Monoclonal antibodies to feline haptoglobin and uses thereof

Abstract

The application discloses a monoclonal antibody of feline haptoglobin and application thereof, and the monoclonal antibody of feline haptoglobin or an antigen binding fragment thereof, and the monoclonal antibody of feline haptoglobin or the antigen binding fragment thereof comprises Hp-mAb8, Hp-mAb9 and / or Hp-mAb10, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody Hp-mAb8 are GFSLDGYD, IYVSDVT and ARDSDVGVSGYTFPDI respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody Hp-mAb8 are ESISTNR, PAS and QSGYATADVPNT respectively. The monoclonal antibody of feline haptoglobin has wider detection linearity, reduced cost, and can meet the budget of most cat-keeping families.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology. More specifically, the present application relates to a monoclonal antibody of feline haptoglobin and uses thereof. BACKGROUND

[0002] Acute phase response (APR) is a complex systemic response that occurs in both acute and chronic inflammatory conditions such as infection, tissue damage, trauma, surgery, neoplasia or immune disorders. The resulting response includes changes in the concentration of acute phase proteins. In the inflammatory response, proteins synthesized by hepatocytes increase under the stimulation of proinflammatory factors, and proteins with a change in plasma concentration of more than 25% are defined as acute phase proteins (APP). During the acute phase of tissue damage, acute phase proteins synthesized by the liver increase significantly. Generally, the degree of change is related to the severity of the disease, and the more severe and complex the case, the more significant the change. Since the acute phase response occurs before the stimulation of specific immune changes, it can be used as an early marker of disease.

[0003] In clinical and laboratory tests, acute phase proteins in cats are a biomarker for monitoring inflammation, which can be used to diagnose subclinical changes, monitor changes and effects of diseases in living organisms, and evaluate the effectiveness of treatment. In cats, serum amyloid A (SAA), alpha 1-acid glycoprotein (AGP) and haptoglobin (Hp) are considered useful indicators for monitoring acute inflammatory responses in cats. SAA is the most important acute phase response protein in cats, with the highest expression in inflammatory responses, followed by AGP and Hp, and the increase in C-reactive protein (CRP) is less significant.

[0004] SAA is a sensitive indicator of early inflammation in infectious diseases, and its concentration is related to the severity of inflammation. The level of acute phase response measured depends on the time of sampling and testing. Since SAA has a very short half-life, the SAA detected at the time of detection may have decayed to a lower concentration, and it is possible that the inflammation is in the process of subsiding. If the subsequent inflammation does not improve, according to the clinical manifestations, SAA and other APPs can be combined for comprehensive diagnosis and monitoring of the course of the disease, and the treatment plan can be optimized.

[0005] According to the degree and duration of the reaction caused by stimulation, APPs are divided into three categories. Positive APPs can be divided into two categories: the first category of APP is the strongest primary APP that increases by 10-1000 times in the human body or 10-100 times in the body of domestic animals in the presence of inflammation, such as C-reactive protein (CRP) and serum amyloid A (SAA); the second category of APP is the secondary APP of moderate intensity that increases by 2-10 times in the inflammatory response, such as haptoglobin and alpha-globulin. The last category of APP is the negative APP, the concentration of which decreases due to inflammation, such as albumin.

[0006] Haptoglobin (Hp) is one of the most important acute phase proteins in cattle, sheep, goats, horses and cats, mainly synthesized by hepatocytes, but also synthesized by other tissues such as skin, lung and kidney. Hp binds to iron molecules, thereby inhibiting the proliferation and invasion of bacteria. Hp can also bind to free hemoglobin to prevent its oxidation with lipids and proteins, thus proving that Hp concentration decreases in the case of hemolysis.

[0007] In cats, Hp concentration under inflammatory conditions usually increases by 2-10 times, and increases significantly when FIP is present. Acute phase protein Hp is a response to systemic inflammation and an increased response to infection and inflammatory conditions. They are useful biomarkers in veterinary medicine for the severity of inflammatory and infectious diseases and treatment response.

[0008] Hp clinical classification (Clinical Classification Hp) (g / L):

[0009] Normal level: 0-2.5

[0010] Intermediate level: 2.5-3

[0011] Acute phase level: >3

[0012] Cat Hp level higher than >3 g / L indicates a significant inflammatory response, while lower than <2.5 g / L is within the normal healthy range. For cats, Hp values between 2.5-3 g / L, the overall health of the animal should be carefully considered. It is recommended that each laboratory should establish its own normal range corresponding to local genetic and environmental factors.

[0013] Currently, ELISA kits are used in clinical practice, but the time and money costs are too high. The purpose of the present application is to provide a combination of genetically recombinant cat Hp monoclonal antibodies, a cat haptoglobin rapid detection kit containing cat Hp monoclonal antibodies, which has a wider detection range and can be used for different clinical diagnostic needs. SUMMARY

[0014] An object of the present application is to solve at least the above problems and / or drawbacks and to provide at least the advantages later described.

[0015] Another object of the present application is to provide a monoclonal antibody of feline haptoglobin and uses thereof.

[0016] To this end, the technical solution provided by the present application is as follows:

[0017] In a first aspect, an expression system for producing recombinant feline haptoglobin or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: a nucleotide sequence as shown in SEQ ID NO: 1 encoding an alpha chain of feline haptoglobin and a nucleotide sequence as shown in SEQ ID NO: 2 encoding a beta chain of feline haptoglobin, the alpha chain and the beta chain being linked into the complete feline haptoglobin or the hemoglobin-binding fragment thereof by a disulfide bond.

[0018] In a second aspect, a monoclonal antibody or an antigen-binding fragment thereof of feline haptoglobin, comprising: Hp-mAb8, Hp-mAb9 and / or Hp-mAb10,

[0019] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody Hp-mAb8 are GFSLDGYD, IYVSDVT and ARDSDVGVSGYTFPDI respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody Hp-mAb8 are ESISTNR, PAS and QSGYATADVPNT respectively,

[0020] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody Hp-mAb9 are GFSLRNYK, IYSNGWT and ARVGYLADNSYWSI respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody Hp-mAb9 are QNIYSGN, AAS and QAGYIDTPIDNV respectively,

[0021] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody Hp-mAb10 are GIDFGSYA, IYPDFGTR and GRGFSTQYGADI respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody Hp-mAb10 are QSISSG, TAS and AGYVGSFGTDIA respectively.

[0022] Preferably, in the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin, the amino acid sequence of the heavy chain variable region of the monoclonal antibody Hp-mAb8 is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region sequence is as shown in SEQ ID NO: 4.

[0023] Preferably, in the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin, the amino acid sequence of the heavy chain variable region of the monoclonal antibody Hp-mAb9 is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6.

[0024] Preferably, in the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin, the amino acid sequence of the heavy chain variable region of the monoclonal antibody Hp-mAb10 is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

[0025] In a third aspect, a gene encoding the feline haptoglobin alpha chain and beta chain, or encoding the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin of any one of the preceding aspects.

[0026] In a fourth aspect, a recombinant vector comprising the gene.

[0027] In a fifth aspect, a composition for detecting Hp protein in feline serum, comprising the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin of any one of the preceding aspects.

[0028] In a sixth aspect, a kit comprising the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin of any one of the preceding aspects, which is a latex-enhanced immunoturbidimetry or chemiluminescent immunoassay kit for in vitro rapid detection of Hp protein in feline serum.

[0029] In a seventh aspect, use of the monoclonal antibody or antigen-binding fragment thereof of feline haptoglobin of any one of the preceding aspects in a medicament or kit for early screening of feline sepsis, fungal infection, feline metritis, plasma cell stomatitis, feline transmissible enteritis, mycoplasma haemofelis, heartworm, liver fluke, babesia, surgical damage, respiratory tract infection, gastroenteritis, tumor, and for therapeutic efficacy monitoring and prognosis and recurrence risk assessment.

[0030] The present application at least includes the following beneficial effects:

[0031] The present application firstly develops an expression system for producing recombinant cat haptoglobin (Hp) or its hemoglobin-binding fragment in mammalian cells. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have higher affinity and specificity, can recognize more epitopes, can recognize mouse antigens, and have good performance and wide application in the fields of scientific research and diagnosis. Therefore, rabbits are immunized with recombinant cat Hp, and three monoclonal antibodies with high affinity to cat Hp protein are screened, which are used for LEIA method for detecting cat Hp.

[0032] The calibration results of the three combinations of Hp-mAb8 and Hp-mAb9, Hp-mAb8 and Hp-mAb10, and Hp-mAb9 and Hp-mAb10 meet the requirements of the kit.

[0033] Compared with the ELISA kit, the cat Hp assay kit containing cat Hp monoclonal antibodies has higher cost performance and wider detection linearity, and can meet the budget of most cat-keeping families, and can be used for different clinical diagnostic needs.

[0034] Other advantages, objects and features of the present application will be partially embodied by the following description, and will be partially understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 For the calibration curve of the cat Hp assay kit prepared by the cat Hp monoclonal antibody in the embodiment of the present application, the cat Hp antigen is prepared into a series of calibrators with a calibrator diluent, and the concentrations are 0 g / L, 0.15 g / L, 0.4 g / L, 1.5 g / L, 4 g / L and 12 g / L.

[0036] Figure 2 For the cat Hp antigen assay kit immunoturbidimetric platform sample compliance rate in the embodiment of the present application. DETAILED DESCRIPTION

[0037] The present application will be further described in detail below, so that those skilled in the art can implement it according to the description.

[0038] The present application will be further described in detail below, so that those skilled in the art can implement it according to the description.

[0039] The present application relates to Anti-Feline Haptoglobin (Feline Hp) monoclonal antibodies and uses thereof, a Feline Hp latex-enhanced immunoturbidimetric assay kit for clinical diagnosis comprising the monoclonal antibodies, nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors carrying the nucleic acids, and methods of using the vectors and host cells to make the Feline Hp monoclonal antibodies or antigen-binding fragments thereof.

[0040] A first aspect of the present application discloses an expression system for producing recombinant Feline Haptoglobin (Hp) or a hemoglobin-binding fragment thereof in a mammalian cell, the expression system comprising:

[0041] (a) a first nucleic acid sequence encoding a Feline Haptoglobin alpha chain, and

[0042] (b) a second nucleic acid sequence encoding a Feline Haptoglobin beta chain;

[0043] wherein, upon introduction of the first nucleic acid sequence and the second nucleic acid sequence into the mammalian cell, and subsequent expression of the Feline Haptoglobin alpha chain and the Feline Haptoglobin beta chain in the cell, the alpha chain and the beta chain are linked into the complete Feline Haptoglobin or a hemoglobin-binding fragment thereof by disulfide bonds through folded assembly. Wherein the Feline Hp protein is as shown in SEQ ID NO: 1-2

[0044] A second aspect of the present application discloses an Anti-Feline Hp high-affinity rabbit monoclonal antibody Hp-mAb8 or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 3, and the light chain variable region amino acid sequence as shown in SEQ ID NO: 4.

[0045] A third aspect of the present application discloses an Anti-Feline Hp high-affinity rabbit monoclonal antibody Hp-mAb9 or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 5, and the light chain variable region amino acid sequence as shown in SEQ ID NO: 6.

[0046] A fourth aspect of the present application discloses an Anti-Feline Hp high-affinity rabbit monoclonal antibody Hp-mAb10 or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 7, and the light chain variable region amino acid sequence as shown in SEQ ID NO: 8.

[0047] A monoclonal antibody, or a variant thereof, or a functional fragment thereof, against Feline Haptoglobin (Feline Hp) protein, comprising:

[0048] Preferably, the Anti-feline Hp protein monoclonal antibody, or a variant thereof, or a functional fragment thereof is a chimeric antibody, a humanized antibody or a fully humanized antibody.

[0049] Further, without substantially affecting the affinity of the antibody, the present application also includes an antibody or an antigen-binding fragment thereof having >90% sequence identity with the above-mentioned antibody variable region sequence, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. They are all considered to be included in the scope of protection of the present application.

[0050] The fifth aspect of the present application discloses a kit comprising any one or more of the above-mentioned rabbit monoclonal antibodies or antigen-binding fragments thereof. Further, the sample used in the kit is blood.

[0051] The sixth aspect of the present application discloses the use of any one or more of the above-mentioned rabbit monoclonal antibodies or antigen-binding fragments thereof in detecting feline Hp.

[0052] The seventh aspect of the present application discloses the use of any one or more of the above-mentioned rabbit monoclonal antibodies or antigen-binding fragments thereof in preparing a kit for detecting feline Hp. The calibration results of the three combinations of Hp-mAb8 and Hp-mAb9, Hp-mAb8 and Hp-mAb10, and Hp-mAb9 and Hp-mAb10 all meet the requirements of the kit.

[0053] Compared with the ELISA kit, the feline Hp assay kit containing feline Hp monoclonal antibody has higher cost performance and wider detection linearity, and can meet the budget of most cat-keeping families, and can be used for different clinical diagnostic needs.

[0054] In order for those skilled in the art to better understand the technical solutions of the present application, the following examples are provided for further illustration:

[0055] Example 1 Construction of feline Hp expression vector and protein transient expression

[0056] The first aspect of the present application discloses an expression system for producing recombinant feline touch globin (Hp) or a hemoglobin-binding fragment thereof in mammalian cells, a first nucleic acid sequence encoding the alpha chain of feline touch globin (the nucleotide sequence of which is shown as SEQ ID NO: 1) and a second nucleic acid sequence encoding the beta chain of feline touch globin (the nucleotide sequence of which is shown as SEQ ID NO: 2) are linked to the expression plasmid pTT5 through the HindIII / EcoRI cloning site to obtain the Hp expression vectors pTT5-Hp-alpha and pTT5-Hp-beta. The feline Hp protein is expressed by using HEK293F host cells.

[0057] Example 2 Obtaining of Anti-feline Hp monoclonal antibody

[0058] 1.1 Immunization of animals

[0059] Two mg / ml of cat Hp antigen (i.e. purified recombinant cat haptoglobin prepared by Example 1) 20 ml were mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into New Zealand white rabbits in multiple sites on the abdomen, 400 μg of antigen per rabbit. After four boosts, the immune system of the rabbits was activated, resulting in the production of antibodies by B cells. The method of boosting was as follows: each time the immunization was separated by two weeks, and the incomplete adjuvant was used for boosting, the antigen dose was 200 μg. The serum titer was determined by immunoturbidimetry, and the rabbits with high serum titers were selected for further immunization (one more time, two weeks later, without adjuvant, the antigen dose was 200 μg), and peripheral blood was collected three days later.

[0060] 1.2 B lymphocyte sorting

[0061] The peripheral blood of the rabbits was collected, and DAPI was used for sorting to remove dead cells. The single cells were screened by flow cytometry to obtain the target lymphocytes.

[0062] 1.3 Cloning of the gene encoding the rabbit monoclonal antibody

[0063] The sorted single B cells were first cultured in vitro in 5-10 plate 96-well plates, and then the supernatant of the cell culture was screened by ELISA or FACS and other early intervention detection methods. The supernatant of the cultured B cells was identified by antigen-coated ELISA to obtain positive clones. The cells of the positive clones were collected, lysed, and the RNA was extracted and reverse transcribed into cDNA. The rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clones by PCR, and the sequences were determined by sequencing.

[0064] Positive clone screening and epitope identification

[0065] Three clones secreting antibodies that can bind to cat Hp antigen were screened from the monoclonal hybridoma cells by ELISA reaction. The epitope was identified by sandwich method, and Hp-mAb8 antibody was captured and fixed to the surface of the enzyme-labeled plate as the primary antibody. Then the cat Hp antigen was injected and combined with the Hp-mAb8 antibody. Then HRP-labeled Hp-mAb9 or Hp-mAb10 antibody was injected as the secondary antibody. The secondary antibody either binds to the cat Hp antigen to form a "sandwich" combination, or is blocked by the primary antibody and does not bind to the cat Hp antigen. The results show that the secondary antibody (Hp-mAb9 or Hp-mAb10) can bind to the cat Hp antigen in the presence of the primary antibody (Hp-mAb8), indicating that they have different epitopes. The method described above uses Hp-mAb9 antibody as the primary antibody and Hp-mAb10 as the secondary antibody, and the results show that they have different epitopes. Therefore, the three antibodies have different bins, respectively.

[0066] Construction of expression vector of anti-feline Hp monoclonal antibody

[0067] The present application provides a monoclonal antibody that can simultaneously specifically bind to three different epitopes of feline Hp protein, the light chain and heavy chain encoding genes of Hp-mAb8 are connected to the expression plasmid pTT5 through HindIII / EcoRI cloning sites to obtain the expression vectors pTT5-Hp-mAb8-LC and pTT5-Hp-mAb8-HC of the monoclonal antibody; the same method is used to obtain pTT5-Hp-mAb9-LC and pTT5-Hp-mAb9-HC; pTT5-Hp-mAb10-LC and pTT5-Hp-mAb10-HC.

[0068] Preparation of anti-feline Hp monoclonal antibody

[0069] Expression of feline Hp monoclonal antibodies Hp-mAb8, Hp-mAb9 and Hp-mAb10 using HEK293F host cells.

[0070] The cell density is diluted to 2x10 6 ml KPM and 100 μg of sterile light chain and heavy chain plasmids, and mix gently; take another centrifuge tube, add 10 ml KPM and 1 ml TA-293 transfection reagent, and mix gently; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and mix gently; prepare the plasmid-carrier complex at room temperature for 10 minutes; remove the cells from the constant temperature shaker, add the prepared plasmid-carrier complex while shaking, and return to the CO2 constant temperature shaker for incubation.

[0071] Add 1.2 ml of 293 cell protein expression enhancer (KE-293) 24 hours after transfection, and add transient transfection nutrient additive (KT-Feed 50x) to improve the expression of the product; measure the expression of the product on the 6th day after transfection.

[0072] Antibody purification

[0073] Centrifuge the cell culture at 5000 rpm, collect the supernatant and filter it with a 0.22 μm filter. Then purify it through a protein A affinity chromatography column, desalt and replace the buffer, and store in aliquots.

[0074] Example 5: Calibration curve of feline Hp antigen detection kit

[0075] Cat Hp test kit calibration product configuration: dilute cat Hp antigen with calibration buffer, and configure calibration concentration points in turn: concentrations are 0 g / L, 0.15 g / L, 0.4 g / L, 1.5 g / L, 4 g / L, and 12 g / L.

[0076] The detection principle of a cat haptoglobin cat Hp assay kit is:

[0077] The kit uses latex-enhanced immunoturbidimetry. The basic principle is that two cat Hp mAbs (Hp-mAb8 and Hp-mAb9) are cross-linked to latex particles, and an antigen-antibody binding reaction occurs with cat Hp in the sample to be tested, forming a large antigen-antibody complex, increasing turbidity, and the cat Hp concentration is proportional to the turbidity formed. At a wavelength of 570 nm, by comparing the turbidity detected with the standard curve, the cat Hp antigen content in the sample can be calculated. The calibration curve is shown in Figure 1 Hp-mAb8 and Hp-mAb10, and the application of Hp-mAb9 and Hp-mAb10 combination were tested according to the same scheme, and the calibration results were similar to Hp-mAb8 and Hp-mAb9.

[0078] The use method of a cat Hp antigen assay kit is:

[0079] The cat Hp antigen kit is tested with a full-automatic biochemical instrument 7180. The parameters are as follows: first add 3 μl of sample, then add 180 μl of reagent R1, incubate at 37°C for 5 min, add 60 μl of reagent R2, read the absorbance A1 after 5 min, and calculate the absorbance difference △A = A1-A0 (initial value); use the matching calibration product for multi-point calibration to obtain the calibration curve and linear fitting, and then the sample concentration (unit g / L) can be calculated by the absorbance difference detected on the calibration curve.

[0080] Example 6 cat Hp antigen test kit performance

[0081] Antibodies are the core raw materials of in vitro diagnostic reagents, and their performance directly affects the quality of in vitro diagnostic reagent products. Sample compliance rate, as one of the most important indicators for verifying the performance of antibody raw materials, is the part that in vitro diagnostic reagent companies pay most attention to when selecting raw materials.

[0082] Sample concordance rate, also known as clinical comparative analysis, reflects the degree of consistency between the measured values ​​of a diagnostic kit and the theoretical values ​​of clinically assigned samples. To evaluate the sample concordance rate of a new antibody raw material, an in vitro diagnostic kit (using platforms such as colloidal gold or immunoturbidimetry) is typically prepared using that antibody. The kit is then used to test actual clinical specimens, and the results are statistically analyzed for comprehensive evaluation. Through standardized comparative studies, analysis, and summarization of the relationship between the kit's test results and the theoretical values ​​of clinical samples, the performance of the antibody raw material can be indirectly inferred. The closer the results are, the better the consistency, and the better the antibody's performance.

[0083] The concentration of *Helicobacter pylori* (Hp) in cats ranged from 0.2 to 13.1 g / L, totaling 26 cases. A scatter plot was created with measured values ​​on the ordinate and theoretical values ​​on the abscissa. A linear regression curve was fitted using a computer, and the R-squared value was calculated. 2 .like Figure 2 The sample concordance rate R of the feline Hp antigen detection kit shown is... 2 >99%.

[0084] SEQ ID NO:1

[0085] VETGNEATDNTEVSLPKPPEIENGYAEHFVRYQCNPLYKLRTEGDGEYTLNSEKHWTNKAIGEKLPECEAVCGKPKNPVDQVQR

[0086] SEQ ID NO:2

[0087] IMGGSVDAKGSFPWQAKMVSHHNLTSGATLISEQWLLTTAKNLFLGHKDDAKAKDIAPTLKLYVGKNQPVEIEKVVLHPNYSNVDIGLIKLKQKVPIDERVMPICLPSKDYAEVGRVGYVSGWGRN TNFNFTELLKYVMLPVADQENCVKHYEGSTVPEKKSSKSPVGVQPILNEHTFCAGLSEFQEDTCYGDAGSAFAVHDEDDNTWYAAGILSFDKSCRVAEYGVYVKVPSILAWIQETTAGNHHHHHHHH

[0088] SEQ ID NO:3

[0089] MNWVRQAPGKGLEWIGFIYVSDVTKYANWAKGRFTISKSSTTVDLKITSPTTEDTATYFCARDSDVGVSGYTFPDIWGPGTLVTVSL

[0090] SEQ ID NO: 4

[0091] LAWYQQKPGQPPKLLIYPASKLASGVPSRFKGSGSGTHFTLTISGVQCDDAATYYCQSGYATADVPNTFGGGTEVMVR

[0092] SEQ ID NO: 5

[0093] LSWVRQAPGKGLEWIGDIYSNGWTSYANWAKGRFTISKTSSTTVDLKITNPTTEDTATYFCARVGYLADNSYWSIWGPGTLVTVSS

[0094] SEQ ID NO: 6

[0095] LAWYQQKPGQPPKLLIYAASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQAGYIDTPIDNVFGGGTEVVVK

[0096] SEQ ID NO: 7

[0097] ISWVRQAPGKGPEWIAHIYPDFGTRFYASWAKGRFTISKTSSTTVDLKVTSPTTEDTATYFCGRGFSTQYGADIWGPGTLVTVSS

[0098] SEQ ID NO: 8

[0099] LAWYQQKPGQPPKLLIYTASTLPSGVPSRFSGSGSETDYTLTISSVQAEDAGIYYCAGYVGSFGTDIAFGGGTEVVVK

[0100] The number of modules and processing stages set forth herein are used to simplify the explanation of the present application. It will be apparent to those skilled in the art that applications, modifications and variations of the application can be practiced based on these teachings.

[0101] While the embodiments of the application have been disclosed as above, it is not limited to the specific embodiments set forth herein, but can be practiced with modifications and alterations, which can be apparent to those skilled in the art. Therefore, the application is not limited to the specific details and embodiments set forth above, but can be practiced with modifications and alterations within the scope of the general concepts disclosed herein.

Claims

1. A composition for detecting haptoglobin in cat serum, characterized in that, Including monoclonal antibody Hp-mAb8 or its antigen-binding fragment and monoclonal antibody Hp-mAb9 or its antigen-binding fragment; The monoclonal antibody Hp-mAb8 or its antigen-binding fragment comprises heavy chains CDR1, CDR2 and CDR3 with amino acid sequences of GFSLDGYD, IYVSDVT and ARDSDVGVSGYTFPDI in sequence, and light chains CDR1, CDR2 and CDR3 with amino acid sequences of ESISTNR, PAS and QSGYATADVPNT in sequence. The monoclonal antibody Hp-mAb9 or its antigen-binding fragment comprises heavy chains CDR1, CDR2 and CDR3 with amino acid sequences of GFSLRNYK, IYSNGWT and ARVGYLADNSYWSI in sequence, and light chains CDR1, CDR2 and CDR3 with amino acid sequences of QNIYSGN, AAS and QAGYIDTPIDNV in sequence.

2. The composition according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody Hp-mAb8 or its antigen-binding fragment is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. The composition according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody Hp-mAb9 or its antigen-binding fragment is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6.

4. A gene combination encoding all monoclonal antibodies or antigen-binding fragments thereof in the composition as described in any one of claims 1 to 3.

5. A recombinant vector combination comprising the gene combination of claim 4.

6. A reagent kit, characterized in that, The kit comprises the composition according to any one of claims 1 to 3, and the kit is a rapid in vitro detection kit for haptoglobin in cat serum by latex-enhanced immunoturbidimetric assay or chemiluminescent immunoassay.

7. Use of the composition according to any one of claims 1 to 3 in the preparation of a kit for detecting cat haptoglobin.

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