CN115873112A - Procalcitonin antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of antibodies, and particularly relates to a procalcitonin antibody and application thereof. The amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or the antigen binding fragment thereof provided by the invention are shown in SEQ ID NO.1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6. The antibody has high affinity to procalcitonin protein, can specifically bind procalcitonin, and has high sensitivity. The immunodetection reagent developed by using the antibody has higher specificity and sensitivity when being used for procalcitonin detection, has higher consistency with a reference detection method, can accurately reflect the procalcitonin content of a real sample, and has wide application prospect.

Description

Procalcitonin antibody and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a procalcitonin antibody and application thereof.

Background

Procalcitonin (PCT) is a precursor of Calcitonin (CT), a hormone-free glycoprotein of 116 amino acids with a molecular weight of 13KD, and is encoded by the CALC-I gene located on chromosome 11. PCT is composed of calcitonin (katacalcin), calcitonin and N-terminal residue fragments, and under the action of special protease, the N-terminal will be sheared at the 57 th position to generate the N-terminal residue at the 1-57 th position and the 60-116 th amino acid containing calcitonin, and at the same time, will also be sheared by protease at the 91 th position to form calcitonin at the 60-91 th position and calcitonin at the 96-116 th position.

PCT is a specific marker of bacterial, fungal infections found in the nineties of the last century. In the absence of infection, the extra-thyroid CALC-I gene expression is suppressed, mainly to a limited extent in the neuroendocrine cells of the thyroid and lungs, and thus the PCT levels in healthy human blood are very low, usually below 0.10ng/mL. When the organism suffers from serious fungal, bacterial and parasitic infection, sepsis, multi-organ function failure and the like, CALC-I gene expression of various cell types of various tissues of the whole body can be induced, so that the PCT concentration is rapidly increased and even reaches hundreds to ten thousands of times of the normal level, but the CT content is kept unchanged or slightly increased. The PCT concentration in the serum of patients with local infection, virus infection, allergy, autoimmune disease, chronic nonspecific inflammation and the like is not increased or slightly increased, so that the PCT has high specificity, can be used as a specific index of bacterial infection, and is used for differential diagnosis of bacterial diseases.

PCT can be detected 2 hours after infection, reaches a peak value 6-12 hours after infection, takes 12-24 hours as a plateau phase, and decreases in concentration after 24 hours, takes a half-life period of 25-30 hours, and can be maintained at a high level for a period of time. Meanwhile, the PCT level is positively correlated with the infection degree, and the higher PCT concentration means the more serious infection degree, so the PCT level has important significance on the aspects of diagnosis of the occurrence and development of inflammation, treatment prognosis and the like. While other inflammatory factors such as IL-6, IL-10, TNF-alpha reach the peak value 2 hours after infection and decline rapidly, and are not easy to detect; c-reactive protein (CRP) peaks 24-48 hours after infection and remains at a high level after inflammation control, which is not conducive to early diagnosis and treatment evaluation. In clinical diagnosis and treatment, the sensitivity and specificity of traditional detection indexes such as body temperature and white blood cell count (WBC) are not high, and PCT has more ideal accuracy and specificity compared with the prior biological markers, is an ideal infection detection marker, and is helpful for clinical early diagnosis and reasonable treatment scheme formulation.

In view of the above, there is a great need for mature PCT detection reagents and methods for diagnosing bacterial or non-bacterial inflammatory responses, differentially diagnosing the severity of sepsis and systemic inflammatory responses, preventing antibiotic abuse, and the like. PCT is currently detected by quantitative, semi-quantitative and qualitative methods. The quantitative detection method mainly comprises an electrochemiluminescence method (ECLIA) and an enzyme-linked immunosorbent assay. The detection specificity, sensitivity and precision of the electrochemical luminescence method and the enzyme-linked immunosorbent assay can reach higher levels. The qualitative detection method is mainly an immunochromatographic method and is commonly used for point-of-care testing (POCT), and is characterized by small and convenient machine and short sample turnover time, but the precision of the method is relatively low. The most commonly used PCT detection methods at present are the electrochemiluminescence method of Rogowski, switzerland, the enzyme-linked immunofluorescence method of Merrier, france, the colloidal gold colorimetric method and the chemiluminescence method of Saimer. Because the same antibody and preparation standard are used in the above 3 methods, the detection results are traceable, and the detection results have higher correlation and consistency.

The antibody is a key influencing factor of the detection effect of the immunological method, and the content of PCT in the plasma of healthy people is extremely low, so that the PCT detection has higher requirements on the sensitivity, affinity and other properties of the antibody. The detection sensitivity of the existing anti-PCT antibody and the correlation between the detection sensitivity and the detection result of Rogowski company are all to be improved. Therefore, an anti-PCT specific antibody with high affinity and high sensitivity needs to be developed, a key raw material is provided for the immunodetection of PCT, and the method has important significance for expanding the clinical application range of PCT detection and reducing the medical cost.

Disclosure of Invention

The invention aims to provide a procalcitonin antibody and application thereof.

In a first aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof are set forth in SEQ ID No. 1-3; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6.

SEQ ID NO.1：SYWMN；

SEQ ID NO.2：WINTNTGRPTYAEDFKG；

SEQ ID NO.3：GGKYGVWFAR；

SEQ ID NO.4：KSSQSLLYSNNQDNYLA；

SEQ ID NO.5：WASTGRS；

SEQ ID NO.6：QQGFGYPST。

The antibody or the antigen binding fragment thereof can be specifically bound with procalcitonin and has high affinity.

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; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8 or has at least 80 percent of similarity with the amino acid sequence shown as SEQ ID NO. 8.

SEQ ID NO.7：

QIHLVQSGPELKKPGETVKISCKASGYTFTSYWMNWVKQTPGKDLKWMGWIN TNTGRPTYAEDFKGRFAFSLETSANTAYLQIDSLKNDDTATYFCARGGKYGVWFAR WGQGTLVTVSA。

SEQ ID NO.8：

DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQDNYLAWYQQKPGQSPKLLI YWASTGRSGVPDRFTGGGSGTDFTLTISGVKAEDLAIYYCQQGFGYPSTFGGGTKLE IR。

In some embodiments of the invention, the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID No.7 and SEQ ID No.8, respectively.

In the case of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and the light chain complementarity determining regions CDR1, CDR2, CDR3 described above, the antibody or antigen-binding fragment thereof having 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 set forth in SEQ ID No.7 and an amino acid sequence corresponding to 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 set forth in SEQ ID No.8 are also within the scope of the present invention.

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

Specifically, the antibody or antigen-binding fragment thereof of the present invention is selected from any one of a monoclonal antibody, fab ', F (ab') 2, fd, fv, dAb, a complementarity determining region fragment, and a single-chain antibody.

The monoclonal antibody includes an animal-derived antibody, a chimeric antibody, a humanized antibody, and the like.

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

In some embodiments of the invention, the antibody further comprises a heavy chain constant region, wherein the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID No. 11.

In some embodiments of the invention, the antibody further comprises a light chain constant region, optionally the light chain type is a kappa chain.

In some embodiments of the invention, the antibody further comprises a light chain constant region, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID No. 12.

In some embodiments of the invention there is provided an anti-procalcitonin antibody having the amino acid sequence of the heavy chain as shown in SEQ ID No.11 and the amino acid sequence of the light chain as shown in SEQ ID No. 12.

In a second aspect, the invention provides a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof described above.

In a third aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above.

The nucleotide sequence of the nucleic acid molecule encoding the above antibody or antigen-binding fragment thereof can be obtained by those skilled in the art based on the amino acid sequence and codon rules of the above antibody or antigen-binding fragment thereof. Due to the degeneracy of the codons, the nucleotide sequence of the nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof is not unique, and all nucleic acid molecules capable of encoding the antibody or the antigen-binding fragment thereof are within the scope of the present invention.

In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule encoding the variable region of the heavy chain is as set forth in SEQ id No. 9; the nucleotide sequence of the nucleic acid molecule for encoding the variable region of the light chain is shown in SEQ ID NO. 10.

In a fourth aspect, the present invention provides a biological material comprising a nucleic acid molecule as described above, said biological material being an expression cassette, a vector or a host cell.

The nucleic acid molecule described above may be operably linked to a promoter and/or a terminator to provide an expression cassette.

The above-mentioned vectors include, but are not limited to, plasmid vectors, viral vectors and the like.

The above-mentioned host cell includes a microbial cell or an animal cell. Wherein the microbial cells include but are not limited to Escherichia coli, yeast, etc., and the animal cells include but are not limited to insect cells, CHO cells, 293T cells, etc.

The antibody or antigen-binding fragment thereof provided by the invention can be prepared by adopting a conventional method in the field, and comprises the following steps: chemical synthesis, host expression, etc.

In some embodiments of the invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof is introduced into a host cell to obtain a recombinant cell, the recombinant cell is cultured, and the antibody or antigen-binding fragment thereof is isolated and purified.

In a fifth aspect, the present invention provides an antibody conjugate obtained by conjugating the above-described antibody or antigen-binding fragment thereof or the bispecific antibody or multispecific antibody with a label or protein; the label is selected from one or more of chemiluminescence dye label, enzyme label, biotin label, fluorescent dye label, colloidal gold label and radioactive label.

In a sixth aspect, the present invention provides an antibody composition comprising 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 sequence of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of the second antibody is represented by SEQ ID nos. 14-16; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 17-19. Preferably, the amino acid sequence of the heavy chain variable region of the second antibody is represented by SEQ ID No.20, and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 21.

Further preferably, the amino acid sequence of the full length of the heavy chain of the second antibody is shown as SEQ ID No.22, and the amino acid sequence of the full length of the light chain is shown as SEQ ID No. 23.

In a seventh aspect, the invention provides any one of the following uses of the antibody or antigen binding fragment thereof described above or the bispecific antibody or multispecific antibody or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition:

(1) Use in the manufacture of a product for detecting the presence or level of procalcitonin in a sample;

(2) Use in the manufacture of a product for the diagnosis of bacterial infections;

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

(4) The application of the composition in preparing products for evaluating the occurrence, development and/or treatment prognosis of inflammation.

In the applications of (1) and (2), the product can be a detection reagent or a kit. (1) The sample mentioned in (1) may be a sample (including blood or the like) derived from a living human or an animal, or may be a sample not derived from a living human or an animal, such as a cell cultured in vitro or a cell culture solution.

The sample described in the above (3) is a sample not derived from a living human or animal, for example: cells cultured in vitro, cell culture solution, procalcitonin standard substance, etc.

In the above applications, the method for detecting the antibody or the antigen-binding fragment thereof provided by the present invention may be a chemiluminescence method, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, a fluorescence immunoassay, an immunochromatography method, and the like.

In an eighth aspect, the invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof described above, or comprising the bispecific or multispecific antibody, or comprising the antibody conjugate, or comprising the antibody composition.

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

The above-described detection reagents may comprise, in addition to the antibody or antigen-binding fragment thereof or the bispecific or multispecific antibody or the antibody conjugate, a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention. Detectable labels include, but are not limited to, chemiluminescent labels, enzymes, colloidal gold, radioisotopes, fluorescent dyes, and the like.

The detection reagent can be any immunodiagnostic reagent, including but not limited to a chemiluminescent detection reagent, an enzyme-linked immunoassay detection reagent, a lateral flow immunochromatographic detection reagent, an immunofluorescence detection reagent, and the like.

In some embodiments of the invention there is provided a chemiluminescent kit for the detection of procalcitonin comprising said antibody or antigen-binding fragment thereof coated with magnetic microparticles.

The invention has the beneficial effects that: the invention provides an antibody of a specific targeting procalcitonin, which has higher affinity to procalcitonin protein, can specifically bind procalcitonin and has higher sensitivity. The immunoassay reagent developed by the antibody has higher specificity and sensitivity when being used for detecting procalcitonin, has higher correlation and consistency with a reference detection method, can accurately reflect the procalcitonin content of a real sample, and has wide application prospect in the field of immunoassay of procalcitonin.

Drawings

In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.

FIG. 1 is a graph showing the binding curves of the antibodies of example 2 of the present invention with PCT antigens respectively under gradient concentrations. Where 0.5. Mu.g (/ mL) is the binding curve for 50ng of antigen coated per well (100. Mu.L per well) and 1. Mu.g (/ mL) is the binding curve for 100ng of antigen coated per well (100. Mu.L per well).

FIG. 2 shows the results of the clinical consistency analysis of the PCT test agent of example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 Procalcitonin monoclonal antibody obtaining

1. Design, preparation and carrier coupling of PCT antigen

PCT protein sequence was obtained from the procalcitonin protein sequence on GenBank (accession number: NP-001029124). Through analysis of immunogenicity, hydrophilicity and hydrophobicity and surface accessibility, a CT region sequence (SEQ ID NO. 13) of PCT protein is finally determined to be used as an antigen through screening, and the peptide and a synthetic peptide (CT-KLH) antigen of a C-end coupling keyhole limpet hemocyanin KLH are synthesized by biological engineering (Shanghai) corporation.

2. Immunization of mice

The synthetic polypeptide protein CT-KLH and Freund complete adjuvant are mixed uniformly according to the ratio of 1 to 500 mu l for emulsification, 4 female Balb/c mice with the age of 6-8 weeks are injected subcutaneously in a multi-point mode, the antigen inoculation dose of each mouse is 50 mu g, the antigen and Freund incomplete adjuvant are mixed uniformly according to the ratio of 1 to 500 mu l for emulsification after three weeks, subcutaneous multi-point injection is carried out, the antigen inoculation dose of each mouse is 50 mu g, 50 mu g of antigen without adjuvant is injected intraperitoneally after three weeks, and the immunization is carried out for 4 times in total.

3. Immune serum potency assay

The immune serum titer is determined by an indirect ELISA method. 50 μ g of synthetic peptide CT-KLH was dissolved in 10mL of 0.05MpH 9.6 phosphate buffer, coated with a polystyrene 96-well plate at 100 μ L/well, overnight at 4 ℃. Washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), 1% BSA blocking solution (1%) with 10mM PBS00. Mu.L/well), blocked at 37 ℃ for 2h, and the plate washed three times with PBS (containing 0.05% (V/V) Tween-20); the tail vein of the mice was collected 10 days after the third immunization, and the mouse immune sera were subjected to 10-day-old concentration in 1% BSA-10mM PBS ^-2 ～10 ^-8 Fold dilution, add 96-well plate, 100 μ L/well incubation at 37 ℃ for 1h, after three washes of PBS (containing 0.05% (V/V) Tween-20), add 1:10000 times diluted horse radish peroxidase labeled goat anti-mouse IgG (Sigma, INC.), 100 μ L/well incubated at 37 deg.C for 30min, washing the plate by the same method, developing with TMB, 100 μ L/well, keeping out of the sun at room temperature for 10min, adding 50 μ L/well 2M H ₂ SO ₄ Stopping reaction, detecting the absorption value of 450nm, taking the serum of the mouse before immunization as negative control, and taking the ratio of the measured value to the control value to be more than or equal to 2.1 as a positive judgment value to determine the titer of the immune serum. The results are shown in Table 1.

TABLE 1

4. Preparation of hybridomas

Mice with serum titer greater than 1. Taking the spleen of a mouse aseptically, preparing a spleen cell suspension and a mouse myeloma cell strain SP2/0 in a logarithmic growth phase according to the ratio of 1:1, centrifuging for 5min at 1000g room temperature, discarding the supernatant, flicking the bottom of the centrifuge tube with a finger to loosen the precipitate, placing the centrifuge tube in a 37 ℃ water bath, dropping 50% polyethylene glycol (PEG, MW4000, sigma) kept in the 37 ℃ water bath into the centrifuge tube with a dropper, shaking the centrifuge tube while dropping for 1min, standing for 2min after dropping, adding serum-free 1640 medium preheated at 37 ℃ every 1min to stop the action of the polyethylene glycol, centrifuging the cell mixture for 5min at 1000g room temperature, discarding the supernatant, adding HAT medium (hypoxanthine (H), aminopterin (a) and thymidine (T) (HAT, sigma)) to gently resuspend the cells, and dividing the cells into 96-well plates with 200 μ L per well. After three days of culture, cell fusion was observed, half of HAT medium was replaced for several days until colonies were formed, and HT medium (hypoxanthine (H) and thymidine (T) (HT, sigma)) was replaced seven days after fusion.

5. Screening hybridoma cells secreting anti-PCT-CT protein monoclonal antibodies

Screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 2-3 times by a limiting dilution method until the cell positive rate reaches 100%, and finally obtaining a cell strain which stably secretes the anti-PCT-CT protein monoclonal antibody and is marked as D1B6. And (5) performing liquid nitrogen freezing storage on the cells with the positive rate reaching 100% after cloning and culturing.

6. Preparation and purification of ascites fluid

Hybridoma cell D1B6 at 1X 10 ⁶ Injecting liquid paraffin pretreated BALB/c female mouse abdominal cavity of 8-10 weeks old, feeding and observing for 10-14 days, and extracting ascites when mouse abdominal cavity is enlarged. The monoclonal antibody D1B6 is obtained by purifying Protein G Sepharose Fast Flow by affinity chromatography, and the purity of the monoclonal antibody is determined by SDS-PAGE and reaches more than 90 percent.

Example 2 Performance testing of Procalcitonin monoclonal antibodies

1. Determination of antibody concentration

Ascites fluid prepared from the hybridoma cell D1B6 was purified to obtain a PCT-CT protein monoclonal antibody D1B6, and the concentration thereof was measured by a Nanodrop nucleic acid protein measuring instrument manufactured by Thermofisor company, and was >1mg/mL.

2. Antibody subtype identification

The subtype of the hybridoma cell strain is identified by adopting a mouse monoclonal antibody subtype identification kit of Thermofish, the subtype of the D1B6 secretion antibody is IgG1 type, and the light chain is kappa chain.

3. Potency assay for purified antibodies

Mu.g of synthetic PCT-CT protein peptide was dissolved in 10mL of 0.05M carbonate-coated buffer pH 9.6, and added to a 96-well plate at 100. Mu.L per well overnight at 4 ℃. PBS (containing 0.05% (V/V) Tween-20) three times, 10mM PBS 1% BSA blocking solution (150. Mu.L/well), 37 ℃ blocking for 2h, PBS (containing 0.05% (V/V) Tween)-20) washing the plate three times, adding 100. Mu.L of purified antibody per well, incubating at 37 ℃ for 1h, washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), adding horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody as a secondary antibody, incubating at 37 ℃ for 30min, washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), adding 100. Mu.L of TMB developing solution per well, incubating at 37 ℃ for 15min, and adding 2M H ₂ SO ₄ The solution is stopped and detected by a microplate reader at the absorbance value of 450 nm. Multiple results show that the titer of the antibody can reach 1 x 10 ⁶ 。

4. Affinity assay

PCT-CT antigen protein is respectively diluted to 0.5 mu g/mL and 1 mu g/mL by 1 XBB, added into the hole of an enzyme-labeled plate by the dosage of 100 mu L/hole, and is subjected to hole duplication and is placed at 4 ℃ overnight or is adsorbed at 37 ℃ for 2 hours. Spin-drying the coated microporous plate, washing once according to the operating program set by a plate washer, adding a sealing solution according to the dosage of 200 mu L/hole, placing in an incubator at 37 ℃ for 2h, and then placing at 4 ℃ overnight. Before use, the sealed microporous plate is taken out from 4 ℃, dried, and added with cleaning solution (1 XPBS-T) to moisten the ELISA plate; monoclonal antibody D1B6 was pre-diluted to 30 μ g/mL with 1 × PBS, and the fold m of the pre-dilution was recorded, and the dilution by 10 times, i.e., 3 μ g/mL, was taken as the maximum concentration (S1) and then further diluted at 1:3 dilution gradients (dilutions in 96-well plates) for a total of 8 dilution gradients (S1-S8).

Adding 100 mu L of diluted monoclonal antibody D1B6 into a 96-hole microporous plate which is cleanly patted on absorbent paper, and incubating for 30min at 37 ℃; after incubation, the ELISA plate is dried by spin-drying, the plate is dried on absorbent paper, the ELISA plate is washed for 3 times by a plate washing machine, and 100 mu L of 1 XPBS is added into each hole of 1-4 rows; adding 200 μ L urea treatment solution into each well of 5 rows and 6 rows, and incubating at 37 deg.C for 30min; spin-drying the ELISA plate after incubation, drying the ELISA plate on absorbent paper, washing the ELISA plate for 3 times by using a plate washing machine, adding 100 mu L of GAM-HRP enzyme-labeled secondary antibody which is diluted by 10000 times by using secondary antibody diluent in advance into each hole, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, drying the ELISA plate on absorbent paper, washing the ELISA plate for 3 times by using a plate washing machine, adding 100 mu L of color developing solution into each hole of TMB color developing solution, and incubating for 5-10min at 37 ℃; after development, 50. Mu.L of stop buffer was added to each well. The reading at 450nm/630nm was set on a microplate reader.

Are respectively atELISA antigen-antibody binding force experiments are tested under the conditions of 0.5 mu g/mL and 1 mu g/mL antigen coating, data (figure 1) are obtained by testing light absorption values OD, corresponding polynomial curves are obtained by fitting, corresponding binding can see obvious gradient along with the change of antibody dilution multiple ratio, meanwhile, affinity binding reflected by the two curves under the conditions of 0.5 mu g/mL and 1 mu g/mL antigen coating reflects binding specificity, and the concentration corresponding to the antibody binding force respectively reaches 1.84E-10mol/L (under the condition of 0.5 mu g/mL antigen coating, the highest OD reading is 50%, namely when the concentration of an antigen-antibody compound accounts for half of the total concentration, the concentration value K corresponding to the antibody is reflected _0.5 ) And 4.46E-10mol/L (50% of the highest OD reading under 1. Mu.g/mL antigen coating, i.e., the concentration of antigen-antibody complex is half of the total concentration), the concentration value K corresponding to the antibody ₁ )。

5. Specificity detection

The specificity of the monoclonal antibody D1B6 is detected by referring to an ELISA antibody titer detection method, and the detection is carried out by using the N-terminal recombinant protein, kata recombinant protein, CRP recombinant protein and the like of PCT, and the result shows that the specificity of the monoclonal antibody D1B6 is good, and the PCT-CT protein is positive.

Example 3 sequencing of Procalcitonin monoclonal antibodies

Sequencing a variable region of a light chain and a variable region of a heavy chain of an antibody by using a monoclonal antibody D1B6 of the PCT-CT protein prepared by the purified hybridoma cell D1B6, wherein the sequencing result shows that the amino acid sequences of CDR1, CDR2 and CDR3 in the complementary determining regions of the heavy chain are shown as SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain complementarity determining region are shown in SEQ ID NO. 4-6. The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, the amino acid sequence of the full length of the heavy chain is shown as SEQ ID NO.11, and the amino acid sequence of the full length of the light chain is shown as SEQ ID NO. 12. The antibody with the variable region sequence has high affinity and good specificity, so the corresponding antibody variable region can be used for the development of recombinant antibodies, single-chain antibodies and bispecific antibodies and the development of related products for diagnostic use.

Example 4 development of a chemiluminescent kit Using procalcitonin monoclonal antibody D1B6

The procalcitonin monoclonal antibody D1B6 can be used for development of immunodiagnosis reagents such as enzyme-linked immunosorbent assay, chemiluminescence assay, lateral flow immunochromatography assay, immunofluorescence assay and the like. This example exemplifies the development of a chemiluminescent kit. And (3) taking the monoclonal antibody D1B6 as a coating antibody, and matching with other high-quality anti-PCT antibodies as a labeled antibody to finally prepare the double-antibody sandwich method chemiluminescence kit.

1. Coating of magnetic particles

The coating antibody D1B6 and the magnetic particles are combined by using a covalent bond: 1 micron carboxyl magnetic beads are activated by EDC and NHS, washed by MES buffer solution with pH 5.0, added with coating antibody of MES buffer solution dialyzed in advance at pH 5.0 for reaction, then washed by MES buffer solution with pH 5.0, and blocked by BSA for standby.

2. Labeling of azetidines

The optimal paired antibody C2F9 (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 heavy chain is shown in SEQ ID NO.22 and the amino acid sequence of the full-length light chain is shown in SEQ ID NO. 23) of the screened coated antibody D1B6 is used as a labeled antibody and is combined with azetidine amide by utilizing a covalent bond: the antibody was first dialyzed into PBS buffer pH 8.5, and azelsulfonamide containing NHS groups was added as 1:10, dialyzing to remove the azetidine ester after the reaction is finished, and using the product for standby.

3. Optimization of reaction systems

Orthogonal experiments are utilized to screen the concentrations of the magnetic particles and the azinyl ester marker, the reaction concentration is determined, a reaction system is optimized, and the specific formula and pH are determined.

4. Tracing of PCT standard substance

And tracing the relation of the quantity values of the standard substance to the measured value of the Roche kit to form the unit quantity value of the standard substance.

5. Detection operation of kit

And (3) testing the detection system by adopting a full-automatic chemiluminescence instrument, wherein the sample adding amount is 15 mu L, the sample adding amount of the detection reagent is 50 mu L respectively, reacting for 10 minutes, and adding the excitation liquid and the pre-excitation liquid for luminescence testing. The luminescent signal and the PCT content of the sample to be detected form a positive correlation.

Example 5 evaluation of the Performance of the chemiluminescent kit

1. Detection limit

And continuously detecting the S0 zero-value sample for 20 times, and calculating the LOB of the kit. As a result, the sensitivity was 0.0035ng/mL, as shown in Table 2.

TABLE 2

2. Accuracy (recovery experiment)

The high value sample (solution A) was added to the low value sample (solution B), each of which was repeatedly tested 3 times, and the recovery rate was calculated according to the formula (1) by taking the average value.

R＝(C×(V0+VS)-C0×V0)/(VS×CS)×100％

Formula (1)

In the formula:

r-recovery rate;

c is the average value of the detection concentration after the solution A is added into the solution B;

v0-volume of liquid B;

VS-volume of liquid A;

c0-average value of the concentration of the solution B;

CS-concentration of solution A.

As a result, the recovery rate R was 93.13%, as shown in Table 3.

TABLE 3

3. Linearity

Diluting high value sample approaching linear interval with a certain proportion to 5 concentrations, wherein the low value sample concentration is near the lower limit of the linear interval, and repeating the measurement for 2 times. The concentrations of 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. And performing straight line fitting on the average value of the measured concentration and the theoretical concentration, and obtaining a linear correlation coefficient r.

As a result, the linear correlation coefficient r was 0.9967 as shown in Table 4.

TABLE 4

4. Repeatability of

The samples were tested 10 times in duplicate and the CV values were calculated for each result.

The results are shown in Table 5.

TABLE 5

5. Precision degree

The experimental requirements are as follows: the coefficient of variation of the detection result should be not more than 15%.

The experimental method comprises the following steps: the samples were tested 5 times each day for 5 consecutive days, with 25 data per sample. The total inaccuracy was calculated.

The results are shown in tables 6 and 7.

TABLE 6

TABLE 7

6、HOOK

The HOOK (1000 ng/mL) samples were tested 3 times repeatedly, and the mean value of the test results was higher than the linear range of the standard curve. The results are shown in Table 8.

TABLE 8

7. Clinical consistency

At least 40 samples were taken, and the high value and the low value were distributed, and the results of the identity analysis with the reference reagent (Roche cobas) are shown in FIG. 2, which indicates that the detection reagent prepared from the antibody D1B6 of the present application has higher serum identity with the reference reagent.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (10)

1. An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain of the antibody or the antigen-binding fragment thereof are shown in SEQ ID NO. 1-3;

the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or the antigen binding fragment thereof are shown in SEQ ID NO. 4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID No.7 or has at least 80% similarity to the amino acid sequence as set forth in SEQ ID No. 7;

the amino acid sequence of the light chain variable region of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.8 or has at least 80% similarity with the amino acid sequence shown as SEQ ID NO. 8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from any one of a monoclonal antibody, fab ', F (ab') 2, fd, fv, dAb, a complementarity determining region fragment, and a single chain antibody.

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

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3;

preferably, the nucleotide sequence of the nucleic acid molecule encoding the variable region of the heavy chain is shown in SEQ ID NO. 9; the nucleotide sequence of the nucleic acid molecule for encoding the variable region of the light chain is shown in SEQ ID NO. 10.

6. Biological material comprising a nucleic acid molecule according to claim 5, wherein the biological material is an expression cassette, a vector or a host cell.

7. An antibody conjugate obtained by conjugating 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 to a label or protein;

the label is selected from one or more of chemiluminescence dye label, enzyme label, biotin label, fluorescent dye label, colloidal gold label and radioactive label.

8. An antibody composition comprising a first antibody according to any one of claims 1 to 3, or an antigen-binding fragment thereof, and a second antibody having the amino acid sequence of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of SEQ ID No. 14-16; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain complementarity determining region are shown in SEQ ID NO. 17-19.

9. Use of any one of the following of the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, or the bispecific or multispecific antibody of claim 4, or the nucleic acid molecule of claim 5, or the biomaterial of claim 6, or the antibody conjugate of claim 7, or the antibody composition of claim 8:

(1) Use in the manufacture of a product for detecting the presence or level of procalcitonin in a sample;

(2) Use in the manufacture of a product for the diagnosis of bacterial infections;

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

(4) The application of the composition in preparing products for evaluating the occurrence, development and/or treatment prognosis of inflammation.

10. A detection reagent comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, or comprising the bispecific or multispecific antibody of claim 4, or comprising the antibody conjugate of claim 7, or comprising the antibody composition of claim 8.

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