阅读说明：本技术 犬腺病毒1型单克隆抗体、可变区序列、杂交瘤细胞株及其应用 (Canine adenovirus 1 type monoclonal antibody, variable region sequence, hybridoma cell strain and application thereof ) 是由 田克恭 邓均华 李凡 于 2018-06-25 设计创作，主要内容包括：本发明提供了具有特异性结合犬腺病毒1型Fiber蛋白的鼠单克隆抗体的可变区序列、特异性结合犬腺病毒1型Fiber蛋白的抗体,该抗体可以用于制备试剂盒和药物组合物。本发明的试剂盒克服了现有技术检测灵敏度低的问题,避免了漏检、假阴性现象发生,可高灵敏检测犬的多种靶标,还能高灵敏检测非犬动物的多种靶标；且具有快速、简便、准确的优势；本发明的药物组合物能有效预防和治疗CAV-1导致的疾病。(The invention provides a variable region sequence of a mouse monoclonal antibody specifically binding to canine adenovirus type 1 Fiber protein, an antibody specifically binding to canine adenovirus type 1 Fiber protein, and the antibody can be used for preparing a kit and a pharmaceutical composition. The kit provided by the invention overcomes the problem of low detection sensitivity in the prior art, avoids the phenomena of omission and false negative, can detect various targets of a dog with high sensitivity, and can detect various targets of non-dog animals with high sensitivity; the method has the advantages of rapidness, simplicity, convenience and accuracy; the pharmaceutical composition of the invention can effectively prevent and treat diseases caused by CAV-1.)

1. A variable region sequence of a monoclonal antibody that specifically binds to canine adenovirus type 1, wherein the monoclonal antibody is monoclonal antibody 4F3 or 2F 9;

the variable region of the heavy chain of the monoclonal antibody 4F3 is a conservative variant obtained by the conservative mutation of the sequence shown in SEQ ID No.1 or the coding of the degenerate sequence of the sequence or through one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 4F3 light chain is a conservative variant obtained by the sequence shown in SEQ ID No.2 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications;

the variable region of the heavy chain of the monoclonal antibody 2F9 is a conservative variant obtained by the conservative mutation of the sequence shown in SEQ ID No.3 or the coding of the degenerate sequence thereof or one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 2F9 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.4 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

2. An antibody or antibody fragment specifically binding to canine adenovirus type 1, wherein the heavy chain variable region of the antibody or antibody fragment is a conservative variant obtained by coding the sequence shown in SEQ ID No.1 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.2 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications;

preferably, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody or a fragment of the antibody that retains the ability to specifically bind canine adenovirus type 1;

more preferably, the antibody is monoclonal antibody 4F 3.

3. An antibody or antibody fragment specifically binding to canine adenovirus type 1, wherein the heavy chain variable region of the antibody or antibody fragment is a conservative variant obtained by coding the sequence shown in SEQ ID No.3 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications; and conservative variants obtained by conservative mutation of the sequence shown in the light chain variable region of the antibody or the antibody fragment or the coding sequence of the degenerate sequence thereof or the sequence thereof through one or more amino acid additions, deletions, substitutions or modifications;

preferably, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody or a fragment of the antibody that retains the ability to specifically bind canine adenovirus type 1;

more preferably, the antibody is monoclonal antibody 2F 9.

4. A hybridoma cell 4F3 strain that secretes the monoclonal antibody 4F3 of claim 2, 4F3 strain.

5. A hybridoma cell 2F9 strain that secretes the monoclonal antibody 2F9 of claim 3, 2F9 strain.

6. A pharmaceutical composition, wherein the pharmaceutical composition comprises an immunizing amount of an antibody that specifically binds canine adenovirus type 1, and a pharmaceutically acceptable carrier;

the antibody that specifically binds canine adenovirus type 1 is selected from one or more of the following antibodies: a single-chain antibody in which the heavy chain variable region and the light chain variable region of monoclonal antibody 4F3, monoclonal antibody 2F9, and monoclonal antibody 4F3 were linked, a single-chain antibody in which the heavy chain variable region of monoclonal antibody 4F3 and the light chain variable region of monoclonal antibody 2F9 were linked, a single-chain antibody in which the light chain variable region of monoclonal antibody 4F3 and the heavy chain variable region of monoclonal antibody 2F9 were linked, and a single-chain antibody in which the heavy chain variable region and the light chain variable region of monoclonal antibody 2F9 were linked.

7. Use of the pharmaceutical composition according to claim 6 for the preparation of a medicament for the prevention and/or treatment of diseases associated with canine adenovirus type 1 infection.

8. A kit comprising an effective amount of the monoclonal antibody 4F3, an effective amount of the monoclonal antibody 2F9, and a detection reagent that detects a canine adenovirus type 1 antigen-antibody reaction;

wherein, the kit includes colloidal gold test paper strip, colloidal gold test paper strip includes the part: a bottom plate (5), wherein the bottom plate (5) is provided with a first end and a second end, and a sample pad (1), a gold-labeled pad (2), a nitrocellulose membrane (3) and a water absorption pad (4) are sequentially arranged along the direction from the first end to the second end, and the nitrocellulose membrane (3) is contacted with the gold-labeled pad (2) or the sample pad (1) and the gold-labeled pad (2) so that the binding body of the canine adenovirus 1 antigen and the monoclonal antibody 2F9 can migrate to the second end of the bottom plate; the gold-labeled pad (2) contains the monoclonal antibody 2F9 labeled by colloidal gold, the nitrocellulose membrane comprises a detection line (6) and a quality control line (7), the monoclonal antibody 4F3 is immobilized on the detection line (6), and a goat-anti-mouse polyclonal antibody or a goat-anti-mouse secondary antibody is immobilized on the quality control line (7);

wherein the fixed content of the monoclonal antibody 4F3 is 0.5-2.5mg/ml, and the content of the monoclonal antibody 2F9 when labeled is 5-70 mug/ml;

preferably, adjacent parts of the sample pad (1), the gold-labeled pad (2), the nitrocellulose membrane (3) and the absorbent pad (4) which are arranged in sequence from the first end to the second end in the kit are contacted with each other, and non-adjacent parts are not contacted with each other; the kit also comprises a sample treatment solution, wherein the sample treatment solution is a phosphate buffer solution;

preferably, the fixed content of the monoclonal antibody 4F3 is 0.8-2.0mg/ml, and the content of the monoclonal antibody 2F9 marked is 10-60 mu g/ml.

9. The kit according to claim 8, wherein the gold-labeled pad (2) contains the monoclonal antibody 2F9, 4H1 and/or 1G5 labeled with colloidal gold thereon, and the nitrocellulose membrane (3) contains two or three detection lines on which the monoclonal antibody 4F3, 2B7 and/or 6E11 is immobilized, respectively;

wherein the content of the monoclonal antibody 4H1 is 20-150 mug/ml when marked, the content of the monoclonal antibody 1G5 is 20-60 mug/ml when marked, the fixed content of the monoclonal antibody 2B7 is 1-3mg/ml, and the fixed content of the monoclonal antibody 6E11 is 1-3 mg/ml; the heavy chain variable region of the canine parainfluenza virus monoclonal antibody 2B7 is a sequence shown in SEQ ID No.5 or a degenerate sequence code thereof, the light chain variable region is a sequence shown in SEQ ID No.6 or a degenerate sequence code thereof, the heavy chain variable region of the canine parainfluenza virus monoclonal antibody 4H1 is a sequence shown in SEQ ID No.7 or a degenerate sequence code thereof, and the light chain variable region is a sequence shown in SEQ ID No.8 or a degenerate sequence code thereof; the canine distemper virus monoclonal antibody 6E11 is prepared from a monoclonal antibody with a preservation number of CCTCC No: c2015202, and the canine distemper virus monoclonal antibody 1G5 is secreted by a mouse bone marrow hybridoma cell 6E11 strain with the preservation number of CCTCC No: c2015201 mouse bone marrow hybridoma cell 1G 5strain secretion; preferably, the distance between the adjacent detection lines and between the detection line closest to the quality control line and the quality control line is more than or equal to 4 mm.

10. An immunohistochemical detection method, wherein the method comprises:

collecting tissue samples of infected animals, carrying out block trimming on the tissue samples, and quickly placing the tissue samples in formalin for fixation; preferably, the tissue sample of the infected animal is liver, gall bladder, kidney, intestine tissue of a dog;

step (2) washing the tissue sample fixed in the step (1) with running water, dehydrating, transparentizing, waxing, embedding, slicing, spreading and mounting on a slide treated by triaminopropyltriethoxysilane, and baking;

dewaxing the glass slide to distilled water in the step (3), and dripping 3% of H₂O₂Standing to inhibit endogenous enzymes;

step (4), using a thermal restoration method or an enzyme digestion method to treat the baked slide to restore the antigen, wherein the thermal restoration comprises high-pressure thermal restoration, boiling thermal restoration or microwave thermal restoration;

washing the repair antigen by using a phosphate buffer solution, and dropwise adding a confining liquid such as horse serum or bovine serum albumin for confining;

adding diluted primary antibody after absorbing the confining liquid in the step (6), incubating, washing by phosphate buffer solution, adding an enzyme-labeled goat anti-mouse antibody as an enzyme-labeled secondary antibody, and incubating; and

washing with a phosphate buffer solution, adding an AEC or DAB color development solution for color development, and stopping with the phosphate buffer solution or distilled water according to the dyeing condition; adding hematoxylin to line-stain the cell nucleus for counterstaining, and performing dehydration, transparence, mounting and microscopic examination if necessary.

Technical Field

The invention relates to a canine adenovirus 1 type monoclonal antibody, a variable region sequence of the monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody, a pharmaceutical composition prepared by using the monoclonal antibody, a kit and application, and belongs to the technical field of biology.

Background

Infectious hepatitis of dogs and encephalitis of bears and foxes are all caused by Canine adenovirus type 1 (CAV-1) and are distributed globally. The infectious hepatitis of dogs can infect dogs of different varieties, ages and sexes, can occur in four seasons, and has the highest infection rate and death rate (up to 25-40 percent and can suddenly die in 1-2 days) of puppies; the clinical symptoms are complex, including vomiting, abdominal pain, diarrhea, body temperature rise and even sudden death, eye injury, and temporary corneal opacity, commonly called blue eye disease; it is susceptible to mixed infection or secondary infection with other viruses such as canine distemper virus, canine parvovirus, and canine parainfluenza virus, thereby aggravating the disease and causing difficulty in clinical diagnosis. The clinical morbidity of encephalitis of the bears and the foxes is about 16.3 percent, and the mortality rate is about 9.7 percent. The disease caused by CAV-1 is one of the most harmful epidemic diseases in the canine industry and the fur animal breeding industry.

At present, the detection method of CAV-1 comprises methods such as virus separation, PCR, hemagglutination test and the like, but the methods have the defects that the virus separation and identification test process is complicated, the time consumption is too long, and the visual observation is subjective; the molecular biological method mainly based on PCR has high requirements on technology and equipment and is complicated to operate; the hemagglutination test requires not only erythrocytes, but also the ready preparation, and is also susceptible to the interference of impurities in secretions or excretions during clinical detection, and the detection methods are difficult to be applied in veterinary clinical fields. The commercial canine adenovirus type 1 colloidal gold test strip currently on the market is only one test strip produced by Shanghai Kuailing, has low detection sensitivity and results with false negative, and thus causes the risk of virus dispersion and mass infection. Therefore, there is an urgent need to develop a fast, simple and accurate product for clinical diagnosis.

The prevention and treatment of the CAV-1 and CAV-2 of the dog mainly comprise immunization of a vaccine containing the canine adenovirus type 2 and hyperimmune serum at present, but the canine adenovirus type 2 strains contained in the vaccine are all low virulent strains, the risk of virus dispersion exists after animals are immunized, antibodies are generated after the immunization, a period of time is required, and the resistance to the virus cannot be quickly established; the preparation process of the hyperimmune serum is complex, and qualified test animals need to be screened, and multiple immunizations with weak toxicity and strong toxicity (Li Shimin et al, the development and application of polyvalent hyperimmune serum for dogs, China immunology journal, 2005,21(7):521-523) are also needed.

In addition, when infected dogs, foxes, particularly puppies or young foxes are attacked, the disease condition of the infected animals needs to be rapidly controlled and treated, even the existing vaccines are immunized, high antibodies are difficult to generate in a short time, and monoclonal antibodies with good neutralizing activity can greatly improve the cure rate of the disease, so that the therapeutic monoclonal antibodies have important significance for treating and controlling the disease.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a pair of canine adenovirus type 1 monoclonal antibodies, a kit containing the monoclonal antibodies, a pharmaceutical composition and application thereof.

The invention relates to a variable region sequence of a monoclonal antibody 4F3 specifically binding to canine adenovirus type 1, wherein the heavy chain variable region of the monoclonal antibody 4F3 is a conservative variant obtained by the sequence shown in SEQ ID No.1 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 4F3 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.2 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

The variable region sequence of the monoclonal antibody 4F3 can be specifically combined with canine adenovirus type 1, and the epitope aimed by the variable region sequence is located in the Fiber protein.

The invention relates to an antibody or an antibody fragment specifically binding to canine adenovirus type 1, wherein a heavy chain variable region of the antibody or the antibody fragment is a sequence shown in SEQ ID No.1 or a degenerate sequence code thereof or a conservative variant thereof obtained by conservative mutation through one or more amino acid additions, deletions, substitutions or modifications; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.2 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications.

As an embodiment of the present invention, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody, or a fragment of the antibody; the antibody or fragment of the antibody still retains the ability to specifically bind canine adenovirus type 1.

In a preferred embodiment of the present invention, the antibody is monoclonal antibody 4F 3.

The combined epitope of the monoclonal antibody 4F3 is the epitope of Fiber protein, the heavy chain variable region is encoded by SEQ ID No.1 or a degenerate sequence thereof, and the light chain variable region is encoded by SEQ ID No.2 or a degenerate sequence thereof; the HI titer to CAV-1 is more than or equal to 1:5120, the IPMA titer is more than or equal to 1:2560, and the reactivity with CAV-1 is good; the neutralizing titer to CAV-1 is 1:320, and the neutralizing activity to CAV-1 is very high.

The invention relates to a variable region sequence of a monoclonal antibody 2F9 specifically binding to canine adenovirus type 1, wherein the heavy chain variable region of the monoclonal antibody 2F9 is a conservative variant obtained by a sequence shown in SEQ ID No.3 or a coding sequence of a degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 2F9 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.4 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

The invention relates to an antibody or an antibody fragment specifically binding to canine adenovirus type 1, wherein the heavy chain variable region of the antibody or the antibody fragment is a sequence shown in SEQ ID No.3 or a degenerate sequence code thereof or a conservative variant thereof obtained by conservative mutation through one or more amino acid additions, deletions, substitutions or modifications; and conservative variants obtained by conservative mutation of the sequence shown by the variable region of the light chain of the antibody or the antibody fragment or the code of the degenerate sequence thereof or the sequence thereof through one or more amino acid additions, deletions, substitutions or modifications.

As an embodiment of the present invention, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody, or a fragment of the antibody; the antibody or fragment of the antibody still retains the ability to specifically bind canine adenovirus type 1.

In a preferred embodiment of the present invention, the antibody is monoclonal antibody 2F 9.

The monoclonal antibody 2F9 is a canine adenovirus type 1 monoclonal antibody, and the combined epitope thereof is an epitope on a canine adenovirus type 1 Fiber protein; the amino acid sequence of the heavy chain variable region is encoded by SEQ.ID No.3 or a degenerate sequence thereof, and the amino acid sequence of the light chain variable region is encoded by SEQ.ID No.4 or a degenerate sequence thereof; the HI titer to CAV-1 is more than or equal to 1: 10240, the IPMA titer is more than or equal to 1: 1280, and the reactivity with CAV-1 is good; the neutralizing titer to CAV-1 is 1: 1280, and the neutralizing activity to CAV-1 is very high.

The invention also relates to a hybridoma cell 4F3 strain, wherein the hybridoma cell 4F3 strain secretes the monoclonal antibody 4F 3.

The invention also relates to a hybridoma cell 2F9 strain, wherein the hybridoma cell 2F9 strain secretes the monoclonal antibody 2F 9.

The invention also relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises an immunizing amount of an antibody that specifically binds canine adenovirus type 1, and a pharmaceutically acceptable carrier; the antibody that specifically binds canine adenovirus type 1 is selected from one or more of the following antibodies: a single-chain antibody in which the heavy chain variable region and the light chain variable region of monoclonal antibody 4F3, monoclonal antibody 2F9, and monoclonal antibody 4F3 were linked, a single-chain antibody in which the heavy chain variable region of monoclonal antibody 4F3 and the light chain variable region of monoclonal antibody 2F9 were linked, a single-chain antibody in which the light chain variable region of monoclonal antibody 4F3 and the heavy chain variable region of monoclonal antibody 2F9 were linked, and a single-chain antibody in which the heavy chain variable region and the light chain variable region of monoclonal antibody 2F9 were linked.

The monoclonal antibody and the single-chain antibody contained in the pharmaceutical composition have high capability of neutralizing the canine adenovirus type 1 virus, and can rapidly control and treat the illness state of the ill animals.

As a preferred embodiment of the present invention, the medicament is administered by intramuscular injection.

As a preferred embodiment of the present invention, the pharmaceutical dosage form includes, but is not limited to, powder, granule, pill, tablet, capsule.

The pharmaceutical composition of the invention makes up the defects of the existing vaccine and avoids the risk of toxin dispersion; solves the inconvenience caused by the complex preparation procedure of the hyperimmune serum, can simultaneously prevent and treat CAV-1, and can also be used for emergently treating the disease, thereby reducing the morbidity and the mortality.

The invention also relates to application of the pharmaceutical composition in preparing a medicament for preventing and/or treating diseases related to canine adenovirus type 1 infection.

The invention also relates to a kit, wherein the kit comprises an effective amount of the monoclonal antibody 4F3, an effective amount of the monoclonal antibody 2F9 and a detection reagent for detecting the canine adenovirus 1 type antigen-antibody reaction; wherein, the kit includes colloidal gold test paper strip, colloidal gold test paper strip includes the part: a bottom plate (5), wherein the bottom plate (5) is provided with a first end and a second end, and a sample pad (1), a gold-labeled pad (2), a nitrocellulose membrane (3) and a water absorption pad (4) are sequentially arranged along the direction from the first end to the second end, and the nitrocellulose membrane (3) is contacted with the gold-labeled pad (2) or the sample pad (1) and the gold-labeled pad (2) so that the binding body of the canine adenovirus 1 antigen and the monoclonal antibody 2F9 can migrate to the second end of the bottom plate; the gold-labeled pad (2) contains the monoclonal antibody 2F9 labeled by colloidal gold, the nitrocellulose membrane comprises a detection line (6) and a quality control line (7), the monoclonal antibody 4F3 is immobilized on the detection line (6), and a goat-anti-mouse polyclonal antibody or a goat-anti-mouse secondary antibody is immobilized on the quality control line (7); wherein the fixed content of the monoclonal antibody 4F3 is 0.5-2.5mg/ml, and the content of the monoclonal antibody 2F9 when labeled is 5-70 mu g/ml.

As an embodiment of the invention, adjacent parts of the sample pad (1), the gold-labeled pad (2), the nitrocellulose membrane (3) and the absorbent pad (4) which are arranged in sequence from the first end to the second end in the kit are contacted with each other, and non-adjacent parts are not contacted with each other; the kit also comprises a sample treatment solution, wherein the sample treatment solution is a phosphate buffer solution.

As an embodiment of the invention, the fixed content of the monoclonal antibody 4F3 is 0.8-2.0mg/ml, and the content of the monoclonal antibody 2F9 marked is 10-60 mug/ml.

As an embodiment of the present invention, the gold-labeled pad (2) contains the monoclonal antibodies 2F9, 4H1 and/or 1G5 labeled with colloidal gold, the nitrocellulose membrane (3) includes two or three detection lines on which the monoclonal antibodies 4F3, 2B7 and/or 6E11 are immobilized, respectively; wherein the content of the monoclonal antibody 4H1 is 20-150 mug/ml when marked, the content of the monoclonal antibody 1G5 is 20-60 mug/ml when marked, the fixed content of the monoclonal antibody 2B7 is 1-3mg/ml, and the fixed content of the monoclonal antibody 6E11 is 1-3 mg/ml; the heavy chain variable region of the canine parainfluenza virus monoclonal antibody 2B7 is a sequence shown in SEQ ID No.5 or a code of a degenerate sequence thereof, the light chain variable region is a sequence shown in SEQ ID No.6 or a code of a degenerate sequence thereof, the heavy chain variable region of the canine parainfluenza virus monoclonal antibody 4H1 is a sequence shown in SEQ ID No.7 or a code of a degenerate sequence thereof, and the light chain variable region is a sequence shown in SEQ ID No.8 or a code of a degenerate sequence thereof; the canine distemper virus monoclonal antibody 6E11 is prepared from a monoclonal antibody with a preservation number of CCTCC No: mouse bone marrow hybridoma cell 6E11 strain secretion of C2015202; the canine distemper virus monoclonal antibody 1G5 is prepared from a monoclonal antibody with a preservation number of CCTCC No: c2015201 mouse bone marrow hybridoma cell line 1G 5.

The preservation number of the mouse bone marrow hybridoma cell 6E11 strain is CCTCC No: c2015202, wherein the preservation number of the mouse bone marrow hybridoma cell strain 1G5 is CCTCC No: c2015201, both disclosed in patent application CN 105695420A.

In one embodiment of the present invention, there are only two detection lines 6A and 6B in a direction from the first end to the second end of the bottom plate (5), the monoclonal antibody 4F3 is immobilized on the detection line 6A or 6B, and the other immobilized monoclonal antibody 4H1 or 1G5 is immobilized on the detection line 6B or 6A.

As an embodiment of the present invention, there are three detection lines 6A, 6B, 6C in a direction from the first end to the second end of the base plate (5), and the monoclonal antibodies 4F3, 4H1 and 1G5 may be immobilized on the detection lines 6A, 6B, 6C, respectively, in any arrangement.

In one embodiment of the invention, the distance between the adjacent detection lines, the detection line closest to the quality control line and the quality control line is not less than 4 mm.

The detection sample of the kit is selected from the group consisting of ocular nasal secretion, throat secretion, anal secretion, feces, serum, urine, liver, intestine or intestinal lymph node, spleen and lung; the detection sample of the kit is from a dog, a fox and a bear.

The kit can be used for detecting various tissues such as liver, gallbladder, kidney, intestine or intestinal lymph nodes, spleen, lung and the like, has good sensitivity and shows wide clinical application adaptability.

The kit can detect not only dog-derived samples but also non-dog animal-derived samples, has wide clinical application, and makes up the defect that the existing commercial diagnostic reagent cannot be used for the non-dog animal-derived samples.

The invention also provides an immunohistochemical detection method, wherein the method comprises the following steps:

collecting tissue samples of infected animals, carrying out block trimming on the tissue samples, and quickly placing the tissue samples in formalin for fixation; step (2) washing the tissue sample fixed in the step (1) with running water, dehydrating, transparentizing, waxing, embedding, slicing, spreading and mounting on a slide treated by triaminopropyltriethoxysilane, and baking; dewaxing the glass slide to distilled water in the step (3), and dripping 3% of H₂O₂Standing to inhibit endogenous enzymes; step (4), using a thermal restoration method or an enzyme digestion method to treat the baked slide to restore the antigen, wherein the thermal restoration comprises high-pressure thermal restoration, boiling thermal restoration or microwave thermal restoration; washing the repair antigen by using a phosphate buffer solution, and dropwise adding a confining liquid such as horse serum or bovine serum albumin for confining; adding diluted primary antibody after absorbing the confining liquid in the step (6), incubating, washing by phosphate buffer solution, adding an enzyme-labeled goat anti-mouse antibody as an enzyme-labeled secondary antibody, and incubating; washing with phosphate buffer solution, adding AEC or DAB color development solution for color development, and stopping with phosphate buffer solution or distilled water according to dyeing condition; adding hematoxylin to line-stain the cell nucleus for counterstaining, and performing dehydration, transparence, mounting and microscopic examination if necessary.

In one embodiment of the present invention, in the immunohistochemical detection method of the present invention, the tissue sample of the infected animal in the step (1) is liver, gall bladder, kidney, or intestine tissue of a dog.

The immunohistochemical detection method of the present invention can be used for not only liver tissues but also biliary, renal and intestinal tissues with high sensitivity.

The invention also relates to a preparation method of the kit, wherein the preparation method comprises the following steps: step 1) labeling the monoclonal antibody 4F3 with colloidal gold as a gold-labeled antibody to prepare a gold-labeled pad, wherein the content of the labeled monoclonal antibody 4F3 is 10-60 mug/ml; step 2) fixing the monoclonal antibody 2F9, a goat anti-mouse secondary antibody or a goat anti-mouse polyclonal antibody to be respectively adsorbed at one end of a nitrocellulose membrane to be used as a detection line T1 and a quality control line, diluting the monoclonal antibody 2F9 to 0.8-2.0mg/ml for fixing to be used as a detection line T1, and fixing the goat anti-mouse polyclonal antibody or the goat anti-mouse secondary antibody to be 1-3mg/ml for being used as a quality control line; step 3), preparing a sample treatment solution, and subpackaging; and step 4) sequentially sticking the gold label pad prepared in the step 1), the nitrocellulose membrane prepared in the step 2) and absorbent paper on a bottom plate, and cutting; assembling the sample processing solution prepared in the step 3) into a kit.

As a preferred embodiment of the present invention, the step 1) further comprises labeling the monoclonal antibody 4H1 and the monoclonal antibody 1G5 with colloidal gold to form a gold-labeled pad, wherein the content of the monoclonal antibody 4H1 is 20 to 150 μ G/ml and the content of the monoclonal antibody 1G5 is 20 to 60 μ G/ml; the step 2) further comprises the step of fixing the monoclonal antibody 2B7 and the monoclonal antibody 6E11 to be adsorbed at one end of the nitrocellulose membrane respectively to serve as a detection line T2, a detection line T3 and a quality control line, and the monoclonal antibody 2B7 and the monoclonal antibody 6E11 are diluted to 1-3mg/ml respectively and serve as detection lines T1, T2 and T3 after being fixed.

In one embodiment of the present invention, the sample processing solution in step 3) is a phosphate buffer solution.

The invention also relates to a detection method of the kit, which comprises the following steps: inserting the collected sample into a sample processing tube to dissolve the sample in the sample processing liquid as much as possible, dripping the processed sample into the center of a sample adding hole of the colloidal gold test strip, and judging the result after 10 minutes.

The invention also relates to the application of the antibody or the antibody fragment in canine adenovirus type 1 epitope identification research; the antibody or antibody fragment is monoclonal antibody 4F3 or monoclonal antibody 2F 9.

The kit containing the monoclonal antibody pair overcomes the problem of low detection sensitivity in the prior art, avoids the phenomena of omission and false negative, can detect various target tissues and samples with high sensitivity, can detect non-canine animal (fox) samples with high sensitivity, has the advantages of rapidness, simplicity, convenience and accuracy, and is more favorable for clinical application.

The invention also relates to the use of said kit for the detection of canine adenovirus type 1 for non-diagnostic purposes. Wherein the non-diagnostic canine adenovirus type 1 detection comprises epidemiological analysis and ex vivo tissue detection.

Drawings

FIG. 1 is a schematic view of a colloidal gold test strip according to a first embodiment of the present invention;

FIG. 2 is a schematic view of a colloidal gold test strip according to a second embodiment of the present invention;

FIG. 3 is a schematic view of a colloidal gold test strip according to a third embodiment of the present invention;

reference numerals: the device comprises a sample pad 1, a gold-labeled pad 2, a nitrocellulose membrane 3, a water absorption pad 4, a bottom plate 5, a detection line 6A, a detection line 6B, a detection line 6C and a quality control line 7.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

The term "Canine Adenovirus type 1" (CAV-1) belongs to the genus Adenovirus (Adenovirus), also called as Canine Infectious hepatitis virus (ICHV), which infects dogs, bears, foxes, etc. and causes Canine Infectious hepatitis and encephalitis of bears and foxes, and clinical symptoms mainly include vomiting, abdominal pain, diarrhea, increased body temperature, even sudden death, eye injury, etc.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except for the possible presence of a small number of possible spontaneous mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies in nature. Preferably, the monoclonal antibodies include monovalent or single chain antibodies, diabodies, chimeric antibodies, caninized antibodies, as well as derivatives, functional equivalents and homologues of the above, as well as antibody fragments and any polypeptides comprising an antigen binding domain. An antibody is any specific binding member which encompasses a binding domain with the desired specificity, and thus, this term encompasses antibody fragments, derivatives, caninized antibodies, and functional equivalents and homologs of antibodies which are homologous thereto, as well as any polypeptide, whether natural or synthetically produced, which contains an antigen binding domain. Examples of antibodies are immunoglobulin subtypes (e.g., IgG, IgE, IgM, IgD and IgA) and subclasses thereof; or a fragment comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies (diabodies). Chimeric molecules comprising an antigen binding domain fused to another polypeptide or an equivalent are also included. Cloning and expression of chimeric antibodies is described in ep.a.0120694 and ep.a.0125023. Antibodies can be modified in a number of ways and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve the introduction of DNA encoding the immunoglobulin variable regions or Complementarity Determining Regions (CDRs) of antibodies into the constant regions or constant region plus framework regions of different immunoglobulins, see ep.a.184187, GB2188638A or ep.a.239400. Genetic mutations or other changes may also be made to the hybridoma cells or other cells that produce the antibody, which may or may not alter the binding specificity of the produced antibody. The "monoclonal antibody" used in the present invention can also be produced by a hybridoma method, since a DNA sequence encoding the murine antibody of the present invention can be obtained by a conventional method well known to those skilled in the art, such as artificially synthesizing a nucleotide sequence based on the amino acid sequence disclosed in the present invention or amplifying it by a PCR method, and thus, a recombinant DNA method can be used, and the sequence can be ligated into an appropriate expression vector by various methods well known in the art. Finally, the transformed host cell is cultured under conditions suitable for the expression of the antibody of the present invention, and then purified by a person skilled in the art using a conventional separation and purification means well known to those skilled in the art to obtain the monoclonal antibody of the present invention. Antibodies comprise polypeptide chain geometries linked together by disulfide bridges, with the two polypeptide backbones, termed light and heavy, constituting all major structural classes (isoforms) of antibodies. Both the heavy and light chains can be further divided into subregions known as variable and constant regions. The heavy chain comprises a single variable region and three different constant regions, and the light chain comprises a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

The term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length whose amino acid sequence corresponds to the amino acid sequence of the heavy chain of a monoclonal antibody of the invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length whose amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the invention starting from the N-terminal amino acid of the light chain. It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody specifically disclosed in the present invention, modifications such as addition, deletion, and substitution of one or more amino acids can be performed by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still retaining specific binding to canine adenovirus type 1. The monoclonal antibodies of the invention also include active fragments or conservative variants thereof.

The term "conservative variant" refers to a variant that substantially retains the characteristics of its parent, such as basic immunological biological, structural, regulatory, or biochemical characteristics. Generally, conservative variants of a polypeptide differ in amino acid sequence from the parent polypeptide, but the differences are limited so that the sequence with the parent polypeptide is very similar to the conservative variant overall and is identical in many regions. The difference in amino acid sequence between the conservative variant and the parent polypeptide can be, for example: substitutions, additions, and deletions of one or more amino acid residues and any combination thereof. The amino acid residue that is substituted or inserted may or may not be encoded by the genetic code. A conservative variant of a polypeptide may occur naturally, or it may be a non-naturally occurring variant. Non-naturally occurring conservative variants of the polypeptide may be generated by mutagenesis techniques or by direct synthesis.

The term "effective amount" when understood as "prophylactically effective amount" refers to an amount sufficient to elicit an immunoprotective response in a vaccinated individual. The skilled artisan will appreciate that the "prophylactically effective amount" will vary with the mode, timing, subject of administration of the immunization and the monoclonal antibody or fragment thereof, and that the skilled artisan, in combination with literature and teachings known in the art and corresponding clinical specifications, should be able to derive a "prophylactically effective amount" of the monoclonal antibody used by limited experimentation.

The term "effective amount" when understood as "therapeutically effective amount" refers to an amount that is capable of producing effective protection and virus neutralization in a subject. One skilled in the art will appreciate that the "therapeutically effective amount" will vary with the treatment regimen, the course of the disease, the condition of the subject being treated, and the monoclonal antibody or fragment thereof used. In combination with literature and teachings known in the art and corresponding clinical protocols, the clinical artisan should be able to derive a "therapeutically effective amount" of the monoclonal antibody used by virtue of his experience.

The term "effective amount" when understood as "detecting an effective amount" refers to an amount that enables a positive sample to be detected with high sensitivity and a negative sample to be distinguished.

The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the body and does not hinder the biological activity and properties of the compound being used.

The term "preventing and/or treating" when referring to a canine adenovirus type 1 infection refers to inhibiting replication of canine adenovirus type 1, inhibiting transmission of canine adenovirus type 1, or preventing colonization of canine adenovirus type 1 in its host, as well as alleviating the symptoms of an epidemic or disorder of canine adenovirus type 1 infection. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.

The terms "quality control line" and "control line" are used interchangeably to refer to the conditions used to determine whether an antigen-antibody reaction is appropriate, and to monitor whether the background of the reaction interferes with the assay results.

The terms "eye-nose swab" and "eye-nose secretion", "throat swab" and "throat secretion" are used interchangeably.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The sample preservation solution used in the examples of the present invention was PBS buffer (pH7.4, 0.01mol/L), and its volume formulation of 1L was: NaCl 8.0g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.24g, but this embodiment is not intended to limit the present invention in any way.

The chemical reagents used in the invention are all analytically pure and purchased from the national pharmaceutical group.

In order that the invention may be more readily understood, reference will now be made to the following examples. It should be understood that these examples are only for the purpose of the present invention and are not intended to limit the scope of the present invention. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.