Preparation and application of anti-porcine erythrocyte membrane antibodies

Abstract

The present invention relates to the field of in vitro diagnostics, and more specifically to the preparation and application of anti-porcine erythrocyte membrane antibodies. Using porcine erythrocytes as immunogens, the present invention prepared a monoclonal antibody, 3B6, that can recognize porcine erythrocytes. The antibody has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2. Experimental results demonstrate that the antibody has strong specificity, high adhesion to erythrocytes, and high application value.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to the preparation and application of anti-porcine erythrocyte membrane antibodies. Background Art

[0002] Rapid testing technologies are often used for swine disease diagnosis, post-drug monitoring, and international pig trade quarantine. Currently, colloidal gold rapid testing methods are popular, such as those for African swine fever (ASFV), porcine reproductive and respiratory syndrome (PRRS), and Mycoplasma hyopneumoniae (Mhp). Because serum or plasma samples require lengthy preparation, colloidal gold testing typically uses whole blood as the test sample. Before applying whole blood directly to colloidal gold test strips, it is typically filtered through a blood filter to remove red blood cells. However, this sample pretreatment method has numerous drawbacks, including the large volume of whole blood required, the high cost of filter membranes, slow separation speeds, and the potential for incomplete red blood cell filtration or hemolysis. During testing, red blood cells and released hemoglobin can reach the detection area due to chromatographic effects, increasing the background color of the detection window and interfering with qualitative, semi-quantitative, and quantitative detection.

[0003] Patent CN206740777U intercepts excess red blood cells by adding an anti-human RBC antibody interception line. The addition of the interception line allows for better membrane running and a cleaner background. It has been demonstrated that red blood cells from various mammals, rodents, and birds possess immune adhesion functions, and CR1 has been confirmed as an animal red blood cell immune adhesion receptor. However, current adhesion receptors are too limited for red blood cell adhesion under different detection backgrounds. Therefore, the development of new, more targeted blood cell adhesion receptors is of great practical significance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the preparation and application of anti-porcine erythrocyte membrane antibodies.

[0005] The present invention provides a monoclonal antibody against porcine erythrocyte membrane, which is a highly specific porcine erythrocyte membrane antibody obtained by immunizing BALB / c mice with porcine erythrocytes and screening through hybridoma technology. The antibody subtype is identified as IgG1, and the antibody titer in the blood sample of the immunized mice is 1:128k.

[0006] The products amplified by primers designed based on the constant region sequence of the antibody gene were identified. The results showed that the anti-porcine erythrocyte membrane monoclonal antibody of the present invention has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2, which are different from currently commercially available blood cell antibodies and have strong specificity.

[0007] Furthermore, the monoclonal antibody of the present invention further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is of mouse IgG type; and the light chain constant region is of mouse IgG type.

[0008] The monoclonal antibodies described in the present invention outperform other commercially available antibodies. Experimental results demonstrate that, when used for blood cell adhesion, the monoclonal antibodies described in the present invention achieve excellent blood cell adhesion at extremely low concentrations. In specific embodiments of the present invention, the monoclonal antibodies were coated onto test strips at concentrations of 15 μg / ml, 30 μg / ml, and 60 μg / ml for observation of blood cell adhesion. The results demonstrated that even at 15 μg / ml, good blood cell adhesion was achieved, surpassing other commercially available blood cell antibodies in terms of blood cell adhesion.

[0009] The present invention provides nucleic acids encoding the monoclonal antibodies.

[0010] Furthermore, in the present invention,

[0011] The nucleic acid encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 7;

[0012] The nucleic acid encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 8.

[0013] In the present invention, the nucleic acid can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid can be linear or circular in topology. Nucleic acid can be, for example, a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0014] The present invention provides an expression module comprising a promoter, a terminator and the nucleic acid of the present invention.

[0015] Furthermore, the expression module includes an expression module composed of the nucleic acid of the present invention in a single or multiple tandem forms with the promoter and terminator, and the present invention is not limited to this.

[0016] The present invention also provides a transcription unit comprising the nucleic acid or the expression module, wherein the transcription unit refers to a DNA sequence starting from a promoter and ending at a terminator. The promoter and terminator may also be flanked or interposed with regulatory segments, which may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site, and homologous recombination site operably linked to the nucleic acid sequence, such as a promoter enhancer, a poly(A) signal, and the like.

[0017] The present invention provides a recombinant vector comprising:

[0018] A vector backbone and the nucleic acid of the present invention;

[0019] Or a vector backbone and the expression module of the present invention.

[0020] Furthermore, the present invention also provides liposome nanoparticles containing the recombinant vector; the lipid nanoparticles include but are not limited to polymer lipid hybrid nanoparticles or solid lipid nanoparticles.

[0021] The recombinant vector described in the present invention refers to a nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences or elements that are essential for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for expression in bacteria include promoters, ribosome binding sites, and possibly other sequences. Bacterial cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0022] The present invention provides a host comprising at least one of the following I) to III):

[0023] 1), secreting the monoclonal antibody of the present invention;

[0024] II), genome integration of the nucleic acid of the present invention or the expression module of the present invention;

[0025] III), transfecting or transforming the recombinant vector of the present invention.

[0026] In the present invention, the recombinant vector is transfected or transformed into a host; the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc. In some specific embodiments, the host is constructed by electroporation.

[0027] Furthermore, the host described in the present invention includes bacteria, fungi, viruses, or animals. Bacteria include Gram-positive and Gram-negative bacteria; Gram-positive bacteria include, but are not limited to, Escherichia coli. Fungi include molds, yeasts, and mushrooms; yeasts include, for example, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris, and Candida. Viruses include, but are not limited to, adenoviruses, adeno-associated viruses, lentiviruses, and prions. Animals include humans, mice, rabbits, pigs, and zebrafish.

[0028] The present invention provides a method for preparing the monoclonal antibody, which comprises culturing the host of the present invention to obtain the monoclonal antibody.

[0029] The present invention provides a product for adhering blood cells, wherein the raw materials thereof include at least one of the following a) to c):

[0030] a) the monoclonal antibody of the present invention;

[0031] b) the nucleic acid of the present invention;

[0032] c) the expression module of the present invention;

[0033] d) the recombinant vector of the present invention;

[0034] e) the host of the present invention;

[0035] f) A culture containing the monoclonal antibody obtained by the preparation method of the present invention.

[0036] Furthermore, the product of the present invention also includes solvents and / or excipients, which are used in the product to maintain the stability of the raw materials or help the raw materials to function.

[0037] Furthermore, the solvent includes, but is not limited to, TB buffer, PBS buffer, Tris buffer, KCl buffer, or NaCl buffer. The excipients include, but are not limited to, proteins, carriers, antioxidants, surfactants, and / or protease inhibitors; the proteins include, but are not limited to, BSA; the carriers include, but are not limited to, auxiliary carriers for viral packaging, auxiliary carriers for phage packaging, and / or auxiliary carriers for eukaryotic or prokaryotic host cell genome integration, which are not limited in the present invention; the antioxidants include, but are not limited to, DTT or β-mercaptoethanol. The surfactants include, but are not limited to, Triton-X-100. The protease inhibitors include, but are not limited to, PMSF.

[0038] In some specific embodiments, the buffer solution of the present invention is a TB buffer solution, and the pH of the TB buffer solution is 7.6.

[0039] Furthermore, the product described herein includes a test strip coated with the monoclonal antibody described herein. In some specific embodiments, the test strip described herein is a colloidal gold test strip, comprising a backing card, a sample pad, a colloidal gold pad, an NC membrane, and a water-absorbing plate affixed in sequence to the upper end of the backing card; the colloidal gold pad is provided with a gold-labeled monoclonal antibody described herein. Experimental results demonstrate that, at a coating concentration of 15 μg / ml, the monoclonal antibody's interception efficiency in porcine whole blood reaches the level achieved by conventional centrifugation methods, outperforming other commercially available porcine whole blood antibodies.

[0040] The product of the present invention also includes a blood cell adhesion agent for adhering blood cells to serum samples. The blood cell adhesion agent also includes a coupling medium and / or a buffer; the coupling medium includes but is not limited to polystyrene plates, magnetic beads, or colloidal gold. The coupling medium is coupled with the monoclonal antibody of the present invention to form a blood cell adhesion agent for processing serum samples.

[0041] The product of the present invention also includes a kit, which uses the monoclonal antibody of the present invention to treat serum.

[0042] Furthermore, the kit may further include a buffer solution, a washing solution, a blocking solution and / or a color developing solution, which is not limited in the present invention.

[0043] The present invention provides application of the product in serum sample processing.

[0044] The method for processing a serum sample comprises adsorbing red blood cells in the blood with the antibody of the present invention to obtain serum.

[0045] The beneficial effects achieved by the present invention are:

[0046] The present invention uses porcine erythrocytes as immunogens to prepare a monoclonal antibody 3B6 that can recognize porcine erythrocytes. The antibody has a titer of 1:128k and has a good blood cell adhesion effect at a concentration of 15 μg / ml, indicating that it has high adhesion to red blood cells. The 3B6 mouse monoclonal antibody obtained by the present invention can be used for rapid detection of colloidal gold in porcine whole blood. This technology can omit the step of porcine plasma / serum separation, save detection cost and time, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the test result of cell supernatant during the hybridoma cell line screening process;

[0048] Figure 2 This is the result of the hemagglutination test (HA) of the mouse monoclonal antibody against porcine erythrocyte membrane;

[0049] Figure 3 A schematic diagram of the colloidal gold test strip prepared by the present invention;

[0050] Figure 4 Application of the 3B6 antibody prepared by the present invention in pig whole blood colloidal gold test strip technology;

[0051] Figure 5 The effect of intercepting pig whole blood by the anti-erythrocyte monoclonal antibody prepared by the present invention was compared with that by the conventional treatment method (Figure A shows the result of sample 1, and Figure B shows the result of sample 2). DETAILED DESCRIPTION

[0052] The present invention provides the preparation and application of anti-porcine erythrocyte membrane antibodies. Those skilled in the art can refer to the disclosure herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify, alter, and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the disclosure, spirit, and scope of the present invention.

[0053] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3B6 is: EVQLQQSGPELVKPGTSVKISCKASGYSFTGHFMNWVMQSHGKSLEWIGRINPYIGDTFYNQKFKGKATLTVDKSSSTAHMELRSLASEDSAVYYCARSSGSDYISYYYAMDYWGQGTSVTVSS (as shown in SEQ ID NO: 1).

[0054] The amino acid sequence of the light chain variable region of monoclonal antibody 3B6 is: NILMTQSPSSLSASLGERVSLTCRTSQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYTSYPFTFGSGTKLEIK (as shown in SEQ ID NO: 2).

[0055] The nucleic acid sequence of 4C-LF from 5′ to 3′ is: gacattgtgatgacccagtctcct (SEQ ID NO: 3).

[0056] The nucleic acid sequence of 4C-LR from 5′ to 3′ is: tggacactgttggggccgcatcggccct (SEQ ID NO: 4).

[0057] The nucleic acid sequence of 4C-HF from 5′ to 3′ is: caggtgcagctgcaggagtcagga (SEQ ID NO: 5).

[0058] The nucleic acid sequence of 4C-HR from 5′ to 3′ is: gataccagatgggggtgtcgttttggc (SEQ ID NO: 6).

[0059] The nucleic acid encoding the heavy chain variable region of monoclonal antibody 3B6 is: gaggtccagctgcagcagtctggacctgagctggtgaagcctgggacttcagtgaagatatcctgcaaggcttctggttactcattcactggccactttatgaactgggtgatgcagagccatggaaagagccttgagtggattggacgtattaatccttacattggtgatactttctacaaccagaagttcaagggcaaggccacattgactgtagacaaatcctctagcacagcccacatggagctccggagcctggcatctgaggactctgcagtctattattgtgcaagatcttccggtagtgactacatatcctattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca (as shown in SEQ ID NO: 7).

[0060] The nucleic acid encoding the light chain variable region of monoclonal antibody 3B6 is: aacattctgatgacccagtctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcggacaagtcaggaaattagtggttacttaagctggcttcagcagaaaccagatggaactattaaacgcctgatctacgccgcatccactttagattctggtgtcccaaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgcagactattactgtctacaatatactagttatccattcacgttcggctcggggacaaagttggaaataaaa (as shown in SEQ ID NO: 8).

[0061] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0062] The present invention will be further described below in conjunction with the embodiments:

[0063] Example 1 Preparation of pig red blood cells

[0064] Collect 20 mL of fresh pig blood and place it in a sterile centrifuge tube (containing anticoagulant). Centrifuge at 4°C, 4000 r / min for 10 min, discard the supernatant, and re-dissolve the precipitate with 20 ml of 0.02 M PBS solution. Repeat the above steps after re-dissolution until the supernatant has no obvious red color. Discard the supernatant for the last time to obtain red blood cells. Finally, resuspend the blood cells with 0.02 M PBS solution to make a 2% red blood cell suspension and store at 4°C for later use.

[0065] Example 2 Preparation of anti-porcine erythrocyte membrane mouse monoclonal antibody

[0066] 1. Mouse Immunization

[0067] Five-week-old female Balb / c mice were intraperitoneally immunized with 2% porcine red blood cells. The initial immunization dose was 0.5 ml of 2% porcine red blood cells per mouse. The second and third immunizations were performed 21 days and 42 days after the first immunization, respectively. The immunization dose was 0.5 ml of 2% porcine red blood cells per mouse. Both immunizations were performed by intraperitoneal injection. Blood was collected from the tail about 7 to 10 days after the third immunization, and the serum titer was measured by hemagglutination method (HA).

[0068] 2. Hybridoma Cell Preparation

[0069] Fusion: Select hemagglutination test to detect serum titer greater than 10 4Mice were given an intrasplenic booster immunization with 0.5 ml of 2% porcine red blood cells (2% porcine erythrocytes) via intraperitoneal injection. Three days after the booster immunization, the mouse spleens were fused with NS1 myeloma cells at an 8:1 PEG ratio. The fused cells were cultured in DMEM medium containing HAT (Gibco), with 200 μl of cell suspension per well placed in a 96-well cell culture plate. The 96-well cell culture plate was not moved for the first three days. On the third and fourth days, one drop of DMEM medium containing HAT (Gibco) was added to each well.

[0070] About 6 to 7 days after fusion, the specific antibody content in the cell culture supernatant was detected by hemagglutination test. There were 10 cell lines that agglutinated. The 10# cell well with the strongest agglutination reaction was selected and subcloned three times by limiting dilution method (see Figure 1 ), HT DMEM medium (Gibco) was selected in the subcloning stage, and finally a hybridoma cell line that can stably secrete anti-porcine erythrocyte membrane was obtained and named 3B6.

[0071] 3. Preparation and purification of anti-porcine erythrocyte membrane antibodies.

[0072] The 3B6 hybridoma cell line was injected into the peritoneal cavity of mice that had been pre-treated with liquid paraffin. The mouse ascites was collected after 7 to 10 days and crudely purified by the octanoic acid-ammonium sulfate method. Then, affinity chromatography (SPA) was used for fine purification to obtain anti-porcine erythrocyte membrane antibodies. The antibody concentration was measured by the ultramicro spectrophotometer A280 method to be 5 mg / ml.

[0073] 4. Identification of anti-porcine erythrocyte membrane monoclonal antibodies

[0074] 4.1 Antibody subtype identification

[0075] The subtypes of the 3B6 mouse monoclonal antibody were determined using a direct ELISA method. A 2 μg / ml working solution of this antibody was prepared in CB buffer. 50 μl was coated onto each well of an ELISA plate and incubated at 4°C for 12-16 hours. The wells were then spun out and washed three times with PBST. 150 μl of 1% casein blocking buffer was added to each well and incubated at 37°C for 2 hours. The wells were spun out and patted dry. The plate was then dried and used for further analysis. Different subtypes of Sigma enzyme were added to the plate column by column. The plate was incubated at 37°C for 30 minutes. After washing six times with PBST, substrate and color development solution were added and color was developed at room temperature for 15 minutes. Stop solution was added to terminate the reaction, and the plate was read at 450 nm on a microplate reader. The results are shown in Table 1 below.

[0076] Table 1. Identification of subtypes of 3B6 mouse monoclonal antibodies

[0077] Subtype reagents 3B6 IgG1 2.494 IgG2a 0.067 IgG2b 0.054 IgG3 0.053 IgM 0.068 IgA 0.066 blank 0.049

[0078] As shown in Table 1, the OD value of the blank control is 0.049, the OD value of the IgG1 subtype reagent is 2.494, and the OD values of the other subtype reagents are all equivalent to the blank control, which shows that the subtype of the antibody is IgG1.

[0079] 4.2 Antibody titer detection

[0080] A direct agglutination test was used to detect the titer of 3B6 mouse monoclonal antibody. Red blood cells were prepared according to the method in Example 1, and then the separated red blood cells were prepared into 1% red blood cell suspension with 0.02M PBS. The 3B6 mouse monoclonal antibody was diluted 1000 times for use. A 96-well V-type blood agglutination plate was prepared, and 25μl of 0.02M PBS was added to wells 1 to 12 in sequence. Then, 25μl of 1000-fold diluted 3B6 mouse monoclonal antibody was added to the first well. After mixing, 25μl of liquid was drawn from the first well and added to the second well. Similarly, after diluting to the 11th well, 25μl of liquid was discarded, and the 12th well was used as a negative control. Then, 25μl of 1% red blood cell suspension was added to each well from 1 to 12, and after shaking, it was placed at room temperature for 15 minutes to observe the results. The highest dilution factor of the antibody that can cause 100% red blood cell agglutination was used as the titer of the antibody. The results are as follows Figure 2 As shown. Figure 2 It can be seen that the titer of this antibody is 1:128k.

[0081] 5. Monoclonal Antibody Sequencing

[0082] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0083] 4C-LF 5′-GACATTGTGATGACCCAGTCTCCT-3′ (SEQ ID NO: 3);

[0084] 4C-LR 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′ (SEQ ID NO: 4);

[0085] 4C-HF 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′ (SEQ ID NO: 5);

[0086] 4C-HR 5'-GATAGACAGATGGGGGTGTCGTTTTGGC-3' (SEQ ID NO: 6).

[0087] 3 × 10 6Total RNA from hybridoma cell 3B6 was reverse transcribed into cDNA. PCR was performed using primers 4C-HF and 4C-HR to amplify the heavy chain variable regions of monoclonal antibodies 4C10 and 6F9. Primers 4C-LF and 4C-LR were used to amplify the light chain variable regions of monoclonal antibodies 4C10 and 6F9. Both PCR reactions used a hot start reaction condition: 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 55°C for 45 seconds, and 72°C for 30 seconds, and finally 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis and purified. The fragments were cloned into the PM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB solid plates. White plaques were inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, plasmids were extracted using a QIAGEN plasmid extraction kit, and sequencing was performed to determine the heavy and light chain variable region sequences of monoclonal antibody 3B6.

[0088] The heavy chain variable region of monoclonal antibody 3B6 has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2.

[0089] Example 3 Application of Anti-Porcine Red Blood Cell Membrane Antibodies in Detecting Colloidal Gold Test Strips in Pig Whole Blood 1. Preparation Method for Directly Detecting Colloidal Gold Test Strips in Pig Whole Blood

[0090] The test strip includes a backing card, which is a sticky setting. A sample pad, a colloidal gold pad, an NC membrane and a water absorption plate are fixed to the upper end of the backing card in sequence. One end of the sample pad overlaps with one end of the gold pad, and the sample pad is located on the upper side with an overlapping length of 2 to 3 mm. The colloidal gold pad is provided with a gold-labeled specific antibody. The two ends of the NC membrane overlap with the colloidal gold pad and one end of the water absorption plate respectively, and the two ends of the NC membrane are located on the lower side with an overlapping length of 2 to 3 mm. The NC membrane is provided with a detection line on the side close to the gold pad and a quality control line on the side close to the water absorption plate. In addition, the sample pad is coated with anti-porcine red blood cell antibodies, such as Figure 3 shown.

[0091] 2. The sample pad is coated with red blood cell antibodies

[0092] Anti-porcine erythrocyte mouse monoclonal antibody 3B6 was diluted to 15 μg / ml in 0.01 M TB coating buffer (pH 7.6) containing 1% BSA blocking protein and 0.01% Triton-X-100 surfactant. The antibody, diluted in the coating buffer, was sprayed onto a nitrocellulose membrane and dried in a drying oven at 37.0°C ± 2.0°C for 10 hours. The membrane was then assembled into a chromatography strip according to the colloidal gold test strip structure, sealed in aluminum foil, and stored at room temperature until needed. A 60 μl sample of anticoagulant-supplemented porcine whole blood was collected and dripped onto the prepared chromatography strip sample pad. A sample pad without anti-porcine erythrocyte membrane mouse monoclonal antibody was also placed as a control. The membrane was then left horizontally at room temperature for 15 minutes, and the sample chromatography was observed.

[0093] See the results Figure 4 Chromatography strip #1 is a sample pad containing anti-erythrocyte antibodies. Red blood cells are fully adsorbed on the sample pad, and the background is clean, indicating good chromatography results. Chromatography strip #2 is a sample pad without anti-erythrocyte antibodies. The background color on the NC membrane in the detection area is darker, interfering with the test results. In summary, the 3B6 mouse monoclonal antibody obtained in this invention can be used for rapid colloidal gold detection in porcine whole blood. This technology eliminates the porcine plasma / serum separation step, saving testing costs and time, and has high application value.

[0094] 3. Comparison of the effect of anti-erythrocyte monoclonal antibody interception on pig whole blood with conventional treatment methods

[0095] Collect 2 portions of fresh pig whole blood (10 ml), take 5 ml of each and place them in a 15 ml sterile centrifuge tube, centrifuge at 3000 r / min for 10 minutes at 4°C, and take the supernatant for later use. Dilute the anti-pig erythrocyte mouse monoclonal antibody 3B6 to 15, 30, and 60 μg / ml with pH=7.6, 0.01M TB coating buffer. The TB coating buffer contains 1% BSA blocking protein and 0.01% Triton-X-100 surfactant. Spray the antibody diluted with coating buffer onto the nitrocellulose membrane, dry it in a drying oven at 37.0°C±2.0°C for 10 hours, and assemble it into a chromatography paper strip according to the structure of the colloidal gold test strip. The chromatography paper strip is sealed with an aluminum foil bag and stored at room temperature for later use (the method is the same as experiment 2 in Example 3). The negative control is a test strip that does not contain anti-pig erythrocyte antibodies. See the results. Figure 5Two porcine whole blood samples, numbered A and B, were analyzed. At a coating concentration of 15 μg / ml, the anti-porcine erythrocyte antibody achieved the same interception efficiency as conventional centrifugation methods, outperforming other commercially available porcine whole blood antibodies. In summary, the 3B6 mouse monoclonal antibody obtained in this invention can be used for rapid colloidal gold detection in porcine whole blood. This technology eliminates the porcine plasma / serum separation step, saving testing costs and time, and possesses high application value.

[0096] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against porcine erythrocyte membrane, characterized in that: It has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO:

2.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is of mouse IgG type; and the light chain constant region is of mouse IgG type.

3. A nucleic acid encoding the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 7; The nucleic acid encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

8.

5. An expression module, characterized in that It comprises a promoter, a terminator and the nucleic acid according to claim 3 or 4.

6. A recombinant vector, characterized in that include: A vector backbone and the nucleic acid according to claim 3 or 4; Or a vector backbone and the expression module according to claim 5.

7. A host, characterized in that It includes at least one of the following I) to III): 1), secreting the monoclonal antibody according to claim 1 or 2; II), genome integration of the nucleic acid according to claim 3 or 4 or the expression module according to claim 5; III), transfecting or transforming the recombinant vector as claimed in claim 6.

8. The method for preparing a monoclonal antibody according to claim 1 or 2, characterized in that: Cultivate the host according to claim 7 to obtain the monoclonal antibody.

9. A product that adheres to blood cells, characterized in that: The raw materials include at least one of the following a) to c): a) the monoclonal antibody according to claim 1 or 2; b) the nucleic acid according to claim 3 or 4; c) The expression module according to claim 5; d) the recombinant vector according to claim 6; e) the host according to claim 7; f) A culture containing the monoclonal antibody obtained by the preparation method according to claim 8.

10. Use of the product according to claim 9 in serum sample processing.

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