CN117285624B - Anti-canine NT-proBNP bispecific antibody and kit - Google Patents
Anti-canine NT-proBNP bispecific antibody and kit Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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Abstract
The present invention discloses an anti-canine NT-proBNP bispecific antibody, or a functional fragment thereof, comprising: scFv1 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv1 are set forth in SEQ ID NO:1-3, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv1 are shown in SEQ ID NO: 4-6. The inventor firstly screens out two monoclonal antibodies with high affinity to the canine NT-proBNP protein, then obtains a gene recombinant canine NT-proBNP bispecific antibody through a genetic engineering means, and uses the antibody for detecting canine NT-proBNP by LETIA method, and the kit of the method has higher sensitivity and wider detection range, and can be used for different clinical diagnosis requirements.
Description
Technical Field
The invention relates to the technical field of immunology. More particularly, the invention relates to a bispecific antibody against canine NT-proBNP and a kit.
Background
Latex immunoturbidimetry is currently a very mainstream immunoassay, whose basic measurement principle is: the antigen-antibody forms antigen-antibody complex in buffer solution, so that turbidity of the reaction solution appears and light transmittance is reduced. In a certain proportion range, the turbidity of the reaction solution and the amount of the antigen-antibody are in linear correlation. In practical use, the excessive amount of antibody in the reaction liquid is generally maintained, the formed complex increases along with the increase of the antigen in the sample, the turbidity of the reaction liquid also increases, and the content of the detected substance can be calculated by comparing with a series of standard substances. However, the latex has longer research and development period than the turbidimetric reagent and complex steps. The most important step in developing latex turbidimetric reagents is the selection of antigen antibodies and latex particles. The antibody has strong specificity and high sensitivity. Latex particles, mainly of comparative particle size: the larger the particle size, the higher the sensitivity; the smaller the particle size, the longer the linearity. In general, particles having a sensitivity of more than 100. Mu.g/L may be tested for particles less than 200nm, and particles having a sensitivity of less than 100. Mu.g/L may be tested for particles greater than 200 nm. When one particle can not meet the sensitivity and linearity requirements at the same time, large spheres and small spheres can be mixed, and most of the small spheres are mixed with the large spheres. In addition to the aforementioned antigens, antibodies and latex particles, the reagent properties are affected by the conditions of the reaction in each step, such as pH, amount of activator, temperature, reaction time, ionic strength of the solution, amount of antibody, choice of blocking buffer or blocking agent, etc. The technology has high automation degree and high measurement efficiency, and is suitable for large-scale clinical analysis. Latex particle enhanced turbidimetric immunoassay (LETIA or LTIA) detection is faster and more convenient than enzyme-linked immunosorbent assay (ELISA), and is suitable for clinical sample detection.
NT-proBNP belongs to the family of natriuretic peptides and regulates fluid homeostasis. Canine type B Natriuretic Peptide (BNP) is produced in the myocardium as a form of a pro-hormone, which then first splits into the pro-hormone proBNP and then becomes the mature active hormone. Thus, proBNP is eventually split by specific serum and cardiac proteases into an active carboxy-terminal fragment BNP (half-life about 90 seconds) and an inactive N-terminal by-product NT-proBNP.
Heart disease is one of the most common diseases in pets, and heart disease prevalence in dogs is high, and as pets age, heart biomarkers should be considered as important as any other biomarker, such as kidney and liver biomarkers. Clinically, the NT-proBNP assay is highly stable and easily assayed, while its specificity is high, accurately reflecting myocardial function, while most biomarkers are indicative of myocardial cell integrity only, such as troponin and CK-MB. Compared with other traditional infection indexes such as PCT, CRP, IL-6, white blood cell number and the like, the infection change of the canine NT-proBNP occurs earlier, and the comprehensive indexes such as sensitivity, specificity, positive and negative detection rate and the like are obviously superior to other infection indexes. One potential indication for NT-proBNP detection is the detection of asymptomatic or occult heart disease. At this stage, early diagnosis is required to monitor disease progression and possibly to intervene before clinical signs appear.
Dogs suspected of suffering from heart disease need to be tested for NT-proBNP: respiratory and/or exercise intolerance; 2, senior dogs (> 9 years of age); 3, dogs with heart murmurs. With respect to differentiating cardiac and respiratory diseases in dogs, they provided the following evaluations in serum NT-proBNP measurements, based on data collected by the london royal veterinary medical college: <900pmol/L: normal or respiratory disease; 900-1800pmol/L: heart disease without heart failure; 1800pmol/L: heart failure.
Currently, methods for detecting canine NT-proBNP concentration are mainly quantitative by enzyme-linked immunosorbent assay (ELISA), and semi-quantitative point-of-care testing (POCT) directly determine whether heart disease is likely to occur by numerical value. The above-mentioned monitoring methods have problems in that they take a long time or the measurement results are not accurate.
Latex particle-enhanced turbidimetric immunoassay consists of a latex coated with antibodies reactive with a specific analyte, and the triggered aggregation of particles can be correlated to analyte concentration by rapid, simple measurement methods such as turbidimetry. The device has high efficiency and high automation degree, and is also suitable for detecting large clinical samples. Polyclonal antibodies are most commonly used for the detection of canine NT-proBNP because of their synergistic effect of having a plurality of specificities and binding to a plurality of epitopes, however, their diagnostic accuracy is questioned due to their lower specificity. Monoclonal antibodies exhibit unprecedented specificity for their antigen targets, but this extreme specificity may hamper the efficacy of any single monoclonal antibody. The concept of binding multiple monoclonal or bispecific antibodies to increase their detection efficacy and overcome the limitation of low neutralization efficacy appears logical.
Disclosure of Invention
The invention provides a bi-specific antibody of anti-canine NT-proBNP or a functional fragment thereof and a kit, wherein the inventor firstly screens out two monoclonal antibodies with high affinity for canine NT-proBNP protein, then obtains a bi-specific antibody of the canine NT-proBNP by genetic engineering means, and uses the bi-specific antibody of the canine NT-proBNP to detect the canine NT-proBNP by a LETIA method.
To achieve these objects and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, there is provided an anti-canine NT-proBNP bispecific antibody or functional fragment thereof comprising:
scFv1 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv1 are set forth in SEQ ID NO:1-3, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv1 are shown in SEQ ID NO: 4-6;
scFv2 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv2 are set forth in SEQ ID NO:7-9, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv2 are shown in SEQ ID NOs: 10-12.
The present invention provides an anti-canine NT-proBNP bispecific antibody or functional fragment thereof comprising:
the amino acid sequence of the VH of the scFv1 is shown as SEQ ID NO. 13; the VL amino acid sequence of the scFv1 is shown as SEQ ID NO. 14;
The amino acid sequence of the VH of the scFv2 is shown as SEQ ID NO. 15; the VL amino acid sequence of the scFv2 is shown as SEQ ID NO. 16.
The invention provides a bispecific antibody against canine NT-proBNP, or a functional fragment thereof,
Linker in the middle of the heavy chain variable region and the light chain variable region of scFv1 and scFv2 is (G 4 S) n, n is 3.
The invention provides a kit comprising the anti-canine NT-proBNP bispecific antibody or a functional fragment thereof described above.
Preferably, the kit comprises a first reagent and a second reagent in a volume ratio of 3:1;
The first reagent comprises the following components in percentage by weight:
the second reagent comprises the following components in percentage by weight:
Latex particles loaded with canine NT-proBNP bispecific antibodies comprise said anti-canine NT-proBNP bispecific antibodies or functional fragments thereof.
The invention also provides an application of the kit in detecting NT-proBNP protein in canine serum, and the kit comprises the anti-canine NT-proBNP bispecific antibody or the functional fragment thereof.
The invention at least comprises the following beneficial effects:
The invention firstly screens out two monoclonal antibodies with high affinity to the canine NT-proBNP protein, then obtains a gene recombinant canine NT-proBNP bispecific antibody or a functional fragment thereof through a genetic engineering means, and uses the antibody or the functional fragment thereof for preparing a detection kit for detecting canine NT-proBNP by a LETIA method, which has short detection time, high sensitivity and wide detection linearity, can be used for different clinical diagnosis requirements, such as rapid diagnosis of the health condition of canine heart disease, and is used for detecting dogs which show healthy but suspected to have heart disease risks and any dogs with respiratory tract disease signs, thereby assisting in removing serious heart disease. The function is as follows: 1) Distinguishing between heart disease and respiratory disease; 2) Staging of Myxomatous Mitral Valve Degeneration (MMVD); 3) Dilated Cardiomyopathy (DCM) was detected in large varieties.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a graph showing the relationship between the sensitivity of canine NT-proBNP and the particle diameter in example 5 of the present invention;
FIG. 2 is a calibration curve of the canine NT-proBNP assay kit of example 6 of the present invention;
FIG. 3 is a linear regression curve of the sample compliance rate of the immunonephelometric plateau of the canine NT-proBNP antigen assay kit of example 6 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Embodiments of the present application provide anti-canine NT-proBNP bispecific antibodies or functional fragments thereof, scFv1 regions thereof, wherein the VH CDR1, CDR2, and CDR3 amino acid sequences of scFv1 are set forth in SEQ ID NOs: 1-3, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv1 are shown in SEQ ID NO: 4-6;
scFv2 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv2 are set forth in SEQ ID NO:7-9, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv2 are shown in SEQ ID NOs: 10-12;
the anti-canine NT-proBNP bispecific antibody or functional fragment thereof has the structure: fc region of VH1-Linker-VL1-mIg G-Linker-VH 2-VL 2.
The bispecific antibody or functional fragment thereof in this embodiment may be a full length antibody, or a fusion protein comprising only two antigen domain binding portions, such as a single chain antibody (scFv), or a full length antibody and an antigen binding portion. One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9 or 10) amino acids to the sequences of the present examples to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the affinity of the antibodies.
A first functional domain of an anti-canine NT-proBNP bispecific antibody or a functional fragment thereof, comprising a scFv1 region of an anti-canine NT-proBN P specific antibody, wherein the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv1 are shown as SEQ ID No. 1-3, respectively, the amino acid sequences of SEQ ID No. 1-3 are GFTFSPYA, ITGGNVNI and ARGEDYRFDVGKGFVY; the amino acid sequences of VL CDR1, CDR2 and CDR3 of the scFv1 are respectively shown in SEQ ID NO: 4-6; the amino acid sequences of SEQ ID NO. 4-6 are QDINKN, KVS and PQGHH VPLT;
A second domain of an anti-canine NT-proBNP bispecific antibody or a functional fragment thereof comprising a scFv2 region of an anti-canine NT-proBN P specific antibody, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv2 being set forth in SEQ ID NO:7-9, SEQ ID NO:7-9 is: GFSLTDYG, IYPDFGVR, AKGPQSALYSDY; the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv2 are respectively shown in SEQ ID NO:10-12, SEQ ID NO:10-12 is: QNVRTN, GAS, QQGYDY PFT.
Linker in the middle of the heavy chain variable region and the light chain variable region of scFv1 and scFv2 is (G4S) n, n being 3.
Wherein the functional domain for specifically recognizing and resisting the canine NT-proBNP consists of a single-chain antibody scFv1 and a single-chain antibody scFv2 which are connected in series through an Fc region and a Linker, and the bispecific antibody or the functional fragment thereof for resisting the canine NT-proBNP is a chimeric antibody, a humanized antibody or a fully humanized antibody.
The canine NT-proBNP bispecific antibody or functional fragment thereof has the structure: fc region of VH1-Linker-VL1-mIgG 1-Linker-VH 2-Linker-VL2.
Embodiments of the invention also provide anti-canine NT-proBNP bispecific antibodies or functional fragments thereof, comprising:
The amino acid sequence of the VH of the scFv1 is shown as SEQ ID NO. 13, and the amino acid sequence of the SEQ ID NO. 13 is
DVQLVESGGGLVQPGGSRKLSCSASGFTFSPYAIHWLRQRPIQGLEWIGCITGGNVNIIYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGEDYRFDVGKGFVYWGQGTTLTVSS; The VL amino acid sequence of the scFv1 is shown as SEQ ID NO. 14, and the amino acid sequence of the SEQ ID NO. 14 is as follows:
DVLMTQIPLSLPVSLGDQASISCKSSQDINKNVSWFQQKPGQSPHQLIFKVSNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCPQGHHVPLTFGAGTKLEVK;
The amino acid sequence of the VH of the scFv2 is shown as SEQ ID NO. 15, and the amino acid sequence of the SEQ ID NO. 15 is as follows:
QVQLQQSGPELVKPGASMKISCKASGFSLTDYGMYWVRQTPEKRLEWVATIYPDFGVRNYNQKFKGKATLTVDKSSSSAFMHLNSLTTEDSAVYYCAKGPQSALYSDYWGTGTTVTVSS; The VL amino acid sequence of the scFv2 is shown as SEQ ID NO. 16, and the amino acid sequence of the SEQ ID NO. 16 is as follows:
DFVMSQSPFSLVVSVGEKVTMSCNSSQNVRTNLHWYQQRPGFSPKLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQQGYDYPFTFGGGTKLEIK;
The canine NT-proBNP bispecific antibody or functional fragment thereof has the structure: fc region of VH1-Linker-VL1-mIgG 1-Linker-VH 2-Linker-VL2.
The anti-canine NT-proBNP bispecific antibody or the functional fragment thereof provided by the invention comprises a first functional domain and a second functional domain, wherein the amino acid sequence of the heavy chain variable region of the first functional domain is shown as SEQ ID NO. 13; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the second functional domain is shown as SEQ ID NO. 15; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16. The first functional domain scFv1 is formed by connecting SEQ ID NO. 13 and SEQ ID NO. 14 through a Linker, and the second functional domain scFv2 is formed by connecting SEQ ID NO. 15 and SEQ ID NO. 16 through a Linker, wherein the Linker is (G 4 S) n, and n is preferably 3.
The embodiment of the application also provides a medicament, which comprises the anti-canine NT-proBNP bispecific antibody or the functional fragment thereof, can be used for preparing medicaments for treating heart diseases such as heart-derived dyspnea diseases, heart injury diseases and the like, and can also comprise pharmaceutically acceptable carriers and excipients.
The embodiment of the application also provides a kit comprising the anti-canine NT-proBNP bispecific antibody or a functional fragment thereof, and the kit uses a latex enhanced turbidimetric immunoassay method and combines a biochemical analyzer for testing.
The kit can be used for detecting NT-proBNP protein in dog serum. The canine NT-proBNP detection kit can be widely applied to distinguishing cardiac dyspnea, early auxiliary diagnosis of heart diseases, heart injury degree evaluation, prognosis evaluation and the like in veterinary clinic; the kit can be used for rapidly diagnosing the health condition of the heart disease of dogs, and is used for detecting dogs which are healthy but suspected to be at risk of heart disease and any dogs with respiratory disease signs, thereby assisting in removing serious heart disease. The function is as follows: 1) Distinguishing between heart disease and respiratory disease; 2) Staging of Myxomatous Mitral Valve Degeneration (MMVD); 3) Dilated Cardiomyopathy (DCM) was detected in large varieties.
Further, the kit comprises a first reagent and a second reagent;
The first reagent comprises the following components in percentage by weight:
wherein the buffer solution is one or two of MES buffer solution pH6.0-7.0 and HEPES buffer solution pH 6.8-8.0; the polyethylene glycol is one or more of PEG6000, PEG8000 and PEG 20000;
the second reagent comprises the following components in percentage by weight:
Wherein the latex particles are carboxylated polystyrene latex particles, and the particle size of the carboxylated polystyrene latex particles is one or more particles with the particle size of 63-300 nm; the blocking agent is one or more of DB1130, BSA, CE210 and CE 510; the preservative is one or more of NaN3, PC-300 and gentamicin; latex particles loaded with canine NT-proBNP bispecific antibodies include the above-described anti-canine NT-proBNP bispecific antibodies or functional fragments thereof.
In the above embodiments, the present invention provides a kit comprising a first reagent (R1) and a second reagent (R2), the first reagent (R1) comprising a sensitizer, a buffer, and a surfactant; the second reagent (R2) comprises latex particles loaded with canine NT-proBNP bispecific antibody, a surfactant, a buffer solution and a preservative, the kit is used for rapidly and dynamically monitoring the canine serum NT-proBNP concentration, and has important indication significance for early diagnosis and treatment of canine heart disease, and the kit is high in measurement accuracy, low in cost and good in stability.
The following is a description of specific examples.
Example 1: obtaining bispecific antibodies against canine NT-proBNP
Animals were immunized.
1Ml of canine NT-proBNP antigen (1 mg/ml) was mixed with an equal volume of Freund's complete adjuvant uniformly, and then injected subcutaneously into 12 BALB/C mice over 4 to 6 weeks. After 1 week, 1ml of canine NT-proBNP protein was mixed homogeneously with an equal volume of Freund's incomplete adjuvant and the mice were subjected to subcutaneous multi-point injection in the abdomen. The above steps were repeated at week 3. The above steps were repeated at week 4. Three days after the fourth injection, 20. Mu.l of blood was collected from the broken rat tail tip, left standing at room temperature for 2.5 hours, centrifuged at 25℃and 11000rpm for 15 minutes, and the supernatant was collected and assayed for titer by ELISA. If the titer is detected, eye blood is taken, standing at room temperature for 2 hr, centrifuging at 25deg.C and 7000rpm for 10min, collecting supernatant, and preserving at-80deg.C.
Hybridoma preparation
Mice were euthanized, the whole mice were sterilized with 75% alcohol, splenocytes from the mice were aseptically removed in a super clean bench and ground to form a cell suspension, 1X 10 8 splenocytes were fused with 4X 10 7 myeloma (SP 2/0) cells, and cultured in HAT selection medium for 4-10 days with 50% PEG as fusion agent, and then in HT medium at 5% CO 2, 37 ℃.
Positive clone screening and epitope identification
Clone 2 strains of antibodies secreting canine NT-proBNP antigen binding antibodies in monoclonal hybridoma cells were screened by ELISA reaction, wherein clone 1 strain was preferred for binding to the first domain and clone 1 strain was preferred for binding to the second domain. The surface position is identified by a sandwich method, and the first antibody is captured and fixed on the surface of the ELISA plate. The canine NT-proBNP antigen was then injected and bound to the first antibody. HRP-labeled secondary antibody was then reinjected. The second antibody either binds to the canine NT-proBNP antigen, creating a "sandwich" binding, or is blocked by the first antibody from binding to the canine NT-proBNP antigen. The results show that the second antibody can bind to canine NT-proBNP antigen in the presence of the first antibody, they have different epitopes and therefore belong to different bins.
Example 2: structural design of bispecific antibodies binding canine NT-proBNP
This example provides a structural model of a bispecific antibody capable of simultaneously specifically binding two different epitopes of canine NT-proBNP protein, a bispecific antibody consisting of the scFv1 region of an anti-canine NT-proBNP specific antibody (VH 1-Linker-VL1 or VL1-Linker-VH 1) -the Fc region of murine IgG 1-the scFv2 region of an anti-canine NT-proBNP specific antibody-Linker- (VH 2-Linker-VL2 or VL2-Linker-VH 2).
Variants and functional fragments of canine NT-proBNP bispecific antibodies include, but are not limited to, ① VH1-Linker-VL1-mIgG1 Fc region-Linker-VH 2-Linker-VL2; ② Fc region of VH1-Linker-VL1-mIgG 1-Linker-VL 2-Linker-VH2; ③ Fc region of VL1-Linker-VH1-mIgG 1-Linker-VH 2-Linker-VL2; ④ Fc region of VL1-Linker-VH1-mIgG 1-Linker-VL 2-Linker-VH2.
Example 3: preparation of anti-canine NT-proBNP bispecific antibodies
Diluting the cell density to 2.5X10 6/ml by using HEK293F host cells to express canine NT-proBNP bispecific antibody, placing a shake flask in a 5% CO 2 constant temperature shaking table, culturing at 37 ℃ with shaking at 150rpm for 10min, starting transfection, preparing two 50ml sterile centrifuge tubes, adding 10ml OPM and 200 μg sterile plasmid DNA into one of the two sterile centrifuge tubes, and gently blowing and mixing; taking the other separation tube, adding 10ml of OPM and 0.5ml of PEI transfection reagent, gently blowing and mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and lightly blowing and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-carrier compound; and (5) taking out the cells from the constant temperature shaking table, adding the prepared plasmid-carrier compound while shaking, and putting the cells back into the CO 2 constant temperature shaking table for shake culture.
After 24 hours of transfection, 1.2ml of cell protein expression enhancer VPA and glucose were added to increase the expression level of the product, and the expression level of the product was measured on day 6 after transfection.
Antibody purification
The cell culture supernatant was centrifuged at 8000rpm for 30min, and the supernatant was collected and filtered with a 0.22 μm filter. Purifying with protein A affinity chromatography column, desalting, replacing buffer, and packaging.
Example 4: research and development of canine NT-proBNP detection kit
The assay reagents were optimized for the study of the best experimental performance of LTIA. The main variables are EDC (coupling reagent) and NHS concentration, amount of antibody, amount of latex immobilized antibody reagent, PEG-6000 concentration and absorption wavelength.
The latex was activated using 0.5mg/mL EDC and NHS and 2.0mg/mL canine NT-proBNP bispecific antibody. The latex particles were then immobilized with 2% PEG-6000 and 100. Mu.L antibody to initiate a turbidimetric immune response and turbidity was monitored at 570 nm. The parameters finally selected after linear and synergistic optimization are as follows.
Chemical coupling step
Polystyrene latex particles (100. Mu.L, 5% wt/vol) with diameters of 107nm and 200nm were suspended in 50mM HEPES buffer (pH 7.0) and then incubated with 1.5mg/mL canine NT-proBNP bispecific antibody on a shaker for 30 minutes at room temperature, followed by addition of 0.5mg/mL EDC and NHS at the same concentration, incubation for 30 minutes to activate the latex, and then addition of 1% BSA. After 30 minutes, the antibody-immobilized latex beads were resuspended in 50mM HEPES (pH 6.8) containing 1% BSA, and then sonicated until it became translucent. The latex-fixing reagent was stored at 4℃and used for the measurement of canine NT-proBNP.
Preparing a calibrator for the canine NT-proBNP detection kit: the canine NT-proBNP antigen was prepared as a series of calibrators using calf serum at concentrations of 0pmol/L,500pmol/L,1000pmol/L,2500pmol/L,5000pmol/L,10000pmol/L, respectively.
The using method of the canine NT-proBNP antigen assay kit comprises the following steps:
Sample processing, pipetting steps and measurements were performed automatically on a BECKMAN AU5400 automated analyzer. Briefly, 10. Mu.L of sample and 240. Mu.L of reagent R1 (50 mM HEPES buffer pH 7.0, 150mM NaCl,0.02% Tween-20, 20g/L PEG-6000 and mouse monoclonal antibody blocker 20 mg/L) were injected into a reaction cuvette. After 5 minutes incubation, reagent 80 μ L R2 (antibody immobilized latex particles) was added to the cuvette to initiate the turbidimetric immune reaction. After a further 5 minutes, the canine NT-proBNP concentration was calculated from the difference in absorbance values between the 5 th and 10 th time points (570 nm wavelength). Calibration curves were obtained using a series of available canine NT-proBNP calibrator and used to calculate the value of serum samples.
Example 5 relationship between canine NT-proBNP sensitivity and particle diameter
As shown in FIG. 1, the effect of different sized particles on sensitivity was tested, and 107nm and 200nm polystyrene latex particles were coupled to canine NT-proBNP bispecific antibody, respectively, and compared to 107nm and 200nm particles when used together; the sensitivity of the low end of 200nm is very good, and the high end cannot be detected; the sensitivity at 107nm is low; the sensitivity and linearity can be both achieved when 107nm and 200nm are combined.
Example 6 Performance test of canine NT-proBNP antigen detection kit
The calibration curve of the canine NT-proBNP assay kit prepared from the canine NT-proBNP bispecific antibody is shown in FIG. 2, and canine NT-proBNP antigen was prepared with calf serum to give a series of calibrators at concentrations of 0pmol/L,500pmol/L,1000pmol/L,2500pmol/L,5000pmol/L,10000pmol/L, respectively. The standard curve is plotted with the expected value of canine NT-proBNP on the X-axis and the measured OD570 on the Y-axis. The OD value increases with increasing canine NT-proBNP concentration over a range.
One of the methods of testing the performance of the test kit is the determination of the sample compliance rate, which may also be referred to as clinical contrast analysis, which represents the degree of agreement between the measured value of the diagnostic kit for the clinically assigned sample and the theoretical value of the clinically assigned sample. The performance of the antibody as a core raw material of an in vitro diagnostic reagent directly influences the quality of in vitro diagnostic reagent products. Sample compliance is one of the most important indicators to verify the performance of antibody raw materials, and is the most interesting part of in vitro diagnostic reagent companies when screening raw materials. The sample coincidence rate of a new antibody raw material is checked, an in-vitro diagnosis kit (a colloidal gold platform, an immune turbidimetry platform and the like) is generally prepared by taking the antibody as the raw material, and the kit is used for detecting an actual clinical specimen and carrying out statistics and analysis on a detection result so as to comprehensively check and evaluate. The performance of the antibody raw material can be indirectly deduced through the relationship between the detection result of the standard comparison research, analysis and summarization kit and the theoretical value of the clinical sample. The closer the results, the better the agreement, and the better the performance of the antibody.
And drawing a scatter diagram by taking the measured value as an ordinate and the theoretical value as an abscissa, fitting a linear regression curve by using a computer, and obtaining R2. The sample compliance rate R2 of the canine NT-proBNP antigen detection kit shown in FIG. 3 is >98%.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. The use, modification and variation of the anti-KL-6 bispecific antibodies of the invention, or variants thereof, or functional fragments thereof, will be apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (5)
1. An anti-canine NT-proBNP bispecific antibody or antigen-binding fragment thereof, comprising:
scFv1 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv1 are set forth in SEQ ID NO:1-3, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv1 are shown in SEQ ID NO: 4-6;
scFv2 region, the amino acid sequences of VH CDR1, CDR2 and CDR3 of scFv2 are set forth in SEQ ID NO:7-9, the amino acid sequences of VL CDR1, CDR2 and CDR3 of scFv2 are shown in SEQ ID NOs: 10-12.
2. The anti-canine NT-proBNP bispecific antibody or antigen-binding fragment thereof according to claim 1, which comprises:
the amino acid sequence of the VH of the scFv1 is shown as SEQ ID NO. 13; the VL amino acid sequence of the scFv1 is shown as SEQ ID NO. 14;
the amino acid sequence of the VH of the scFv2 is shown as SEQ ID NO. 15; the VL amino acid sequence of the scFv2 is shown as SEQ ID NO. 16.
3. The anti-canine NT-proBNP bispecific antibody or antigen-binding fragment thereof of claim 2, wherein Linker intermediate the heavy and light chain variable regions of scFv1 and scFv2 is (G 4 S) n, n is 3.
4. A kit comprising an anti-canine NT-proBNP bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
5. The kit of claim 4, comprising a volume ratio of the first reagent to the second reagent of 3:1;
The first reagent comprises the following components in percentage by weight:
Buffer solution 20-60 mM
NaCl 100-200 mM
Tween-20 0.01-0.03%
Polyethylene glycol 1-20g/L
20-40Mg/L of a mouse monoclonal antibody blocker;
the second reagent comprises the following components in percentage by weight:
20-60 mM parts of buffer solution;
Tween-20 0.01-0.03%；
Coupling reagent EDC 0.5-1g/L;
0.5-1g/L of coupling reagent NHS;
1-5 g/L of sealing agent;
1.2-3.0g/L of latex particles loaded with canine NT-proBNP bispecific antibody;
0.1-3.5g/L preservative;
Latex particles loaded with canine NT-proBNP bispecific antibodies comprising the anti-canine NT-proBNP bispecific antibody or antigen binding fragment thereof of any one of claims 1 to 3.
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