccine immunogenicity or immune effect detection;
(3) Use in quality control of a product containing porcine IgM.
In the above (1), the detection for the purpose of non-disease diagnosis includes detecting the content of pig IgM in the in vitro-produced pig IgM-containing product for the purpose of production of the product, quality control, etc. Wherein, the pig IgM-containing products comprise medicines, feeds, feed additives and the like.
In the above (2), the immunogenicity or immune effect of the vaccine can be detected by detecting the content of pig IgM in a biological sample derived from a pig.
In the invention, the method for detecting the pig IgM can be detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography and the like.
In an eighth aspect, the invention provides a kit comprising an antibody or antigen binding fragment thereof as described above, or comprising the antibody conjugate, or comprising the antibody composition.
Preferably, the kit is a double-antibody sandwich ELISA detection kit.
Preferably, the kit comprises a coated antibody and a labeled antibody, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the coated antibody are shown as SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region are shown as SEQ ID NO.4, 5 and 6 respectively; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the labeled antibody are shown as SEQ ID NO.7, 8 and 9 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.10, 11 and 12 respectively.
For ease of detection, the labeled antibodies may also include a detectable label (e.g., an HRP label); the kit may further comprise a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof.
The kit may also contain other reagents for ELISA detection including, but not limited to, ELISA plates, pig IgM standards, PBST washes, blocking solutions, chromogenic solutions, stop solutions, and the like.
The current commonly used immunological detection methods comprise a serum neutralization test (SN), an indirect hemagglutination test (IHA) and ELISA, wherein the neutralization test is complicated in operation; the indirect hemagglutination is simple and convenient to operate, is suitable for clinical use, and has low sensitivity; the ELISA method has the advantages of simple operation, high sensitivity, suitability for large-scale sample detection and the like, and is widely applied clinically. The double-antibody sandwich ELISA method established by the invention has the advantages of specificity, sensitivity and good repeatability, and provides a powerful tool for detecting the antibody level after pig immunization and detecting the early infection of bacteria or viruses.
The principle of detecting the content of the pig IgM by adopting the ELISA method based on the double antibody sandwich is as follows: coating antibodies of pig IgM on an ELISA plate; respectively adding a gradient diluted standard substance and a pre-diluted sample, wherein pig IgM in the standard substance and the sample can be fully combined with a coating antibody on an ELISA plate; after washing the plate, adding an antibody of HRP-labeled pig IgM, wherein the antibody can be specifically combined with a standard product captured by the coated antibody on the ELISA plate and the pig IgM in the sample; adding a chromogenic agent substrate TMB after washing the plate, if pig IgM with different concentrations exists in the sample in the reaction hole, the HRP can change colorless TMB into blue substances with different depths (positive correlation), and after adding a stopping solution, the reaction hole can change into yellow; finally, the absorbance (OD) of the reaction well sample was measured at λmax=450 nm (od=450 nm), the concentration of porcine IgM in the sample was proportional to the OD, and the concentration of porcine IgM in the sample was calculated from the standard curve. The method uses an enzyme color amplification system, has higher detection sensitivity, and can detect samples with lower pig IgM content by using high-affinity antibodies.
Serological tests play a central role in assessing adaptive immune responses, not only are critical to assessing the immunogenicity and immune effects of vaccines, but also have instructive significance for contacting pathogens and cross-reacting with other viruses. The antibody or antigen binding fragment, antibody conjugate, antibody composition and kit provided by the invention can realize quantitative detection of pig IgM, have higher specificity and sensitivity, and are important tools for diagnosing early infection of pigs and evaluating vaccine immunity effects.
The kit disclosed by the invention is subjected to an accelerated stability experiment at 37 ℃, the kit is not obviously changed within 13 days, and the stability is high; the results of similar protein crossing experiments show that the kit is specific in expression, does not identify pigs IgA, igG, igE, people IgM, igG, igA,IgE, mouse IgM, igG, igE, rat IgM, igG, igA, igE, cow IgM, igG, igA, chicken IgM, igG, igA, and rabbit IgM, igG, igA. Compared with the existing similar kit, the kit has obviously better fitting curve and sample detection accuracy. The detection range of the kit is 3.125-200ng/mL, R 2 The lowest detectable pig IgM concentration was 0.9999 up to 132pg/mL. The results of detecting the serum and the cell culture supernatant of the healthy pigs by using the kit show that the standard adding recovery rate and the linear dilution recovery rate are both in the normal range of 70-130 percent.
In a ninth aspect, the present invention provides a method for detecting porcine IgM for non-disease diagnostic purposes, the method comprising: detecting the content of the pig IgM in the sample to be detected by using the antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition or the kit.
The detection method can be selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay or immunochromatography.
The beneficial effects of the invention at least comprise: the antibody of the pig IgM provided by the invention can specifically bind the pig IgM, has no cross reaction with other similar proteins, has higher affinity with the pig IgM, and has higher stability; the detection of the pig IgM by using the double-antibody sandwich ELISA method based on the antibody has higher sensitivity, specificity and accuracy, can realize accurate and high-flux detection of the IgM content in pig serum or other serum analogues or products containing the pig IgM, and has better application prospect in pig IgM detection.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the SDS-PAGE results of monoclonal antibodies 4B9 and 6A11 of example 1 of the present invention, wherein M is a protein molecular weight standard.
FIG. 2 is a standard curve of the detection of pig IgM by the double antibody sandwich ELISA kit of example 3 of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of anti-porcine IgM monoclonal antibodies
1. Immunization of animals
The naturally extracted pig serum IgM protein is used as antigen (100 mug/mouse) to be emulsified with the Freund's complete adjuvant with the same volume, and the emulsified pig serum IgM protein is subcutaneously injected into the back of a female BALB/c mouse with the age of 4-6 weeks to immunize 4 mice in total. After 3 weeks interval, the antigen is emulsified by incomplete Freund's adjuvant and immunized for 3 times, and the titer is measured. Impact immunization antigen was injected into the left abdominal cavity and three days later cell fusion was performed.
2. Cell fusion
The mice immunized by impact were sacrificed by orbital exsanguination, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in dishes. The prepared syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes and myeloma cells are fused to form hybridoma cells, and the specific operation is as follows:
(1) Preparation of feeder cells
One non-immunized BALB/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. 10mL of HAT medium is injected into the abdominal cavity of the mouse, the mice are slowly sucked out and placed in a 96-well culture plate for standby, and the abdominal cavity macrophages are contained in the liquid.
(2) Myeloma (SP 2/0) cell activation
Resuscitating the frozen SP2/0 cells in liquid nitrogen, re-suspending in nutrient solution (RPMI-1640 containing 20% calf serum), and placing at 37deg.C and 5% CO 2 Culturing in an incubator under the condition, observing the growth condition of cells, collecting cells and suspending in RPMI-1640 culture solution when the cells grow in a circular transparent and slightly adherent state, counting and taking out 0.5-1×10 6 The mice were injected subcutaneously into the back of BALB/c mice and cultured continuously for 9-10 days. After the tumor volume at the back is increased to about 0.8/cm, the mice are killed by pulling the neck, immersed in 75% alcohol for 5min, and then sterilized for tumor taking.
Cutting off tumor mass, placing in a sterilized homogenizer, adding 5mL of RPMI-1640 culture solution, fully grinding, adding 10mL of RPMI-1640 culture solution, standing for 2 min, sucking the cell suspension at the upper layer after larger tissue mass is settled at the bottom of a tube, placing in another centrifuge tube, adding 10mL of RPMI-1640 culture solution, repeatedly grinding twice, centrifuging the obtained cell suspension at 1000 r/min for 10 min, removing the supernatant, re-suspending with the RPMI-1640 culture solution, and fixing the volume to 30 mL.
Adding 15mL of lymphocyte separation liquid into another centrifuge tube, and slowly adding the cells on the separation liquid (the ratio is 1:2-1:1); then, the mixture is centrifuged for 15min at 1200 r/min, the suction tube is deeply sucked into a compact white cell layer, then the cells are washed by RPMI-1640 culture solution for 2 times and resuspended in 10mL of RPMI-1640 culture solution, and the cells are counted for later use.
(3) Preparation of immune spleen cells
The BALB/c mice after the impact immunization are subjected to orbital exsanguination and sacrifice (serum is collected and is positive serum), then are soaked in 75% alcohol for 5min for sterilization, then are fixed on an anatomical plate by using a sterile knife and forceps, taken out, the spleen is cut through an outer membrane, and are placed in a sterilized homogenizer; adding 30mL of RPMI-1640 culture solution into a homogenizer, grinding (not too severe to damage spleen cells, a relatively loose homogenizer is selected), extruding spleen cells, taking out a homogenizing rod, supplementing 10mL of RPMI-1640 culture solution, centrifuging at 1200 r/min for 5min, removing the supernatant, and cleaning the cells for 2 times.
(4) Cell fusion
SP2/0 and splenocytes inAccording to the following steps: 1 in a 50mL centrifuge tube, and centrifuging at 1200 r/min for 5min. The supernatant was discarded and placed in a 37℃water bath, the bottom of the tube was gently tapped to loosen the cell pellet slightly (to allow PEG to act on the cells sufficiently), 50% PEG (Sigma) pre-warmed to 37℃at 1mL was slowly added and mixed well, stirred and allowed to stand for 30s. Then, 1mL of the RPMI-1640 medium preheated at 37℃was added dropwise over 2 minutes, 1mL was added dropwise over 1 minute, 5mL was added dropwise over 1 minute, and the volume was set to 40 mL to terminate the fusion. Centrifuging at 1000 r/min for 5min, removing supernatant, and standing in water bath at 37deg.C for 5-8 min. Then, the cells were seeded at 100. Mu.L/well in the above feeder cells and placed at 37℃in 5% CO 2 Culturing and observing in an incubator. Colonies growing to a certain size on day 5 after fusion are changed into HT medium for continuous culture. When the fused cell colony grows to 1/4 of the culture hole, the cell cluster is good in state, and antibody titer detection is carried out.
3. Screening of hybridoma-positive clones, cloning of cells and preparation of monoclonal antibodies
The indirect ELISA method is adopted to screen positive hybridoma cells, and the steps are as follows:
(1) Coating known antigens: diluting the purified coating antigen to 2 mug/mL with coating buffer; 100. Mu.L of each well was added to the microwells and the wells were cooled overnight at 4 ℃; the liquid in the hole is photographed the next time; phosphate Tween buffer (PBST) was washed 4 times for 2-3 minutes each.
(2) Blocking the positions of the enzyme-labeled wells not coated with antigen: adding 250 mu L of 2% BSA to each hole to seal the ELISA plate, standing for 1h at room temperature, and throwing away liquid in the holes;
(3) Sample adding: PBST is washed for 4 times, hybridoma cell supernatant to be detected is added after beating, 50 mu L of each hole is added into enzyme-labeled holes in sequence, incubation is carried out for 1h at 37 ℃ in an incubator, washing is carried out, and beating is carried out.
(4) Adding enzyme-labeled anti-antibody: secondary antibody was diluted to 1 with goat anti-mouse IgG, PBST: 5000, 100 mu L of each well is added, gently shaken, and incubated at 37 ℃ for 1h; then washing and beating to dry.
(5) Adding a color development solution and a termination solution: 100. Mu.L of TMB color development solution was added to each well, and after 5min at room temperature, 50. Mu.L of stop solution was added to each well.
(6) Determination result: the ELISA plate is arranged on the OD of the ELISA instrument 450 Readings were taken at the bottom and were judged positive 2.1 times greater than the negative wells.
The method for cloning the hybridoma cells by adopting a limiting dilution method comprises the following specific steps:
preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture hole, and counting the number of living cells by using a blood cell counting plate; diluting cells to 5, 10, 30 cells/ml with complete medium; the three concentrations of cell suspension were added to the prepared 96-well culture plates of feeder cells at 100. Mu.L/well, so that each well contained 0.5, 1 and 3 cells, respectively. One drop of the fluid was added to the culture on day 4, and the growth of cells in each well was carefully observed on days 5-6 and recorded.
Detection of specific antibodies: the cell clone can be detected when 1/3-1/2 of the visual field is full of the cell clone on the 7 th to 9 th days after the cloning; and (3) selecting single cell clusters with positive holes for recloning, selecting single cell clusters with high titer and good state for expansion culture after 2-3 times of cloning, freezing for standby, inoculating into abdominal cavities of mice to prepare ascites, and generating and recovering antibodies.
Preparation of monoclonal antibodies in large quantities: the cells were washed and suspended in 0.5. 0.5 mL Phosphate Buffered Saline (PBS), and were intraperitoneally injected into paraffin-sensitized mice, and ascites were collected after the bellyband had risen and become blue. The ascites was collected and centrifuged at 3500 r/min for 5min at 4 ℃. Carefully aspirate ascites and collect in centrifuge tube and store at-20℃for further use.
Two monoclonal antibodies which specifically bind to the pig IgM are obtained through screening and are respectively named monoclonal antibodies 4B9 and 6A11.
4. Variable region sequencing of monoclonal antibodies 4B9 and 6A11
Collecting hybridoma cell number greater than 10 6 And (5) sending the sample to the Beijing engine family organism for subsequent construction and sequencing. Sequencing results showed: the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody 4B9 are shown as SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4, 5 and 6 respectively; heavy chain variable regionThe amino acid sequence of the light chain variable region is shown as SEQ ID NO.13 and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14; the coding gene sequence of the heavy chain variable region is shown as SEQ ID NO.17, and the coding gene sequence of the light chain variable region is shown as SEQ ID NO. 18; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody 6A11 are shown as SEQ ID NO.7, 8 and 9 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.10, 11 and 12 respectively; the amino acid sequence of the heavy chain variable region 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; the coding gene sequence of the heavy chain variable region is shown as SEQ ID NO.19, and the coding gene sequence of the light chain variable region is shown as SEQ ID NO. 20.
5. Purification of monoclonal antibodies 4B9 and 6A11
The hybridoma cells after the strain establishment are injected into the abdominal cavity of a mouse, ascites is collected for about 7 days, antibodies are purified by Protein G affinity chromatography, and the purity of the antibodies is identified by SDS-PAGE, and the result is shown in figure 1.
Example 2 affinity detection of monoclonal antibodies
The relative affinity constants of monoclonal antibodies 4B9 and 6a11 were determined as follows:
porcine IgM antigen was coated onto the ELISA plate and blocked. After washing the plates with PBST, monoclonal antibodies 4B9 and 6A11 were diluted to saturation concentration and added to the ELISA plate at 100. Mu.L/well, and incubated at room temperature for 2h. After PBST plate washing, 60 μl/well of NaSCN (sodium thioglycolate) solution of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0mol/L was added sequentially, and the plates were left standing at room temperature for 15 min. After PBST plate washing, HRP-labeled goat anti-mouse IgG is added, and the plate is incubated at room temperature for 45min for chromogenic detection. The concentration of sodium thiocyanate corresponding to the decrease of OD value at 450nm after elution to 50% without elution is the relative affinity constant of the antibody, which is expressed in mol/L. The results showed that the relative affinity constants of the monoclonal antibodies 4B9 and 6A11 were not less than 1.5mol/L (Table 1), and the affinities were good.
TABLE 1
Example 3 establishment of double antibody sandwich ELISA kit for pig IgM content detection
1. HRP labelling of monoclonal antibody 6A11
Monoclonal antibody 6a11 was added to a dialysis bag and dialyzed overnight at 4 ℃ in 0.01M Carbonate Buffer (CBS). 10mg of HRP was dissolved in 2mL of water. Fresh 0.1mol/L sodium periodate (NaIO) was prepared 4 ) Solution, take 0.4mL NaIO of 0.1mol/L 4 Added into 2mL of horseradish peroxidase (HRP) solution, mixed well, and soaked in 10mM sodium acetate (NaAc) buffer solution for 45min at room temperature and kept away from light, and dialyzed overnight at 4 ℃. The overnight dialyzed antibody and HRP solution was removed, 0.1mL of 0.2mol/L CBS pH 9.5 was added, and immediately after the removal of the antibody, the HRP solution was gently stirred at room temperature for 2 hours in the absence of light. 0.04g of sodium borohydride (NaBH) 4 ) Dissolving in 10mL of water, adding 0.4mL into the reaction solution, mixing, and standing at 4 ℃ for 2 hours. Taking out, placing in a dialysis bag, dialyzing in 0.01mol/L PBS, changing the solution once after 2 hours, and dialyzing at 4 ℃ overnight. Taking out the overnight dialyzed labeling solution, adding equal volume of glycerol, and storing at-20deg.C.
2. Preparation of monoclonal antibody 4B9 coated ELISA plate
Monoclonal antibody 4B9 was diluted to 2. Mu.g/mL with 0.05M carbonate coating buffer pH 9.6. 0.1mL of the sample was added to a 96-well polystyrene reagent plate reaction well at 4℃overnight. The next day, the solution in the wells was discarded, and washed 3 times with wash buffer for 3 minutes each. After the above procedure, each well of the reaction plate was blocked by adding a 2% BSA solution, 0.3mL per well, and left at room temperature for 2 hours. The solution in the hole is discarded, and the solution is placed in a dry room for drying, vacuumized and stored at 4 ℃.
3. Establishment of double antibody sandwich ELISA method
And taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Pig IgM standards were added at 100. Mu.L of different dilution concentrations of 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, and a blank was placed. After the sealing plate is arrangedAnd standing at room temperature for incubation for 2 hours, washing the plate for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed in the dark for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. And drawing a standard curve by taking pig IgM standard substances with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The result shows that the detection range is 3.125-200ng/ml, R 2 0.9999 (fig. 2).
4. Sensitivity detection of double-antibody sandwich ELISA kit
And taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Pig IgM standards were added at 100. Mu.L of different dilution concentrations of 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, and 20-well blank was set. After sealing the plates, standing and incubating for 2 hours at room temperature, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and dual wavelength detection (within 5 min) was performed immediately using an ELISA reader. The average of 20 zero standard concentrations OD was measured, plus two standard deviations, and the corresponding detectable concentration was calculated, with a sensitivity of 132pg/mL (Table 2).
TABLE 2
5. Specific detection of double-antibody sandwich ELISA kit
And taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. 100. Mu.L of porcine IgM structurally similar proteins, including porcine IgA, igG, igE, human IgM, igG, igA, igE, mouse IgM, igG, igE, rat IgM, igG, igA, igE, bovine IgM, igG, igA, chicken IgM, igG, igA, rabbit IgM, igG, igA were added. After sealing the plates, standing and incubating at room temperature for 2h, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The results showed that monoclonal antibodies 4B9 and 6a11 did not react with other similar proteins (table 3).
TABLE 3 Table 3
6. Stability detection of double-antibody sandwich ELISA kit
The ELISA plate coated with monoclonal antibody 4B9, HRP-labeled monoclonal antibody 6A11, and standard porcine IgM were subjected to an accelerated stability test at 37℃for 13 days (corresponding to 15 months at 4 ℃). Then taking out for detection, wherein the detection method is as follows: and taking out the ELISA plate, washing the plate for 3 times, and spin-drying. Pig IgM standards were added at 100. Mu.L of different dilution concentrations of 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, and a blank was placed. After sealing the plates, standing and incubating for 2 hours at room temperature, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. And drawing a standard curve by taking pig IgM standard substances with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results showed that the kit OD changed little and the gradient was good within 13 days of the test, indicating that the kit stability was good (table 4).
TABLE 4 Table 4
7. Labeling recovery rate detection
And taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Pig serum 4 parts (numbered 1, 2, 3, 4) and pig IgM-containing cell supernatant 1 part were selected, three different concentrations of pig IgM were added, 80ng/mL, 20ng/mL, 5ng/mL, respectively, and a blank control was set. After sealing the plates, standing and incubating for 2 hours at room temperature, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The detection results show that the standard adding recovery rate is in the normal range of 70% -130% (table 5).
TABLE 5
8. Linear dilution recovery detection
And taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Pig serum 4 parts (numbered 1, 2, 3, 4) and cell supernatant 1 part were selected, high-concentration pig IgM 80ng/mL was added, dilution was performed in the standard curve dynamic range, and linearity was evaluated. After sealing the plates, standing and incubating for 2 hours at room temperature, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The test results showed that the linear dilution recovery was in the range of 94% -119%, the kit was linear over a wide dilution range and the content of IgM samples with different levels of analyte could be flexibly determined (table 6).
TABLE 6
Example 4 comparison with test results of other brands of kits of the same type
A commercial pig IgM detection kit, the X brand kit (Genorise, cat# GR 113235-1), was selected for analogy testing, and experiments were performed according to the method best suited for the brand kit (operating fully according to its instructions). The performance of the kit of the invention was compared to the branded kit by the following two aspects.
1. Standard curve comparison: the results show that the fitting curve of the kit of the invention is better than that of the X brand kit (R 2 =0.9990), and the background of the kit of the invention performed well (zero well value 0.012), below the background of the X brand kit (zero well value 0.017) (table 7).
TABLE 7
2. Sample measurement result comparison: 12 pig serum samples were randomly selected and assayed simultaneously (in mg/mL), and IgM content in normal pig serum was reported to be in the range of 0.3-1.6mg/mL according to literature. The measurement results show that the measurement content of the kit disclosed by the invention is consistent with the normal range, the IgM content measured by the X brand kit is lower and is between 0.4 and 1.5mg/mL, the detection value of the X brand kit is close to the lower limit value of the detection range of the standard curve, the accuracy is poor, the difference between the detection value and the detection value of the kit disclosed by the invention is about 100 times, and the difference between the detection value and the reference range is larger (Table 8).
TABLE 8
Example 5 pig serum IgM content determination
The method for measuring the IgM content in the pig serum by using the double-antibody sandwich ELISA kit comprises the following specific steps:
and taking out the ELISA plate coated with the monoclonal antibody 4B9 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Healthy pig serum 4 parts (No. 1, 2, 3 and 4) are selected, 100 mu L pig serum samples/standard substances with different dilution factors are added, the dilution factors of the samples are 8000 times and 10000 times, the dilution concentration of the pig IgM standard substances is 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL, and blank control is arranged. After sealing the plates, standing and incubating for 2 hours at room temperature, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-6A11 working solution into the reaction holes, standing at room temperature for incubation for 45min after sealing the plates, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution (2M sulfuric acid solution) was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The detection result shows that the kit can accurately determine the content of IgM in pig serum under different dilution factors, and has good linear relation in the standard curve range (Table 9).
TABLE 9
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (11)
1. The antibody of the pig IgM is characterized in that the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the antibody are respectively shown in SEQ ID NO.7, 8 and 9, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region are respectively shown in SEQ ID NO.10, 11 and 12.
2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No.15 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 16.
3. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 80% similarity to the sequence set forth in SEQ ID No.15 and the amino acid sequence of the light chain variable region has at least 80% similarity to the sequence set forth in SEQ ID No. 16.
4. The antibody according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody, fab ', F (ab') 2 Fv or single chain antibodies.
5. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 4.
6. A biological material comprising the nucleic acid molecule of claim 5;
the biological material is an expression cassette, a vector or a host cell.
7. An antibody conjugate, which is characterized in that the antibody conjugate is obtained by coupling the antibody according to any one of claims 1-4 with a label, wherein the label is one or more selected from the group consisting of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
8. Use of an antibody according to any one of claims 1 to 4 or a nucleic acid molecule according to claim 5 or a biomaterial according to claim 6 or an antibody conjugate according to claim 7 in the preparation of a product for detecting the presence or level of pig IgM in a sample.
9. Use of the antibody of any one of claims 1-4 or the antibody conjugate of claim 7 for any one of the following:
(1) Use of a detection of the presence or level of porcine IgM in a sample for non-disease diagnostic purposes;
(2) Use in the preparation of a product for the detection of vaccine immunogenicity or immune effects.
10. A kit comprising the antibody of any one of claims 1 to 4, or the antibody conjugate of claim 7.
11. A method of detecting porcine IgM for non-disease diagnostic purposes, the method comprising: detecting the content of pig IgM in a sample to be detected by using the antibody according to any one of claims 1 to 4 or the antibody conjugate according to claim 7 or the kit according to claim 10.
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