CN117805402B

CN117805402B - Cat blood typing detection method and kit

Info

Publication number: CN117805402B
Application number: CN202410231392.6A
Authority: CN
Inventors: 赵方圆; 赵荣茂; 巩玉洁; 毕铭钰; 宋铮; 杨晓霞; 李川敏; 张琼林; 陈娟; 魏单平; 袁婷婷
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2024-03-01
Filing date: 2024-03-01
Publication date: 2024-05-28
Anticipated expiration: 2044-03-01
Also published as: CN117805402A

Abstract

The invention discloses a cat blood typing detection method and a kit, and belongs to the technical field of rapid biological detection of pets. In particular, the present invention relates to a feline type A red blood cell monoclonal antibody and a feline type B red blood cell monoclonal antibody that recognize specific antigens on feline red blood cells. Wherein the amino acid sequence of the heavy chain variable region of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 1; the amino acid sequence of the cat A type red blood cell monoclonal antibody light chain variable region is shown as SEQ ID No. 3. The cat blood typing detection kit provided by the invention is used for detecting cat blood types, is simple to operate, greatly improves the detection sensitivity, reduces the detection cost and shortens the detection time.

Description

Cat blood typing detection method and kit

Technical Field

The invention relates to the technical field of rapid biological detection of pets, in particular to a cat blood typing detection method and a kit.

Background

Similar to the human familiar ABO blood group system, the blood group of cats also mainly comprises A, B and AB. Among these, type a blood is most common, accounting for about 90% or more of the total number of cats worldwide (siamesed, burmese, russian blue, oxes, eastern shorthair cats are all essentially type a blood). Type B blood is rare (the most abundant varieties of type B blood are deven cat, british cat, ke Nisi cat, puppet cat, turkish cat, foreign cat, etc.). AB type blood is less common and has a lower proportion than panda blood in humans. Unlike dogs, cats have antibodies to "non-self" or foreign erythrocytes, which can elicit a fatal immune response.

For example, a strong accessory anti-type a antibody in a type B cat causes a strong immune response when blood from a type a cat is delivered to a type B cat, and the half-life of the delivered red blood cells is only a few minutes to a few hours, which in turn causes acute or subacute hemolysis of the transfusion, resulting in death of the transfused cat. In addition, it is also particularly important to determine the blood group of cats during breeding, and clinical hemolysis of newborn cats occurs because the milk of the female cats contains antibodies of 'non-self' blood group, and when the female cats of the B blood group breast-feed a or AB blood type kittens, the kittens can generate hemolysis, jaundice and brown urine symptoms within two weeks, and even die. Thus, the identification of the blood group of cats is of great importance.

Disclosure of Invention

Therefore, the embodiment of the invention provides a cat blood typing detection method and a kit.

In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:

In a first aspect, the invention provides a kit for detecting cat blood typing, comprising a detection card, a dropper filled with diluent, a stirring rod and a quantitative binaural dropper, wherein the detection card comprises a cat A type red blood cell monoclonal antibody and a cat B type red blood cell monoclonal antibody;

The monoclonal antibody contains a heavy chain variable region named V _H and a light chain variable region named V _L;

the amino acid sequence of V _H of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 1 in a sequence table;

The amino acid sequence of V _L of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 3 in a sequence table;

The amino acid sequence of V _H of the cat type B red blood cell monoclonal antibody is shown as SEQ ID No. 5 in a sequence table;

The amino acid sequence of V _L of the cat type-B red blood cell monoclonal antibody is shown as SEQ ID No. 7 in a sequence table.

In a second aspect, the nucleotide sequence of V _H of the A-type red blood cell monoclonal antibody is shown as SEQ ID No. 2 in a sequence table;

The nucleotide sequence of V _L of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 4 in a sequence table;

The nucleotide sequence of V _H of the cat B-type red blood cell monoclonal antibody is shown as SEQ ID No. 6 in a sequence table;

the nucleotide sequence of V _L of the cat B-type red blood cell monoclonal antibody is shown as SEQ ID No. 8 in a sequence table.

In a third aspect, V _H and V _L of the above-described type a red blood cell monoclonal antibody are each composed of a complementarity determining region and a framework region, the complementarity determining regions being each composed of CDR1, CDR2 and CDR 3;

the amino acid sequence of CDR1 of V _H of the A-type red blood cell monoclonal antibody is shown as 63-69 th position in SEQ ID No. 1;

The amino acid sequence of CDR2 of V _H of the A-type red blood cell monoclonal antibody is shown in 87 th-99 th positions of SEQ ID No. 1;

The amino acid sequence of CDR3 of V _H of the A-type red blood cell monoclonal antibody is shown in 130 th-141 th positions of SEQ ID No. 1;

The amino acid sequence of CDR1 of V _L of the A-type red blood cell monoclonal antibody is shown in the 38 th-46 th positions of SEQ ID No. 3;

the amino acid sequence of CDR2 of V _L of the A-type red blood cell monoclonal antibody is shown in the 64 th-66 th positions of SEQ ID No. 3;

the amino acid sequence of CDR3 of V _L of the A-type red blood cell monoclonal antibody is shown as 101 th-107 th positions in SEQ ID No. 3.

In a fourth aspect, V _H and V _L of the above-described type B red blood cell monoclonal antibody are each composed of a complementarity determining region and a framework region, the complementarity determining regions being each composed of CDR1, CDR2 and CDR 3;

The amino acid sequence of CDR1 of V _H of the B-type red blood cell monoclonal antibody is shown in 66 th to 76 th positions in SEQ ID No. 5;

The amino acid sequence of CDR2 of V _H of the B-type red blood cell monoclonal antibody is shown in 94 th-101 th positions of SEQ ID No. 5;

The amino acid sequence of CDR3 of V _H of the B-type red blood cell monoclonal antibody is shown in 137 th-148 th positions in SEQ ID No. 5;

the amino acid sequence of CDR1 of V _L of the B-type red blood cell monoclonal antibody is shown in 58 th-68 th positions of SEQ ID No. 7;

The amino acid sequence of CDR2 of V _L of the B-type red blood cell monoclonal antibody is shown as 85 th-88 th positions in SEQ ID No. 7;

The amino acid sequence of CDR3 of V _L of the B-type red blood cell monoclonal antibody is shown as 124 th-133 th positions in SEQ ID No. 7.

In a fifth aspect, the kit is a kit for detecting blood type in cats, wherein the kit comprises a type A red blood cell monoclonal antibody and/or a type B red blood cell monoclonal antibody. Antibodies may be used alone or as part of a kit.

In a sixth aspect, the invention provides the use of a kit as described above for detecting the blood group of a cat.

In a seventh aspect, the present invention provides a method for detecting cat blood typing, comprising the steps of:

(a) Collecting sample EDTA anticoagulated cat whole blood;

(b) Adding the collected fresh sample into the diluent by using a double-ear dropper, uniformly stirring, and dripping the mixture onto a detection card;

(c) Uniformly spreading the mixture by using a stirring rod;

(d) And judging the result.

The embodiment of the invention has the following advantages:

The invention provides a rapid and simple method for detecting cat blood type; meanwhile, the cat A type red blood cell monoclonal antibody and the cat B type red blood cell monoclonal antibody are provided, are applied to cat blood type detection, are simple to operate, and have the advantages of rapid response and high accuracy of a measurement result. The cat blood typing detection kit prepared by the two monoclonal antibodies can remarkably improve the convenience and accuracy of cat blood typing detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.

FIG. 1 is a SDS-PAGE identification chart of cat A type red blood cell monoclonal antibodies provided by the embodiment of the invention;

FIG. 2 is a SDS-PAGE identification of a cat type-B red blood cell monoclonal antibody according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a screening procedure for binding to cat type A red blood cell monoclonal antibodies using a tear drop agglutination assay according to an embodiment of the present invention.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but 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 Mao A type Red blood cell monoclonal antibody

1. Preparation of antigens

And (3) collecting EDTA anticoagulated A-type blood cat erythrocytes, washing the blood cat erythrocytes three times with normal saline respectively, and re-suspending the blood cat erythrocytes by using sterile Ab solution with the same proportion as the erythrocytes to obtain erythrocyte suspension, thereby being used as an immunogen for preparing the cat A-type blood monoclonal antibody.

2. Immunization of animals

Mice of appropriate age are immunized with the immunogen of the prepared cat type A blood monoclonal antibody. The first immunization dose was 200. Mu.l per BALB/C mouse, followed by subcutaneous multipoint immunization in the same way, 200. Mu.l per mouse. Each immunization interval is 1-2 weeks. Mice were bled from their tail tips on day 7 after immunization at day 5, and tested for clotting potency with cat blood type a and cat blood type B, respectively, using the tear drop method. The collected mouse tail blood was allowed to stand at 4℃for 30min to precipitate serum, which was diluted with PBS in a double ratio, and serum aggregated with cat type B blood and blank mouse serum were used as controls. The maximum dilution of serum with which cat type a hemagglutinated is the titer of mouse serum.

Mice with high titers were selected and boosted by intraperitoneal injection of 200 μl of antigen. Mice were sacrificed 3 days later and cell fusion was performed.

Culture of SP2/0 myeloma cells

One SP2/0 myeloma cell frozen in liquid nitrogen is taken out, immediately transferred into a constant temperature water bath kettle at 37 ℃, the frozen storage tube is gently rocked from time to time, and taken out when the cell is melted to a semi-ice crystal state. And (3) operating in a sterile environment, transferring SP2/0 cells in the cryopreservation tube into a 50mL sterile centrifuge tube, taking 10mL of preheated 1640 complete culture medium, slowly dripping into the centrifuge tube, centrifuging at 1000r/min for 5min, and discarding the supernatant. The cell pellet was gently broken up, 5mL of medium was taken to resuspend the cells and transferred to T75 cell flasks. In addition, 5mL of culture medium is added, the cell bottle is rocked in the cross direction, and the cell bottle is placed into a CO ₂ cell incubator for culture at 37 ℃. The cell status was observed under a microscope, and SP2/0 cells were subcultured at a density of about 80%.

4. Cell fusion

(1) Serum was collected: the boosted mice were collected by orbital blood collection, collected in an EP tube, allowed to stand at 37℃for 2h, centrifuged at 4000rpm for 10min, and serum was collected as a positive control for subsequent screening of monoclonal antibodies. Mice were sacrificed by cervical removal and sterilized by soaking in 75% alcohol.

(2) Preparation of spleen cells: the outer skin of the mouse is cut off by using sterilized scissors and tweezers in the biosafety cabinet, the abdominal cavity of the mouse is cut off by replacing a set of new sterilized scissors and tweezers, and the whole spleen is carefully taken out by using the set of sterilized scissors and tweezers, so that redundant fat is removed. A sterile 15mL centrifuge tube was prepared, 10mL DMEM medium was added, the spleen was placed in the tube, and after wetting the spleen, the excess medium was carefully discarded. Then 10mL of DMEM culture medium is sucked and placed in a sterile plate, the spleen is ground by the tail end of a sterile syringe to prepare single-cell suspension, the single-cell suspension is filtered into a sterile centrifuge tube through a 200-mesh nylon net, 30mL of DMEM is added into a 50mL sterile centrifuge tube, the nylon net is washed by a suction tube, the centrifuge tube filled with the spleen cell suspension is centrifuged at 1500rpm for 5min, the supernatant is discarded, the cell mass is gently dispersed by hands, 30mL of DMEM culture medium is added for one time after being resuspended, the supernatant is discarded, the cell mass is gently dispersed by hands, and 10mL of DMEM culture medium is added for resuspension.

(3) Cell fusion: the well-grown SP2/0 cells were centrifuged at 1000rpm for 5min, collected in a 50mL centrifuge tube, gently dispersed in SP2/0 cell mass, resuspended in 30mL DMEM medium, centrifuged again, and resuspended in 10mL DMEM medium. Mixing the spleen cell suspension and the SP2/0 cell suspension according to the cell number ratio of 8:1, reversing the mixture, and centrifuging at 1000rpm for 4min; beating the precipitated cells in a water bath at 37 ℃ to uniformly distribute the cells at the bottom of the tube, standing for 1min, adding 1ml of PEG1450 into the centrifuge tube within 1min, standing for 1min, and adding 1ml of incomplete DMEM culture solution preheated at 37 ℃ along the tube wall within 30s to terminate the cell fusion reaction; extending the gun head under the liquid level, adding 1ml of incomplete DMEM for 1min, and repeating the steps until 20ml of incomplete DMEM culture solution is added; after slowly adding 30ml of incomplete DMEM medium, the supernatant was discarded, and then 50ml of incomplete DMEM medium was added and centrifuged at 800rpm for 4min. Removing the supernatant, adding HAT culture medium, gently blowing up cells and adding 96-well cell culture plates (200 μl/well), and marking; after 7 days, erythrocyte agglutination was tested by the tear drop method.

5. Screening positive hybridoma cells

Hybridoma cell supernatants were collected and screened by tear drop agglutination. As shown in FIG. 3, wells with high titer of hemagglutination with cat type A, no hemagglutination with cat type B, and only single cell mass were selected, medium was discarded, 200. Mu.l HT medium was added, cells were blown and counted, approximately 200 cells were plated in half 96 well plates, and the remaining cells were passaged to 48 well plates for continued expansion culture and frozen. After 7d, the monoclonal cells were subjected to agglutination assay, subcloned again by the method described above, and after 3 subcloning, single cell mass having high titer of agglutination with cat type a and non-agglutination with cat type B was selected, and then cloned as described above. And (3) performing expansion culture on the hybridoma cell strain which is identified after subcloning and can stably secrete the monoclonal antibody, and preparing ascites for collection.

6. Preparation of monoclonal antibody ascites

Injecting the positive hybridoma cell strain obtained by culture into the abdominal cavity of a mouse, culturing the mouse, and extracting ascites from the abdominal cavity of the mouse for purification. The specific operation steps are as follows:

The mice were injected intraperitoneally with 500. Mu.l Freund's incomplete adjuvant, and after 24 hours, about 1X 10 ⁷ hybridoma cells were injected into the abdominal cavity of the mice, and after 7 days, ascites were collected. The antibody was purified using a commercial antibody purification kit, specifically as follows: centrifuging the ascites at 10000rpm for 10min, collecting supernatant, and collecting 20 μl to obtain sample; 60 μl 1M Tris-HCl (pH=9.0) was added to the centrifuge tube; filtering Binding buffer and the partition buffer for standby by using a filter with the diameter of 0.45 mu m; ascites diluted twice by Binding buffer; filling 10ml Binding buffer with a syringe, connecting the syringe to a purification column, removing bubbles, slowly pushing a piston, and removing the storage liquid; sucking 10ml Binding buffer, balancing the column, wherein the flow rate is 1 ml/min; sucking diluted ascites by the injector at a flow rate of 0.2ml/min to combine the antibody with the column; absorbing 10ml Binding buffer fully, washing out unbound antibody until the effluent liquid is colorless; the antibody bound to the column was eluted by pipetting 5ml Elution buffer and added dropwise to the above Tris-HCl added centrifuge tube, 8 drops/tube. Samples of 20. Mu.l each tube were prepared for use and the purified cat type A red blood cell monoclonal antibodies were characterized.

EXAMPLE 2 preparation of feline B-type Red blood cell monoclonal antibody

The preparation of a cat type B red blood cell monoclonal antibody was performed in the same manner as in example 1 using red blood cells of cat type B blood as an immunogen, and the characterization was performed after purification.

Characterization of monoclonal antibodies prepared in example 3, example 1 and example 2

1. Identification of monoclonal antibody concentration

And (3) carrying out concentration measurement on the purified monoclonal antibody by adopting a nucleic acid protein concentration measuring instrument, wherein the concentration of the cat A type red blood cell monoclonal antibody is 7.12mg/ml, and the concentration of the cat B type red blood cell monoclonal antibody is 5.76mg/ml.

2. Determination of monoclonal antibody purity

SDS-PAGE analysis was performed on the purified monoclonal antibodies. As shown in fig. 1 and 2, 2 distinct bands appeared around 25kDa and 65kDa, with an antibody light chain at 25kDa and an antibody heavy chain at 65kDa, with almost no other bands, and purity meeting the expected requirements.

3. Determination of monoclonal antibody specificity

Specific identification was performed by a paper sheet agglutination method. The method comprises the following specific steps: and (3) diluting the purified monoclonal antibody in gradient ratio, taking 5 mu l of the diluted monoclonal antibody onto a paperboard, and placing the paperboard in a baking oven at 37 ℃ for 20-30min for baking. 50 μl of fresh A-type and B-type EDTA anticoagulated whole blood is sucked by a dropper and a pipette, added into 600 μl of diluent, and mixed uniformly to prepare red blood cell suspensions, and 1 drop (30-50 μl) of red blood cell suspension is respectively suspended and added on an antibody reagent. The reagent coated position is stirred vigorously by a stirring rod and mixed for 15 seconds until the reagent and blood are completely mixed at the coated position. After standing for 2 minutes, the card is lifted up and shaken up and down uniformly to judge whether granular agglutination exists or not. The results showed that type a antibodies: to form a blood clot with type A cats and to form a blood clot with type B cats; type B antibody: to form a blood-clotting group with cat type B and not to form a blood-clotting group with cat type A. This indicates that the type a and type B monoclonal antibodies are well specific.

4. Monoclonal antibody subclass identification

Monoclonal antibodies prepared in example 1 and example 2 were detected using a commercial ELISA kit for IgG subclass identification. The results show that the heavy chain of the cat A type red blood cell monoclonal antibody is of an IgG1 type, and the light chain is of an L chain; the heavy chain of the cat type B red blood cell monoclonal antibody is of an IgG1 type, and the light chain is of an L chain.

Example 4 PCR amplification and sequencing of monoclonal antibody variable region genes

RNA extraction

The hybridoma cells secreting cat A type red blood cell monoclonal antibody and cat B type red blood cell monoclonal antibody are cultured in RPMI 1640 complete medium at 37 ℃ under the condition of 5% carbon dioxide, and total RNA in the cells is extracted by using a total RNA extraction kit (purchased from the root of the heaven) when the number of the cells reaches 1×10 ⁷.

2. Gene amplification

The specific upstream and downstream universal primers of the murine heavy chain antibody gene and the light chain antibody gene are designed.

Heavy chain upstream primer (SEQ ID No. 9): TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC;

Heavy chain downstream primer (SEQ ID No. 10): AGGTSMARCTGCAGSAGTCWGG.

Light chain upstream primer 1 (SEQ ID No. 11): CCGTTTGATTTCCAGCTTGGTGCC;

Light chain upstream primer 2 (SEQ ID No. 12): CCGTTTTATTTCCAGCTTGGTCCC;

light chain upstream primer 3 (SEQ ID No. 13): CCGTTTTATTTCCAACTTTGTCCC;

light chain upstream primer 4 (SEQ ID No. 14): CCGTTTCAGCTCCAGCTTGGTCCC;

Light chain downstream primer 5 (SEQ ID No. 15): GACATTGAGCTCACCCAGTCTCCA.

3. Sequencing

Heavy and light chains are amplified by using an RT-PCR kit and are connected to a PLB cloning vector for gene sequencing.

Sequencing results were as follows:

cat A type red blood cell monoclonal antibody Heavy chain amino acid sequence (SEQ ID No. 1)

LKTLTLIMECVWILPCILSVHSNVYSELQLQTWGTSLQSGTVLRRPGASVVMSCKASQYTFSGSYMWMGHWVKQRVQPGQGLEWIGAGSMGSTTYCKFPKAKLVAVTSTSTLYMEDLTVEDSAVYYCTRRVVNQLDWTFDCWGAGDVTVPSAKTWPPSVYPVAPGSAQYNSVVTLCLVEGYFPQPVC.

Cat A type red blood cell monoclonal antibody Heavy chain nucleotide sequence (SEQ ID No. 2)

CTGAAAACATTGACTCTAATCATGGAATGTGTCTGGATACTTCCTTGCATTCTGTCGGTACATTCAAATGTCTACTCAGAGTTGCAGCTCCAGACATGGGGAACGTCACTGCAGTCTGGGACTGTGCTGCGAAGGCCTGGGGCTTCAGTGGTAATGTCCTGCAAGGCTTCTCAGTACACCTTTTCCGGTAGCTATATGTGGATGGGTCACTGGGTAAAACAGAGGGTTCAGCCTGGACAGGGTCTGGAATGGATTGGCGCTGGTTCTATGGGAAGTACTACTTACTGCAAGTTCCCTAAGGCCAAACTGGTTGCAGTCACATCCACCAGCACTCTGTACATGGAGGACCTGACAGTAGAGGACTCTGCGGTCTATTACTGTACAAGACGTGTTGTTAACCAGCTTGATTGGACTTTCGATTGCTGGGGCGCAGGGGACGTCACCGTCCCATCAGCCAAAACGTGGCCCCCATCTGTCTATCCAGTCGCCCCTGGATCTGCCCAATACAACTCCGTCGTGACCCTGTGCCTGGTCGAAGGCTATTTCCCTCAGCCAGTGTGTGT.

Cat A type red blood cell monoclonal antibody Light chain amino acid sequence (SEQ ID No. 3)

NLPNSRNETVSGAAVVTQESALTTKPGETVTLNSRSSTKAVTFSNYPNWVQEKPDLLFTFLIGGENDRLPGVTPRFKGSLGDKSALTIGAQTLDEEIYFCALWKSNHTFGATLEMVSSLETAKLRQFTLAIKRA.

Cat A type red blood cell monoclonal antibody Light chain nucleotide sequence (SEQ ID No. 4)

AATCTGCCCAATAGTAGAAACGAGACAGTCAGTGGGGCGGCTGTGGTAACCCAGGAGAGCGCACTGACTACCAAACCTGGGGAAACCGTCACACTCAACTCCCGCTCCAGTACCAAGGCAGTAACCTTTTCCAACTATCCGAACTGGGTGCAGGAAAAGCCTGATCTGCTGTTCACATTCCTGATCGGCGGAGAAAACGATCGGTTACCTGGAGTAACTCCTAGATTCAAAGGGTCTCTCGGCGATAAGTCGGCTCTGACTATCGGCGCACAGACTTTGGATGAAGAAATCTACTTCTGCGCACTGTGGAAGTCCAACCATACATTCGGGGCAACATTGGAAATGGTCTCCTCCCTGGAGACAGCTAAACTGCGTCAATTCACTCTCGCCATCAAACGGGCTT.

Cat B-type red blood cell monoclonal antibody Heavy chain amino acid sequence (SEQ ID No. 5)

MSTDSTPGGSSHCMSSEHQHLTGNFFFRLSFLLLVVKGSQCEKHVELGTGLHKPGPSLKLSCAPSGFGTFSYSLYDMSFVRDTPGKRVEWLASISGWGGSTRYPDVKGLFTLRDKARQILYDQMQLRCEDAAMYYCAGRSTHYPYKDYWPQQTAETVPSAKATPPQYPDAPISAGQTNGMTLGLLEGYGPERVTVTW.

Cat B-type red blood cell monoclonal antibody Heavy chain nucleotide sequence (SEQ ID No. 6)

ATGTCAACGGACAGTACACCAGGGGGATCCTCACATTGTATGAGCTCAGAACACCAGCACCTCACCGGAAACTTCTTCTTCCGATTGTCCTTCCTTCTGCTTGTTGTAAAAGGTTCGCAGTGTGAAAAGCACGTGGAGCTCGGGACCGGCTTACACAAGCCTGGACCGTCCCTGAAGCTCTCCTGTGCACCGTCTGGATTCGGGACTTTCAGTTATTCCCTGTATGACATGTCTTTCGTTCGCGACACTCCAGGCAAGAGGGTCGAGTGGCTGGCATCCATTAGTGGTTGGGGTGGTTCAACCCGCTATCCAGACGTGAAGGGCTTGTTCACCCTAAGAGATAAAGCCAGGCAAATCCTATACGACCAAATGCAGCTGCGGTGTGAGGACGCTGCCATGTATTACTGTGCAGGCCGCTCCACCCATTACCCCTACAAGGACTACTGGCCGCAACAAACCGCTGAGACAGTCCCTTCAGCCAAAGCAACACCCCCACAGTATCCAGACGCCCCTATTTCTGCTGGCCAAACTAACGGTATGACCCTGGGATTGCTGGAAGGCTATGGTCCTGAGAGGGTGACAGTGACCTGG.

Cat B-type red blood cell monoclonal antibody Light chain amino acid sequence (SEQ ID No. 7)

TWGTIIEKARPPGIMRWISFISLLALSSPAISQAVVTQSSALTTSPGETVTLTCRSSAGAMVTTQSNYANWVNEKPILFHGSIGGQTNNRSPGVPPRFSGSLGGDKALLTHGRQTEQESIYFCAMWYNHDFGVRWRNQSDCPRTAVRIHPPSTLLREARDE.

Cat B-type red blood cell monoclonal antibody Light chain nucleotide sequence (SEQ ID No. 8)

ACATGGGGGACCATTATTGAAAAGGCTAGACCTCCGGGAATTATGCGCTGGATTTCATTCATATCTCTCCTGGCTCTCAGCTCACCTGCCATTTCCCAGGCTGTTGTGACTCAGTCGTCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTGCTGGGGCTATGGTTACAACTCAAAGTAACTATGCCAACTGGGTCAACGAAAAACCAATCTTATTCCATGGTTCAATAGGTGGTCAGACAAACAACCGATCGCCAGGTGTTCCTCCGAGATTCTCAGGCTCCCTGGGAGGAGACAAGGCTCTCCTCACCCATGGGCGACAGACTGAGCAGGAGTCAATATATTTCTGTGCTATGTGGTACAACCATGACTTCGGTGTTCGGTGGAGGAACCAATCAGACTGTCCTAGGACCGCCGTCCGGATCCACCCTCCTTCCACCCTTCTCCGAGAAGCTCGAGACGAA.

4. Analysis of amino acid sequence homology in variable region

The heavy chain gene Sequence and the light chain gene Sequence are respectively compared and analyzed in NCBI database, and the analysis result shows that the heavy chain amino acid Sequence of the cat A type red blood cell monoclonal antibody has the highest homology with the heavy chain amino acid Sequence of the mouse e0D01 (Sequence ID:5NUZ A), the homology is 112/169, and the homology percentage is 66%. The light chain amino acid Sequence of the cat A type hemoglobus monoclonal antibody has the highest homology with the L chain amino acid Sequence of the mouse immunoglobulin (Sequence ID: BAA 83393.1), the homology is 78/105, and the homology percentage is 74%. The results of amino acid sequence homology analysis of the heavy and light chains of the cat A-type red blood cell monoclonal antibody indicate that the same sequences as those of the present invention are not found.

The homology of the heavy chain amino acid Sequence of the cat B type red blood cell monoclonal antibody and the heavy chain amino acid Sequence of the mouse antibody gamma 1 (Sequence ID: BAB 33403.1) is highest, the homology is 105/182, and the homology percentage is 58%. The light chain amino acid Sequence of the cat B type hemoglobus monoclonal antibody has the highest homology with the amino acid Sequence of the mouse immunoglobulin lambda chain (Sequence ID: AAO 53371.1), the homology is 87/120, and the homology percentage is 73%. The results of amino acid sequence homology analysis of the heavy and light chains of the monoclonal antibody encoding cat type B red blood cells indicate that the same sequences as the present invention are not found.

Cdr region analysis

Monoclonal antibodies V _H and V _L are each composed of a complementarity determining region consisting of CDR1, CDR2 and CDR3, and a framework region. The sequences of the heavy chain variable region and the light chain variable region were analyzed at HTTP:// www.novopro.cn/tools/cdr.html to obtain the CDR regions.

Cat a type red blood cell monoclonal antibody heavy chain CDR region:

CDR-H1：GSYMWMG；

CDR-H2：AGSMGSTTYCKFP；

CDR-H3：RVVNQLDWTFDC。

cat a type red blood cell monoclonal antibody light chain CDR region:

CDR-L1：TKAVTFSNY；

CDR-L2：GEN；

CDR-L3：ALWKSNH。

Cat B-type red blood cell monoclonal antibody heavy chain CDR regions:

CDR-H1：GFGTFSYSLYD；

CDR-H2：ISGWGGST；

CDR-H3：AGRSTHYPYKDY。

Cat B-type red blood cell monoclonal antibody light chain CDR regions:

CDR-L1：AGAMVTTQSNY；

CDR-L2：GQTN；

CDR-L3：AMWYNHDFGV。

example 5 preparation and use of a Mao blood typing kit Using monoclonal antibodies as raw materials

The kit consists of a detection card coated with the cat A type red blood cell monoclonal antibody prepared in the example 1 and the cat B type red blood cell monoclonal antibody prepared in the example 2, a dropper filled with a diluent, a stirring rod and a quantitative binaural dropper, wherein the detection card comprises an A hole, a B hole and a control hole.

1. Concrete fabrication

And (3) detecting card: taking a customized paper detection card, diluting the purified A, B monoclonal antibody 10 times, respectively taking 5 mu l points to A, B holes on the paper card, and placing in a 37 ℃ oven for 20-30min for drying.

Dilution liquid: A0.01M PBS pH7.2 solution containing 0.05% Tween-20.

Stirring rod: commercial wood stirring rod.

Quantitative binaural dropper: commercial droppers.

2. Application method

Step one: sample EDTA-anticoagulated cat whole blood was collected.

Step two: taking out the detection card (the detection card should be used as soon as possible after unpacking), putting the detection card on a horizontal table top, and marking; the fresh sample was sucked up to about 50. Mu.L using a binaural dropper, and added to a diluent tube, followed by thoroughly mixing to prepare a red blood cell suspension.

Step three: respectively suspending and dripping 1 drop of red blood cell suspension on the detection card A, B and the quality control hole, and covering A, B coating points; the reagent coating position is stirred by a stirring rod forcefully for 15 seconds until the reagent and blood are completely mixed at the coating position, and the mixture is uniformly spread to exceed 80% of the detection window.

Step four: interpretation of results

Type a blood: control does not agglutinate, a well agglutinates, B well does not agglutinate;

type B blood: control does not agglutinate, pore a does not agglutinate, pore B Kong Ningji;

type AB blood: control is not agglutinated, A hole agglutinates, B Kong Ningji;

Invalid card 1: control agglutination, a-well agglutination, B Kong Ningji;

Invalid card 2: control did not agglutinate, a well did not agglutinate, B well did not agglutinate.

Characterization of the Cat blood typing kit in example 6, example 5

1. Accuracy identification

100 Serum samples are detected simultaneously by using the cat blood typing kit and the commercial test paper card prepared by the invention, the detection results show that 89 cases of A types and 11 cases of B types are all detected, and the comparison results show that the coincidence rate of the cat blood typing kit prepared by the invention and the detection of the whole sample of the outsourcing test paper card is 100%, which indicates that the cat blood typing kit has good accuracy.

2. Specificity identification

The cat blood typing kit prepared by the invention is used for testing cat blood, dog blood and cow blood, and only has specific agglutination reaction with cat blood, which shows that the kit has better specificity.

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The kit for detecting cat blood typing comprises a detection card, a dropper filled with diluent, a stirring rod and a quantitative binaural dropper, and is characterized in that the detection card comprises a cat A type red blood cell monoclonal antibody and a cat B type red blood cell monoclonal antibody;

The amino acid sequence of V _H of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 1;

the amino acid sequence of V _L of the cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 3;

the amino acid sequence of V _H of the cat type B red blood cell monoclonal antibody is shown as SEQ ID No. 5;

The amino acid sequence of V _L of the cat type B red blood cell monoclonal antibody is shown as SEQ ID No. 7.

2. The kit of claim 1, wherein the nucleotide sequence of V _H of the type a red blood cell monoclonal antibody is shown in SEQ ID No. 2 of the sequence listing;

the nucleotide sequence of V _L of cat A type red blood cell monoclonal antibody is shown as SEQ ID No. 4;

The nucleotide sequence of V _H of cat type B red blood cell monoclonal antibody is shown as SEQ ID No. 6;

The nucleotide sequence of V _L of cat type B red blood cell monoclonal antibody is shown as SEQ ID No. 8.

3. The kit of claim 1, wherein V _H and V _L of the type a red blood cell monoclonal antibody are each comprised of a complementarity determining region and a framework region, the complementarity determining regions being comprised of CDR1, CDR2, and CDR 3;

4. The kit of claim 1, wherein V _H and V _L of the type B red blood cell monoclonal antibody are each comprised of a complementarity determining region and a framework region, the complementarity determining regions being comprised of CDR1, CDR2, and CDR 3;

5. Use of a type a and/or type B red blood cell monoclonal antibody in the kit of claim 1 for the preparation of a product for detecting cat blood typing.

6. Use of the kit of claim 1 for detecting blood group in cats.

7. A method for detecting cat blood typing, characterized in that the kit of claim 1 is used, comprising the steps of:

(a) Collecting sample EDTA anticoagulated cat whole blood;

(c) Uniformly spreading the mixture by using a stirring rod;

(d) And judging the result.