CN117801111B

CN117801111B - Specific antibody combined with canine red blood cells and application thereof

Info

Publication number: CN117801111B
Application number: CN202410231436.5A
Authority: CN
Inventors: 巩玉洁; 赵方圆; 杨晓霞; 赵荣茂; 毕铭钰; 邓西; 姜斯嘉; 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: CN117801111A

Abstract

The invention discloses a specific antibody combined with canine erythrocytes and application thereof. Wherein, the specific antibody combined with the canine red blood cell comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. According to the invention, through separating and extracting canine red blood cell antigens, an immunized mouse screens monoclonal antibodies with high sensitivity and high specificity by utilizing a hybridoma cell technology, canine red blood cells of different canine species can be specifically combined, and the monoclonal antibodies are not combined with red blood cells of other species, can be used as specific antibodies for identifying canine blood type test strips, and can also be used as sample pad treatment fluid components in colloidal gold test strips for identifying canine whole blood samples. The prepared canine blood grouping test strip provides convenience for domestic veterinary clinical canine blood transfusion treatment, and avoids blood transfusion reaction of dogs caused by blood group mismatch.

Description

Specific antibody combined with canine red blood cells and application thereof

Technical Field

The invention relates to the technical field of rapid biological detection of pets, in particular to a specific antibody combined with canine erythrocytes and application thereof.

Background

The blood group of dogs was first discovered in 1910. Dogs, like humans, have a wide variety of blood types, now internationally recognized as 8 blood types, designated canine erythrocyte antigen 1 (DEA 1), DEA3, DEA4, etc., respectively. Among them, DEA1 has 4 subtypes: 1.1, 1.2, 1.3 and negative. DEA1 dogs do not contain natural antibodies of DEA1 blood group, the first transfusion does not cause serious transfusion reaction due to blood group incompatibility, but the dogs undergo transfusion reaction due to blood group mismatch when transfused again, and the dogs are serious and even fatal, wherein the transfusion reaction of DEA1 is the fastest and the most severe. Therefore, special attention should be paid to whether the DEA1 blood group is matched or not at the time of transfusion, and the DEA 1-positive blood-receiving dogs can receive DEA 1-positive or negative blood, and the DEA 1-negative blood-receiving dogs can receive DEA 1-negative blood.

The blood banks of animals are established in developed countries such as the united states and australia in the form of a central blood bank and have correspondingly perfected blood group testing procedures. There is no corresponding animal blood bank in China, blood transfusion is rarely performed in veterinary clinic, only temporary blood supply dogs are found, simple cross blood matching tests are performed before blood transfusion, few blood tests also depend on foreign import test paper or antibodies, but the import test paper and the antibodies have poor value, and huge resistance is caused for domestic veterinary clinic blood transfusion treatment.

Disclosure of Invention

In order to solve the problems, the invention provides a specific antibody combined with canine erythrocytes and application thereof.

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

According to a first aspect of embodiments of the present invention, there is provided a canine red blood cell-binding specific antibody comprising a heavy chain variable region and a light chain variable region;

The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2;

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 48-52, 67-79 and 111-119 of SEQ ID No. 1;

the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 68-80, 96-101 and 132-140 of SEQ ID No. 2.

Further, the specific antibody combined with the canine red blood cells is a murine monoclonal antibody obtained by separating and extracting canine red blood cell antigen as an immunogen and screening.

According to a second aspect of embodiments of the present invention there is provided the use of an antibody specific for binding canine erythrocytes in a detection reagent.

Further, the specific antibody combined with the canine red blood cells is coated on the canine blood group identification test strip quality control line C line.

Further, the specific antibody combined with the canine red blood cells is added into a sample pad treatment liquid to prepare a mixed liquid which is used as a canine blood group identification colloidal gold test strip sample pad treatment mixed liquid.

Further, in the sample pad treatment mixed solution, the concentration of the specific antibody is 2-8 mg/ml.

The invention has the following advantages:

The invention acquires EDTA anticoagulated canine erythrocytes, washes the canine erythrocytes three times with normal saline, and then uses sterile Ab solution with equal proportion to erythrocytes to resuspend the obtained erythrocyte suspension as immunogen for preparing canine erythrocytes specific antibody. After mice are immunized, monoclonal antibodies with high sensitivity and high specificity are screened by a limiting dilution method and a tear drop agglutination test method, so that the monoclonal antibodies can be specifically combined with canine erythrocytes of different canine species, and are not combined with erythrocytes of other species, and the monoclonal antibodies can be used as specific antibodies for identifying canine blood type test strips and can also be used as sample pad treatment fluid components in colloidal gold test strips for identifying canine whole blood samples. The prepared canine blood grouping test strip provides convenience for domestic veterinary clinical canine blood transfusion treatment, and avoids blood transfusion reaction of dogs caused by blood group mismatch.

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.

FIG. 1 is a diagram of screening specific antibodies binding canine erythrocytes for different canine species types using a tear drop agglutination assay provided by an embodiment of the present invention;

FIG. 2 is a schematic diagram of a screening process for binding canine erythrocyte specific antibodies using the tear drop agglutination assay according to an embodiment of the present invention;

FIG. 3 is a graph showing the purity of purified bound canine erythrocyte specific antibodies provided in the examples of the present invention;

FIG. 4 is a diagram showing the identification of the specificity of the purified canine red blood cell-binding antibodies provided in the examples of the present invention;

FIG. 5 is a graph showing the detection results of positive and negative samples of a canine blood typing test strip provided by an embodiment of the present invention;

Fig. 6 is a diagram showing the detection results of samples before and after the treatment of the sample pad of the rabies virus colloidal gold antibody detection test strip according to the embodiment of the 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 canine erythrocyte antigen

EDTA anticoagulated diesel and canine erythrocytes are collected, washed three times by normal saline, and then the erythrocyte suspension is obtained by resuspension of sterile Ab liquid with equal proportion to the erythrocytes and is used as immunogen for preparing canine erythrocyte antibody.

Example 2 preparation and screening of antibodies specific for canine erythrocytes

1. Immunization of mice

3 Female Balb/c mice of 6-8 weeks old were taken and the red blood cell suspension prepared in example 1 was used as immunogen. 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. The blood sampling of the tail tip of the mouse is carried out on the 7 th day after the 5 th immunization, the hemagglutination titer of the red blood cells of 4 dogs (beagle dogs, demu, firewood dogs and Thai) is respectively tested by adopting a tear drop agglutination test method, the tail blood of the mouse is collected and then is put into a temperature of 4 ℃ for standing for 30min, serum is separated out, PBS (0.01 mol/L, pH value is 7.4) is used for carrying out multiple dilution on the serum, and the maximum dilution multiple of the serum agglutinated with the dog, namely the titer of the anti-canine red blood cell antibody of the mouse serum.

The tear drop agglutination test method comprises the following steps:

(1) Preparation of erythrocyte suspension:

A. taking fresh EDTA anticoagulated whole blood 2500 rpm, centrifuging for 10 minutes, and sucking the supernatant by a pipette;

B. Adding normal saline with the same volume as the red blood cells, mixing the mixture upside down, and centrifuging the mixture for 10 minutes again at 2500 rpm;

C. repeating the step B2 times;

D. Adding the normal saline with the same volume again, reversing and uniformly mixing to prepare the erythrocyte suspension for standby.

(2) The experimental steps are as follows:

A. adding 25 μl of antibody/serum to be detected into the reaction well;

B. The prepared red blood cell suspension is diluted 100 times by PBS (0.01 mol/L, pH value is 7.4) to prepare 1 percent red blood cell suspension;

C. adding 25 μl of the 1% erythrocyte suspension prepared in step B into the reaction well;

D. shaking the reaction plate, mixing uniformly, and standing at room temperature for 30 minutes;

E. after the timing is finished, the reaction plate is lifted up, inclined by 45 degrees and observed.

(3) Interpretation of the results: the red blood cells were allowed to flow in the form of teardrop, indicating that the red blood cells were not aggregated, and if the red blood cells were not allowed to flow down or were spread in a diffuse manner at the bottom of the well, indicating that the red blood cells were aggregated.

The results are shown in FIG. 1, with the dilution gradient of 2, 4, 8, 16, 32, 64, 128, 256-fold ratios from top to bottom for different mouse sera; from left to right: the number 1 mouse serum and beagle blood agglutination titer, the number 1 mouse serum and dewra blood agglutination titer, the number 1 mouse serum and firewood dog blood agglutination titer, the number 1 mouse serum and tad blood agglutination titer, the number 2 mouse serum and beagle blood agglutination titer, the number 2 mouse serum and dewra blood agglutination titer, the number 2 mouse serum and firewood dog blood agglutination titer, the number 2 mouse serum and tad blood agglutination titer, the number 3 mouse serum and beagle blood agglutination titer, the number 3 mouse serum and dewra blood agglutination titer, the number 3 mouse serum and firewood dog blood agglutination titer, the number 3 mouse serum and the tad blood agglutination titer.

Mice number 2, which have high titers of hemagglutination with canine erythrocytes, were selected and boosted by intraperitoneal injection of 200 μl of erythrocyte antigen. Mice were sacrificed 3 days later and cell fusion was performed.

2. Cell fusion

The boosted mice were eyeballs were collected and serum was isolated as a positive control. After the mice were sacrificed by cervical fracture, the mice were immersed in 75% ethanol for 10min and fixed on an dissecting table in an ultra clean bench. The abdomen skin was lifted with sterilized forceps, small pieces of skin were cut upward from below the abdomen with sterile scissors, the skin and peritoneum were separated, other viscera were carefully pulled apart, and the spleen was gently removed and placed in a petri dish containing 20ml of incomplete DMEM broth. The spleen cells were blown out in a petri dish by puncturing the spleen from the top with a syringe filled with 20ml of incomplete DMEM broth, penetrating the spleen, gently pushing the syringe, repeating several times until the spleen no longer discolored, and filtering the spleen cells with a filter screen. Counting SP2/0 and spleen cells, mixing the spleen cells with SP2/0 according to the cell number ratio of 8:1, reversing and mixing uniformly, 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; slowly adding 30ml of incomplete DMEM culture medium, centrifuging at 800rpm for 4min, discarding supernatant, adding 50ml of incomplete DMEM culture medium, centrifuging at 800rpm for 4min, discarding supernatant, adding HAT culture medium, gently blowing up cells, adding 96-well cell culture plate (200 μl/well), and marking; after 7 days, the hemagglutination was tested by the tear drop agglutination test.

3. Screening of positive hybridoma cells

Hybridoma cell supernatants were collected and screened by tear drop agglutination. Selecting a hole which has high agglutination titer with canine erythrocyte and has only single cell mass, discarding the culture medium, adding 200 mu l of HT culture medium, blowing and counting cells, spreading about 200 cells into a half 96-well plate, and passaging the rest cells to a 48-well plate for further expansion culture and freezing. And (3) performing agglutination identification on the monoclonal cells after 7d, performing subcloning again by adopting the method, selecting single cell aggregates which are agglutinated with the canine blood of multiple dogs and have high titers after 3 subcloning, and performing cloning according to the method. As shown in FIG. 2, a schematic diagram of a positive hybridoma cell screening process is shown, and the most suitable specific antibody hybridoma cell strain as canine erythrocytes is finally selected.

4. Ascites preparation and purification

The cell line obtained above was injected into the abdominal cavity of a mouse, the mouse was cultured, and ascites was collected from the abdominal cavity of the mouse and purified. 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 monoclonal antibodies were identified.

Example 3 identification of specific antibodies that bind canine erythrocytes

1. Concentration measurement

And (3) carrying out concentration measurement on the purified specific antibody by adopting a nucleic acid protein concentration measuring instrument, wherein the concentration of the canine erythrocyte specific antibody is 8.59mg/ml.

2. Purity determination

SDS-PAGE electrophoresis analysis was performed on the purified canine erythrocyte specific antibodies. As shown in the results of FIG. 3, 2 clear bands appear near 20kDa and 50kDa, the 20kDa is the antibody light chain, the 50kDa is the antibody heavy chain, and almost no other miscellaneous bands exist, so that the purity meets the expected requirement.

3. Specificity identification

Specific identification was performed by a paper sheet agglutination method. The method comprises the following specific steps: and (3) diluting the purified canine erythrocyte specific antibody in gradient ratio, taking 5 mu l of the diluted antibody onto a paperboard, and placing the paperboard in a baking oven at 37 ℃ for 20-30min for baking. And (3) sucking 50 mu l of fresh EDTA anticoagulated whole blood from different canine species, feline species and bovine species by using a dropper and a pipette, adding the 50 mu l of the blood into 800 mu l of diluent, and uniformly mixing the two to prepare erythrocyte suspensions, and respectively suspending and dropwise adding 1 drop (30-50 mu l) of erythrocyte suspension 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 are shown in FIG. 4, which shows the hemagglutination patterns of dogs, cats, cattle and pigs in order from left to right, and the results indicate that the antibodies bind to canine erythrocytes with good specificity, while not to feline, bovine and porcine blood.

EXAMPLE 4 cloning of heavy and light chain variable region genes of specific antibodies that bind canine erythrocytes

1. Hybridoma cell culture and total RNA extraction

The hybridoma cells secreting the specific antibody of canine erythrocytes 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 sky) when the number of the cells reaches 1×10 ⁷.

2. PCR 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: TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC the process of the preparation of the pharmaceutical composition,

Heavy chain downstream primer: AGGTSMARCTGCAGSAGTCWGG.

Light chain upstream primer 1: CCGTTTGATTTCCAGCTTGGTGCC the process of the preparation of the pharmaceutical composition,

Light chain upstream primer 2: CCGTTTTATTTCCAGCTTGGTCCC the process of the preparation of the pharmaceutical composition,

Light chain upstream primer 3: CCGTTTTATTTCCAACTTTGTCCC the process of the preparation of the pharmaceutical composition,

Light chain upstream primer 4: CCGTTTCAGCTCCAGCTTGGTCCC the process of the preparation of the pharmaceutical composition,

Light chain downstream primer 5: GACATTGAGCTCACCCAGTCTCCA.

3. Sequencing vector cloning and sequencing

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

The nucleotide sequence of the coding heavy chain variable region is shown as SEQ ID No. 3:

ATGGGCTTCATGCTGAACTTTCCTTTCCTGATGACGGCTCCACAAAGTTGGCAAACTATCCAGTTGCACCAGTATTGGCCTGAGTGGAAGTCTGGAGATACAGTCAAGATATCCTGCAAGTCTGGATTTTCCTTCACAAAGAATGGAAAAATGAACTGGGTGAAGCAGTCGGCAGGAGAAGATTTACAGTGGATGGGCTGGAACGGTTCCGGAGAGTCAACATATGACTTCAAGGGCCGGTTTCCCTTATCTGAAGCAACCTCCCAAAACACTCCCTATAAAATCACCAACGTGAAGAATACAGACATGGCTACATATTTCTGTGCAAGAGGGGGACGTGGCTTTAGGTCTCAGTACTGGGGTCAAGGGTCCTCAGTCAGCATCTCCTCAGCCTTTACACCATCAAAGCCCCTGCCCAGCTGGACCGATACACAGGGTTCCCCTGTG.

The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1:

MGFMLNFPFLMTAPQSWQTIQLHQYWPEWKSGDTVKISCKSGFSFTKNGKMNWVKQSAGEDLQWMGWNGSGESTYDFKGRFPLSEATSQNTPYKITNVKNTDMATYFCARGGRGFRSQYWGQGSSVSISSAFTPSKPLPSWTDTQGSPV.

The nucleotide sequence of the coding light chain variable region is shown as SEQ ID No. 4:

GGGCAGAGTTCTTGGGAGCAATATCAAAGAAGATCGGGGCTGAATAATATTGAAAATAATCCTCCTGGTTTGAGTATTATGTGGTGGATTACTCTTCTCCTCTGGCTCGCTGCTCTCTCATCAGGGGCCTTATCCCAGTCGGTTACTACTCCTGAAGAAGCACTCCCAACAACTCCTGGTAAGACAGTCGAGCTCTCATGTCGCTCAAATAGTACTACAACAGGTCTAAACTATTGCAACTGGAACCAAGAATTTCCAGATATCTTAACTACTGGTATCATATCCGCTCCAAACCGATGTAGTCCAAGTCCTCCTAGAAGATTCACTTCCCTGTTAGGACAGAAGGCCTCGATCACACTCGCACAGTCAGAGGATCACGCAATAATGTTCTGTGCTCTATGGTTCAACCATTGGTGGGTGTTCGGTACTGGAACCAGACTGACTGTCCTGCAGCCCAAGTCGCCACAGGTCACCCCTTTTCCACCTAGGTCTGAAGAGGTAGAGAACGAA.

The amino acid sequence of the light chain variable region is shown in SEQ ID No. 2:

GQSSWEQYQRRSGLNNIENNPPGLSIMWWITLLLWLAALSSGALSQSVTTPEEALPTTPGKTVELSCRSNSTTTGLNYCNWNQEFPDILTTGIISAPNRCSPSPPRRFTSLLGQKASITLAQSEDHAIMFCALWFNHWWVFGTGTRLTVLQPKSPQVTPFPPRSEEVENE.

The sequences of the heavy chain variable region and the light chain variable region were analyzed to obtain the CDR regions.

Wherein, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively 48-52, 67-79 and 111-119 of SEQ ID No.1, and NGKMN, WNGSGESTYDFKG, GGRGFRSQY respectively;

The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are 68-80, 96-101 and 132-140 of SEQ ID No.2 respectively, and RSNSTTTGLNYCN, APNRCS, ALWFNHWWV respectively.

Application example 1 application of canine blood typing kit

1. The kit comprises the following components:

(1) A canine blood group identification test strip with a quality control line C coated with a canine erythrocyte specific antibody and a detection line T coated with a monoclonal antibody resisting DEA1 antigen;

(2) A dropper filled with a diluent;

(3) Quantitative binaural dropper.

2. The preparation method of the kit components comprises the following steps:

(1) Canine blood typing test strip:

A. And (3) dividing NC films by the specific antibody of the purified canine erythrocytes and the monoclonal antibody of the DEA1 antigen. Wherein the quality control line C line: diluting the specific antibody of the canine erythrocytes by 1000 times, wherein the dividing amount is 1 mu l/cm; and (3) detecting a line T: diluting a monoclonal antibody against DEA1 antigen by 1500 times, wherein the membrane dividing amount is 1 mu l/cm;

B. And (3) drying: placing the NC film in a 37 ℃ oven for drying for 4 hours;

C. Cutting: cutting the NC film into test strips with the width of 3 mm;

D. And (3) assembling: and (5) outsourcing the commercial card shell and assembling the test strip with the cut test strip to form the test strip.

(2) Dilution liquid: PBS (0.01 mol/L, pH 7.4) solution containing 0.05% Tween-20.

(3) Quantitative binaural dropper: commercial droppers.

3. Kit sample detection results

As shown in fig. 5, the detection results of the canine DEA1 positive sample and the canine DEA1 negative sample are sequentially from left to right.

Application example 2 application of rabies virus colloidal gold antibody detection test strip

1. Sample pad treatment step:

(1) Diluting the purified specific antibody combined with the canine red blood cells to 5mg/ml by using PBS (0.01 mol/L, pH value is 7.4) to obtain a solution for combining the specific antibody combined with the canine red blood cells;

(2) Cutting the blood filtering membrane to be 8mm wide;

(3) Spraying the solution combined with the canine erythrocyte specific antibody in the step (1) onto a hemofilter membrane by a gold spraying instrument in a spraying amount of 5 μl/cm;

(4) Placing the hemofilter membrane in a 37 ℃ oven for drying for 3 hours;

(5) And combining the dried hemofilter film with the specific antibody combined with the canine red blood cells onto a sample pad of a colloidal gold antibody detection test strip, cutting and assembling.

2. Sample detection results after sample pad treatment:

The background is very deep when the test strip without the blood filtering membrane and without the canine erythrocyte specific antibody treatment detects the whole blood sample; test strips with hemofilter but without canine erythrocyte specific antibody treatment have a pale red background; when the whole blood sample is detected by adding the hemofilter treated by the canine erythrocyte specific antibody, the background of the detection window is clear.

As shown in fig. 6, from left to right, the steps are as follows: a test strip for serum sample without blood filtering film and dog erythrocyte specific antibody treatment; a test strip for whole blood sample without blood filtering membrane and dog erythrocyte specific antibody treatment; the whole blood sample is provided with a blood filtering membrane and a test strip without canine erythrocyte specific antibody treatment; the whole blood sample is provided with a blood filtering membrane and a test strip for treating the canine erythrocyte specific antibody.

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. A canine erythrocyte-binding specific antibody, wherein the canine erythrocyte-binding specific antibody comprises a heavy chain variable region and a light chain variable region;

2. The specific antibody binding to canine erythrocytes according to claim 1, characterized in that the specific antibody binding to canine erythrocytes is a murine monoclonal antibody obtained by isolating and extracting canine erythrocyte antigen as an immunogen and screening.

3. Use of the canine erythrocyte-binding specific antibody of claim 1 or 2 in the preparation of a detection reagent.

4. The use according to claim 3, wherein the canine red blood cell-binding specific antibody is coated on canine blood typing test strip quality control line C.

5. The use according to claim 3, wherein the canine red blood cell-binding specific antibody is added to a sample pad treatment solution to prepare a mixed solution as a canine blood group identification colloidal gold test strip sample pad treatment mixed solution.

6. The use according to claim 5, wherein the concentration of the specific antibody in the sample pad treatment mixture is 2-8 mg/ml.