Anti-procalcitonin antibody and its application

Abstract

The present invention relates to the field of antibody technology, and in particular to anti-procalcitonin antibodies and applications thereof. The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or antigen-binding fragment thereof provided by the present invention are shown in SEQ ID NO.1-3, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.4-6. The antibody can specifically bind to procalcitonin and has a high affinity. The immunoassay reagent developed using the antibody has high specificity and sensitivity when used for procalcitonin detection, and has a high consistency with the reference detection method, can more accurately reflect the procalcitonin content of real samples, and has a wide range of application prospects in the field of immunoassay of procalcitonin.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, in particular to an anti-procalcitonin antibody and an application thereof. Background Art

[0002] Procalcitonin (PCT) is a hormone-free glycoprotein containing 116 amino acids and a molecular weight of 13KD. It is the precursor of calcitonin (CT) and is encoded by the CALC-I gene located on chromosome 11. PCT is a specific marker for bacterial and fungal infections. The PCT content in healthy human blood is extremely low, usually less than 0.10 ng / mL. This is because in the absence of infection, the expression of the CALC-I gene outside the thyroid gland is suppressed, and it is mainly limited to a certain degree of expression in the neuroendocrine cells of the thyroid gland and lungs. When the body suffers from severe fungal, bacterial, parasitic infections, sepsis, and multiple organ failure, it will induce the expression of CALC-I genes in various cell types of various tissues throughout the body, causing the PCT concentration to rise rapidly, even to hundreds to tens of thousands times the normal level, but the CT content remains unchanged or slightly increased. However, the PCT concentration in the serum of patients with local infection, viral infection, allergy, autoimmune disease, chronic nonspecific inflammation, etc. does not increase or increases slightly. Therefore, PCT can be used as a specific indicator of bacterial infection for the differential diagnosis of bacterial diseases.

[0003] Compared with a variety of biological markers, PCT has higher accuracy and specificity and is an ideal marker for infection detection. PCT can be detected 2 hours after infection, reaches a peak 6-12 hours after infection, is in a plateau phase 12-24 hours, and decreases in concentration after 24 hours. The half-life is 25-30 hours, and it can be maintained at a high level for a period of time. Moreover, the PCT level is positively correlated with the degree of infection. The higher the PCT concentration, the more serious the infection. Therefore, the PCT level is of great significance for the diagnosis of the occurrence and development of inflammation and the treatment prognosis. Other inflammatory factors such as IL-6, IL-10, and TNF-α reach a peak 2 hours after infection and decline rapidly, making them difficult to detect; C-reactive protein (CRP) reaches a peak 24-48 hours after infection, and the concentration remains at a high level after inflammation is controlled, which is not conducive to early diagnosis and treatment evaluation. In addition, in clinical diagnosis and treatment, the sensitivity and specificity of traditional detection indicators such as body temperature and white blood cell count (WBC) are not high.

[0004] Currently, PCT can be detected by quantitative, semi-quantitative and qualitative methods. The main quantitative detection methods are electrochemiluminescence (ECLIA) and enzyme-linked immunofluorescence. The most commonly used PCT detection methods are electrochemiluminescence from Roche, Switzerland, enzyme-linked immunofluorescence from BioMerieux, France, colloidal gold colorimetry and chemiluminescence from Thermo Fisher Scientific. Since the above three methods use the same antibodies and preparation standards, their test results are traceable, and there is a high correlation and consistency between the test results.

[0005] The content of PCT in healthy human plasma is extremely low, so PCT detection has high requirements on the sensitivity and affinity of antibodies. The detection sensitivity of existing anti-PCT antibodies and their correlation with Roche's test results need to be improved. Therefore, it is necessary to develop high-affinity and high-sensitivity anti-PCT specific antibodies. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-procalcitonin antibody and its application.

[0007] The present invention provides an anti-procalcitonin antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the anti-procalcitonin antibody or the antigen-binding fragment thereof are as shown in SEQ ID NOs.1-3, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs.4-6.

[0008] SEQ ID NO.1: NYAMN;

[0009] SEQ ID NO.2: EITLESNNYATHYAESVKG;

[0010] SEQ ID NO.3:MINMD;

[0011] SEQ ID NO.4: KSSQSLLYSSNQENYLA;

[0012] SEQ ID NO.5: WASTHES;

[0013] SEQ ID NO.6: HQYYTYPDT.

[0014] The above-mentioned antibodies or antigen-binding fragments thereof can specifically bind to procalcitonin with high affinity.

[0015] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence 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 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.8.

[0016] SEQ ID NO.7:

[0017] EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYAMNWVRQSPEKGLEWVAEITL ESNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTSMINMDWGQG TTLMVSS.

[0018] SEQ ID NO.8:

[0019] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQENYLAWYQQKLGQSPKLLI YWASTHESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYYTYPDTFGGGTKLE IK.

[0020] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0021] In the case of having the heavy chain complementary determining regions CDR1, CDR2, CDR3 and the light chain complementary determining regions CDR1, CDR2, CDR3 described above, the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. The antibodies or antigen-binding fragments thereof corresponding to the amino acid sequences having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in NO.8 are also within the scope of protection of the present invention.

[0022] In the present invention, the antigen-binding fragment refers to a polypeptide comprising a partial sequence fragment of the light chain variable region and / or heavy chain variable region of an antibody, which retains the ability to specifically bind to procalcitonin, or can compete with the full-length antibody for specific binding to procalcitonin.

[0023] Specifically, the antibody or antigen-binding fragment thereof described in the present invention is selected from any one of a monoclonal antibody, Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementary determining region fragment, and a single-chain antibody.

[0024] Among them, monoclonal antibodies include animal-derived antibodies, chimeric antibodies or humanized antibodies.

[0025] In some embodiments of the present invention, the antibody further comprises a heavy chain constant region, and the optional heavy chain type comprises IgG1 type.

[0026] In some embodiments of the present invention, the antibody further comprises a heavy chain constant region, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.11.

[0027] In some embodiments of the present invention, the antibody further comprises a light chain constant region, and the optional light chain type is a κ chain.

[0028] In some embodiments of the present invention, the antibody further comprises a light chain constant region, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.12.

[0029] In some embodiments of the present invention, an anti-procalcitonin antibody is provided, the amino acid sequence of the heavy chain of which is shown in SEQ ID NO.11, and the amino acid sequence of the light chain is shown in SEQ ID NO.12.

[0030] The present invention provides a bispecific antibody or a multispecific antibody comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0031] The present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0032] According to the above-mentioned amino acid sequence and codon rules of the antibody or its antigen-binding fragment, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding and producing the above-mentioned antibody or its antigen-binding fragment are within the protection scope of the present invention.

[0033] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown as SEQ ID NO.9; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO.10.

[0034] The present invention provides a biological material comprising the above-mentioned nucleic acid molecule, wherein the biological material is an expression cassette, a vector or a host cell.

[0035] The above-mentioned nucleic acid molecule can be operably linked to a promoter and / or a terminator to obtain an expression cassette.

[0036] The vectors mentioned above include but are not limited to plasmid vectors, viral vectors, etc.

[0037] The host cells mentioned above include microbial cells or animal cells, wherein microbial cells include but are not limited to Escherichia coli, yeast, etc., and animal cells include but are not limited to insect cells, CHO cells, 293T cells, etc.

[0038] The antibodies or antigen-binding fragments thereof provided by the present invention can be prepared by conventional methods in the art, including chemical synthesis, host expression, etc.

[0039] In some embodiments of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof is introduced into a host cell to obtain a recombinant cell, and the recombinant cell is cultured to obtain the antibody or antigen-binding fragment thereof through separation and purification.

[0040] The present invention provides an antibody conjugate, which is obtained by coupling the above-mentioned antibody or its antigen-binding fragment or the bispecific antibody or multispecific antibody with a marker or protein; the marker is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0041] The present invention provides an antibody composition, which comprises a first antibody and a second antibody, wherein the first antibody is the antibody or antigen-binding fragment thereof described above, and the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the second antibody are as shown in SEQ ID NOs.14-16; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs.17-19.

[0042] Preferably, the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.20, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.21.

[0043] Further preferably, the full-length amino acid sequence of the heavy chain of the second antibody is as shown in SEQ ID NO.22, and the full-length amino acid sequence of the light chain is as shown in SEQ ID NO.23.

[0044] The present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the bispecific antibody or multispecific antibody, the nucleic acid molecule, the biomaterial, the antibody conjugate or the antibody composition:

[0045] (1) Use in the preparation of a product for detecting the presence or level of procalcitonin in a sample;

[0046] (2) Use in the preparation of products for diagnosing bacterial infections;

[0047] (3) Use in detecting the presence or level of procalcitonin in a sample for non-diagnostic and therapeutic purposes;

[0048] (4) Use in the preparation of products for evaluating the occurrence and development of inflammation and / or the prognosis of treatment.

[0049] In the applications described in (1) and (2) above, the product may be a detection reagent or a test kit. The sample described in (1) may be a sample from a living human or animal (including blood, etc.), or may be a sample not from a living human or animal, such as a cell cultured in vitro or a cell culture fluid.

[0050] The samples described in (3) above are samples that are not from living humans or animals, such as in vitro cultured cells, cell culture fluid, procalcitonin standard products, etc.

[0051] In the above applications, the detection method using the antibody or antigen-binding fragment thereof provided by the present invention can be chemiluminescence, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent immunoassay, immunochromatography, etc.

[0052] The present invention provides a detection reagent, which comprises the above-mentioned antibody or antigen-binding fragment thereof, or comprises the bispecific antibody or multispecific antibody, or comprises the antibody conjugate, or comprises the antibody composition.

[0053] The detection reagents described above can be used to detect the presence or level of procalcitonin in a sample, or to diagnose bacterial infection, or to assess the occurrence and development of inflammation and / or the prognosis of treatment.

[0054] In addition to the antibody or antigen-binding fragment thereof or the bispecific antibody or multispecific antibody or the antibody conjugate, the above detection reagent may also contain a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention. The detectable label includes but is not limited to chemiluminescent label, enzyme, colloidal gold, radioisotope, fluorescent dye, etc.

[0055] The above detection reagents may be any immunodiagnostic reagents, including but not limited to chemiluminescence detection reagents, enzyme-linked immunosorbent assay reagents, lateral flow immunochromatography detection reagents, immunofluorescence detection reagents, and the like.

[0056] In some embodiments of the present invention, a chemiluminescent kit for detecting procalcitonin is provided, comprising the antibody or antigen-binding fragment thereof labeled with a chemiluminescent dye.

[0057] The beneficial effects of the present invention are as follows: the present invention provides an antibody specifically targeting procalcitonin, the antibody has a high affinity for procalcitonin protein, can specifically bind to procalcitonin, and has a high sensitivity. The immunoassay reagent developed using the antibody has a high specificity and sensitivity when used for the detection of procalcitonin, and has a high correlation and consistency with the reference detection method, can more accurately reflect the procalcitonin content of the real sample, and has a wide range of application prospects in the field of immunoassay of procalcitonin. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The graphs are the binding curves of the antibody in Example 2 of the present invention with the PCT antigen at gradient concentrations. Among them, 0.5 μg ( / mL) is the binding curve of 50 ng of antigen coated per well (100 μL per well), and 1 μg ( / mL) is the binding curve of 100 ng of antigen coated per well (100 μL per well).

[0060] Figure 2 This is the clinical consistency analysis result of the PCT detection reagent in Example 5 of the present invention. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1 Obtaining anti-procalcitonin monoclonal antibodies

[0063] 1. Design, preparation and carrier coupling of PCT antigen

[0064] The PCT protein sequence was obtained based on the procalcitonin protein sequence on GenBank (accession number: NP_001029124). After immunogenicity, hydrophilicity and surface accessibility analysis, the PCT protein kata region sequence was finally determined as the antigen (SEQ ID NO.13) after screening. The peptide and the synthetic peptide antigen (kata-KLH) with C-terminal coupling to keyhole limpet blood carrier protein KLH were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0065] 2. Immunization of mice

[0066] The synthetic polypeptide protein kata-KLH was mixed with Freund's complete adjuvant at a ratio of 1:1 to 500 μL for emulsification, and then subcutaneously injected into 4 female Balb / c mice aged 6-8 weeks, with an antigen vaccination dose of 50 μg for each mouse. Three weeks later, the antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 to 500 μL for emulsification, and then subcutaneously injected into multiple points, with an antigen vaccination dose of 50 μg for each mouse. After an interval of three weeks, 50 μg of antigen without adjuvant was intraperitoneally injected, for a total of 4 immunizations.

[0067] 3. Immune serum titer determination

[0068] The indirect ELISA method was used to determine the titer of immune serum. 50 μg of synthetic peptide kata-KLH was dissolved in 10 mL of 0.05 M pH 9.6 phosphate buffer and coated on a polystyrene 96-well plate at 100 μL / well at 4°C overnight. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), blocked with 100 μL / well of 10 mM PBS containing 1% BSA blocking solution at 37°C for 2 h, and washed three times with PBS (containing 0.05% (V / V) Tween-20). Blood was collected from the tail vein of mice 10 days after the third immunization, and the mouse immune serum was incubated with 10 mM PBS containing 1% BSA for 10 -2 ~10 -8 The plate was diluted 1:10000, added to a 96-well plate, incubated at 37°C for 1h at 100μL / well, washed three times with PBS (containing 0.05% (V / V) Tween-20), and then added with 100μL / well of horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) at 1:10000, incubated at 37°C for 30min, washed according to the above method, developed with TMB, 100μL / well, incubated at room temperature in the dark for 10min, and terminated with 50μL / well of 2M H2SO4, and the absorbance at 450nm was measured, and the pre-immune mouse serum was used as a negative control, and the ratio of the measured value to the control value ≥2.1 was used as a positive judgment value to determine the immune serum titer. The results are shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072] 4. Preparation of hybridomas

[0073] Take mice with serum titers greater than 1:160000. Three days before fusion, take synthetic peptide kata-KLH and mix it with an equal volume of PBS, and then inject it intraperitoneally into BALB / c mice to be fused at a dose of 50μg / 500μL per mouse for booster immunization. Take the spleen of the mouse aseptically, make a spleen cell suspension and mix it with the mouse myeloma cell line SP2 / 0 in the logarithmic growth phase at a ratio of 1:1, centrifuge at 1000g for 5 minutes at room temperature, discard the supernatant, flick the bottom of the centrifuge tube with your fingers to loosen the precipitate, place the centrifuge tube in a 37℃ water bath, and add 50% polyethylene glycol (PEG, MW4000, Sigma) kept in a 37℃ water bath drop by drop into the centrifuge tube with a dropper, shake the centrifuge tube while dropping, and drip it within 1 minute. After dripping, After standing for 2 minutes, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 10 mL of serum-free 1640 medium preheated at 37°C were added every 1 minute to terminate the effect of polyethylene glycol. The cell mixture was centrifuged at 1000g for 5 minutes at room temperature, the supernatant was discarded, and HAT culture medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) was added to gently resuspend the cells, and the cells were divided into 96-well plates, 200 μL per well. After three days of culture, the cell fusion was observed, and half of the HAT culture medium was replaced for several consecutive days until clones were formed. Seven days after fusion, HT culture medium (hypoxanthine (H) and thymidine (T) (HT, Sigma)) was replaced for culture.

[0074] 5. Screening of hybridoma cells secreting anti-PCT-kata protein monoclonal antibodies

[0075] The cell culture supernatant was screened by indirect ELISA, and the positive cloned hybridoma cells with higher titers were selected for subcloning, and cloned 2-3 times continuously by limiting dilution method until the cell positive rate reached 100%, and finally a stable anti-PCT-kata monoclonal antibody secreting cell line was obtained, which was labeled as C2F9. The cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

[0076] 6. Preparation and purification of ascites

[0077] Hybridoma C2F9 cells were cultured at 1×10 6 / mouse was injected into the peritoneal cavity of 8-10 week old BALB / c female mice pretreated with liquid paraffin, and after 10-14 days of feeding and observation, ascites was extracted when the abdomen of the mice was swollen. Monoclonal antibody C2F9 was purified by affinity chromatography Protein G Sepharose Fast Flow, and the purity of the monoclonal antibody was determined by SDS-PAGE, which was more than 90%.

[0078] Example 2 Performance test of anti-procalcitonin monoclonal antibody

[0079] 1. Determination of antibody concentration

[0080] The ascites prepared by hybridoma cell C2F9 was purified to obtain anti-PCT-kata protein monoclonal antibody C2F9, and its concentration was determined by Nanodrop nucleic acid protein assay produced by Thermofisher, and was >1 mg / mL.

[0081] 2. Antibody subtype identification

[0082] The subtype of hybridoma cell line was identified by using Thermofisher's mouse monoclonal antibody subtype identification kit. The subtype of antibody secreted by C2F9 was IgG1, and the light chain was κ chain.

[0083] 3. Identification of the potency of purified antibodies

[0084] 50 μg of synthetic PCT-kata protein peptide was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6 and added to a 96-well plate, 100 μL per well, at 4°C overnight. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), and then blocked at 37°C for 2h with 10mM PBS containing 1% BSA blocking solution 150μL / well. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), and 100μL of purified antibody diluted in gradient was added to each well, and incubated at 37°C for 1h. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), and goat anti-mouse IgG polyclonal antibody labeled with horseradish peroxidase was added as the secondary antibody, and incubated at 37°C for 30min. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), and 100μL TMB colorimetric solution was added to each well. After incubation at 37°C for 15min, 2MH2SO4 solution was added to terminate the reaction, and the microplate reader was used to detect the absorbance at 450nm. Multiple results showed that the titer of the antibody could reach 1×10 6 .

[0085] 4. Affinity test:

[0086] PCT-kata antigen protein was diluted to 0.5μg / mL and 1μg / mL with 1×CB, and added to the wells of the ELISA plate at a volume of 100μL / well, and duplicated, and placed at 4℃ overnight or 37℃ for adsorption for 2 hours. The coated microplate was dried, washed once according to the operating procedure set by the plate washer, and the blocking solution was added at a volume of 200μL / well, placed in a 37℃ incubator for 2h, and then placed at 4℃ overnight. Before use, the blocked microplate was taken out from 4℃, dried, and the cleaning solution (1×PBS-T) was added to wet the ELISA plate; the monoclonal antibody C2F9 was pre-diluted to 30μg / mL with 1×PBS, and the pre-dilution multiple m was recorded. The dilution of 10 times, that is, 3μg / mL, was taken as the highest concentration (S1), and then diluted in a 1:3 gradient (diluted in a 96-deep well plate), with a total of 8 dilution gradients (S1-S8).

[0087] Add 100 μL of diluted monoclonal antibody C2F9 to a 96-well microplate that has been patted clean on absorbent paper and incubate at 37°C for 30 min. After incubation, spin dry the ELISA plate and pat dry on absorbent paper. Wash the plate three times with a plate washer. Add 100 μL of 1×PBS to each well in columns 1-4. Add 200 μL of urea treatment solution to each well in columns 5 and 6. Incubate at 37°C for 30 min. Spin dry the ELISA plate and pat dry on absorbent paper. Pat dry, wash the ELISA plate 3 times with a plate washer, add 100μL of GAM-HRP enzyme-labeled secondary antibody diluted 10000 times with secondary antibody diluent to each well, incubate at 37℃ for 30min; after incubation, spin dry the ELISA plate, pat dry on absorbent paper, wash the ELISA plate 3 times with a plate washer, take TMB color development solution, add 100μL color development solution to each well, incubate at 37℃ for 5-10min; after color development, add 50μL stop solution to each well. Set the ELISA reader to read at 450nm / 630nm.

[0088] The ELISA antigen-antibody binding experiment was tested under the conditions of 0.5μg / mL and 1μg / mL antigen coating, and the data were obtained by testing the absorbance value OD ( Figure 1 ), and the corresponding polynomial curve was fitted. The corresponding binding showed obvious gradient with the change of antibody dilution ratio. At the same time, the affinity binding reflected by the two curves under the conditions of 0.5μg / mL and 1μg / mL antigen coating reflected the specificity of binding and reflected that the concentration corresponding to the antibody binding force reached 1.45E-9mol / L (under the condition of 0.5μg / mL antigen coating, the highest OD reading was 50%, that is, the concentration of antigen-antibody complex accounted for half of the total concentration, and the antibody concentration value K 0.5 ) and 9.08E-10 mol / L (the concentration value K1 corresponding to 50% of the highest OD reading under 1 μg / mL antigen coating conditions, that is, when the concentration of the antigen-antibody complex accounts for half of the total concentration).

[0089] 5. Specificity detection

[0090] The specificity of monoclonal antibody C2F9 was detected by referring to the ELISA antibody titer detection method. The N-terminal recombinant protein of PCT, recombinant protein of CT, and recombinant protein of CRP were used for detection. The results showed negative, and PCT-kata protein was positive, indicating that the monoclonal antibody C2F9 had good specificity.

[0091] Example 3 Sequence determination of anti-procalcitonin monoclonal antibody

[0092] The monoclonal antibody C2F9 against PCT-kata protein prepared by the purified hybridoma cell C2F9 was sequenced for the variable region of the antibody light chain and the variable region of the heavy chain. The sequencing results showed that the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 were as shown in SEQ ID NO.1-3; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 were as shown in SEQ ID NO.4-6. The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, the amino acid sequence of the full length of the heavy chain is as shown in SEQ ID NO.11, and the amino acid sequence of the full length of the light chain is as shown in SEQ ID NO.12. The antibodies with the above variable region sequences have high affinity and good specificity, so the corresponding antibody variable regions can be used for the development of recombinant antibodies, single-chain antibodies, bispecific antibodies, and for the development of related products for diagnostic purposes.

[0093] Example 4 Development of a chemiluminescent kit using anti-procalcitonin monoclonal antibody C2F9

[0094] The anti-procalcitonin monoclonal antibody C2F9 can be used for the development of immunodiagnostic reagents such as enzyme-linked immunosorbent assay, chemiluminescence, lateral flow immunochromatography and immunofluorescence detection. This embodiment takes a chemiluminescent reagent as an example. The anti-PCT-kata protein monoclonal antibody C2F9 is used as a labeled antibody, and other high-quality anti-PCT antibodies are used as coating antibodies. In this embodiment, the optimal paired antibody D1B6 of C2F9 obtained by screening (the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.14-16; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.17-19. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.20, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21, the amino acid sequence of the full length of the heavy chain is shown in SEQ ID NO.22, and the amino acid sequence of the full length of the light chain is shown in SEQ ID NO.23) is used as a coating antibody, and a double antibody sandwich chemiluminescence kit is finally prepared.

[0095] 1. Coating of magnetic particles

[0096] The coated antibody D1B6 and magnetic particles were bound by covalent bonds: 1 μm carboxyl magnetic beads were activated with EDC and NHS, washed with MES buffer at pH 5.0, and coated antibodies pre-dialyzed in MES buffer at pH 5.0 were added to react, then washed with MES buffer at pH 5.0, blocked with BSA and set aside.

[0097] 2. Labeling of acridinium

[0098] The labeled antibody C2F9 and acridinium amide are covalently bonded: first, the antibody C2F9 is dialyzed into a PBS buffer solution of pH 8.5, and acridinium amide containing an NHS group is added and reacted at a ratio of 1:10. After the reaction is completed, the acridinium ester is dialyzed and set aside.

[0099] 3. Optimization of reaction system

[0100] Orthogonal experiments were used to screen the concentrations of magnetic particles and acridinium markers to determine the reaction concentration, and the reaction system was optimized to determine the specific formula and pH.

[0101] 4. Traceability of PCT reference materials

[0102] The quantity relationship of the standard substance is traced back to the measured value of the Roche test kit to form the unit quantity value of the standard substance.

[0103] 5. Test kit operation

[0104] The detection system was tested using a fully automatic chemiluminescence instrument, with a sample volume of 15 μL and a detection reagent volume of 50 μL each, reacted for 10 minutes, and then added with excitation solution and pre-excitation solution for luminescence testing. The luminescence signal was positively correlated with the PCT content of the sample to be tested.

[0105] Example 5 Performance Evaluation of Chemiluminescent Kit

[0106] 1. Detection limit

[0107] The S0 zero value sample was tested 20 times continuously and the LOB of the kit was calculated. The results are shown in Table 2, and the sensitivity is 0.0035 ng / mL.

[0108] Table 2

[0109]

[0110] 2. Accuracy (recovery experiment)

[0111] The high-value sample (liquid A) was added to the low-value sample (liquid B), and the test was repeated 3 times. The average value was taken and the recovery rate was calculated according to formula (1).

[0112] R=(C×(V0+VS )-C0×V0) / (VS×CS )×100% Formula (1)

[0113] Where:

[0114] R——recovery rate;

[0115] C——the average value of the detected concentration after adding liquid A to liquid B;

[0116] V0——volume of liquid B;

[0117] VS——volume of liquid A;

[0118] C0——average value of concentration of liquid B;

[0119] CS——Concentration of liquid A.

[0120] The results are shown in Table 3, and the recovery rate R is 93.13%.

[0121] Table 3

[0122]

[0123] 3. Linear

[0124] The high-value samples close to the upper line of the linear interval were diluted to 5 concentrations in a certain proportion, among which the low-value sample concentration was close to the lower limit of the linear interval, and each sample concentration was repeated twice. The concentrations of the 7 samples were as follows: L, 0.833L+0.167H, 0.667L+0.333H, 0.5L+0.5H, 0.333L+0.667H, 0.167L+0.833H, H. The average value of the measured concentration was linearly fitted with the theoretical concentration, and the linear correlation coefficient r was obtained.

[0125] The results are shown in Table 4, and the linear correlation coefficient r is 0.9967.

[0126] Table 4

[0127]

[0128] 4. Repeatability

[0129] Each sample was tested 10 times, and the CV value of each result was calculated.

[0130] The results are shown in Table 5.

[0131] Table 5

[0132]

[0133] 5. Precision

[0134] Experimental requirements: The coefficient of variation of the test results should not be greater than 15%.

[0135] Experimental method: Repeat the test 5 times a day for 5 consecutive days, and obtain 25 data points for each sample. Calculate the total imprecision.

[0136] The results are shown in Tables 6 and 7.

[0137] Table 6

[0138]

[0139]

[0140] Table 7

[0141]

[0142] 6. HOOK

[0143] The HOOK (1000 ng / mL) sample was tested three times, and the average value of the test results was higher than the linear range of the standard curve. The results are shown in Table 8.

[0144] Table 8

[0145]

[0146] 7. Clinical consistency

[0147] Take at least 40 samples, with both high and low values, and perform consistency analysis with the reference reagent (Roche cobas). Figure 2 As shown, it is shown that the detection reagent prepared by the antibody C2F9 of the present application has a high consistency with the serum of the reference reagent.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-procalcitonin antibody or an antigen-binding fragment thereof, characterized in that The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs.1-3, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence 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 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is selected from any one of monoclonal antibody, Fab, Fab', F(ab')2, Fv, and single-chain antibody.

4. A bispecific antibody or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO.

10.

7. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule according to claim 5 or 6, and the biological material is an expression cassette, a vector or a host cell.

8. An antibody conjugate, characterized in that It is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the bispecific antibody or multispecific antibody according to claim 4 with a marker or protein; The label is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

9. An antibody composition, characterized in that The antibody composition comprises a first antibody and a second antibody, wherein the first antibody is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the second antibody are shown in SEQ ID NOs.14-16; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs.17-19.

10. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the bispecific antibody or multispecific antibody according to claim 4, the nucleic acid molecule according to claim 5 or 6, the biomaterial according to claim 7, the antibody conjugate according to claim 8, or the antibody composition according to claim 9: (1) Use in the preparation of a product for detecting the presence or level of procalcitonin in a sample; (2) Use in the preparation of products for diagnosing bacterial infections; (3) Use in detecting the presence or level of procalcitonin in a sample for non-diagnostic and non-therapeutic purposes; (4) Use in the preparation of products for evaluating the occurrence and development of inflammation and / or the prognosis of treatment.

11. A detection reagent, characterized in that: The detection reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or comprises the bispecific antibody or multispecific antibody according to claim 4, or comprises the antibody conjugate according to claim 8, or comprises the antibody composition according to claim 9.

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