CN116284382B

CN116284382B - Anti-procalcitonin antibody and its application

Info

Publication number: CN116284382B
Application number: CN202211667710.0A
Authority: CN
Inventors: 罗海峰; 奚建红; 王文峰; 鲍勇刚; 滕宏; 陈超; 周昊; 万欢
Original assignee: Cnpair Biotech Co ltd
Current assignee: Cnpair Biotech Co ltd
Priority date: 2022-12-23
Filing date: 2022-12-23
Publication date: 2025-02-11
Anticipated expiration: 2042-12-23
Also published as: CN116284382A

Abstract

本发明涉及抗体技术领域，具体涉及抗降钙素原抗体及其应用。本发明提供的抗体或其抗原结合片段的重链互补决定区CDR1、CDR2、CDR3的氨基酸序列如SEQ IDNO.1‑3所示，轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO.4‑6所示。该抗体能够特异性结合降钙素原，具有较高的亲和力。利用该抗体开发的免疫检测试剂在用于降钙素原检测时具有较高的特异性和灵敏度，且与参考检测方法具有较高的一致性，能够较为准确地反映真实样本的降钙素原含量，在降钙素原的免疫检测领域具有广泛的应用前景。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 immunodetection 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 immunodetection of procalcitonin.

Description

Procalcitonin-resistant antibodies and uses thereof

Technical Field

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

Background

Procalcitonin (procalcitonin, PCT) is a hormone-free glycoprotein of 116 amino acids, has a molecular weight of 13KD, is a precursor of Calcitonin (CT), and is produced by coding the CALC-I gene located on chromosome 11. PCT is a specific marker for bacterial, fungal infections. PCT levels in healthy human blood are very low, typically below 0.10ng/mL, since extra-thyroid CALC-I gene expression is inhibited in the absence of infection, primarily limited to only some degree of expression in neuroendocrine cells of the thyroid and lung. When the organism suffers from serious fungus, bacteria, parasite infection, sepsis, multiple organ 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, even reaching millions to millions of times of the normal level, but the CT content is kept unchanged or slightly increased. In contrast, PCT concentration in serum of patients suffering from local infection, viral infection, allergy, autoimmune disease, chronic nonspecific inflammation, etc. does not increase or slightly increases, so PCT can be used as a specific indicator of bacterial infection for differential diagnosis of bacterial diseases.

PCT has higher accuracy and specificity than a variety of biological markers, and is an ideal infection detection marker. PCT can be detected 2 hours after infection, peaks at 6-12 hours after infection, plateau at 12-24 hours, concentration decreases after 24 hours, and half-life is 25-30 hours, and can be maintained at high level for a period of time. Furthermore, PCT levels are positively correlated with the infection level, and a higher PCT concentration means a more severe infection, so PCT levels are important for diagnosis of the occurrence and development of inflammation, prognosis of treatment, and the like. While other inflammatory factors such as IL-6, IL-10, TNF-alpha peak at 2 hours post-infection and decrease rapidly and are not easily detected, C-reactive protein (CRP) peaks only 24-48 hours post-infection and remains at a higher level after inflammatory control, which is detrimental to early diagnosis and therapeutic assessment. In addition, in clinical diagnostic treatment, the sensitivity and specificity of traditional detection indicators such as body temperature, white blood cell count (WBC) are not high.

PCT is currently detectable by quantitative, semi-quantitative and qualitative methods. The quantitative detection method mainly comprises an electrochemical luminescence method (ECLIA) and an enzyme-linked immunosorbent assay. The most commonly used PCT detection methods at present are the electrochemical luminescence method of Swiss Roche, the enzyme-linked immunofluorescence method of French Mei Liai, the colloidal gold colorimetric method and the chemiluminescent method of Siemens in the United states. The 3 methods use the same antibody and the same preparation standard, so that the detection results have traceability and higher correlation and consistency.

PCT is extremely low in healthy human plasma, so PCT detection has high performance requirements on antibody sensitivity, affinity and the like. The detection sensitivity of the existing anti-PCT antibodies and the correlation between the anti-PCT antibodies and the detection results of the Roche company are to be improved. Therefore, there is a need to develop high affinity, high sensitivity anti-PCT specific antibodies.

Disclosure of Invention

The object of the present invention is to provide anti-procalcitonin antibodies and uses thereof.

The invention provides an anti-procalcitonin antibody or an antigen binding fragment thereof, wherein the amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the anti-procalcitonin antibody or the antigen binding fragment thereof are shown as SEQ ID NO.1-3, and the amino acid sequences of light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 4-6.

SEQ ID NO.1:NYAMN;

SEQ ID NO.2:EITLESNNYATHYAESVKG;

SEQ ID NO.3:MINMD;

SEQ ID NO.4:KSSQSLLYSSNQENYLA;

SEQ ID NO.5:WASTHES;

SEQ ID NO.6:HQYYTYPDT。

The antibody or the antigen binding fragment thereof can specifically bind procalcitonin and has higher affinity.

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

SEQ ID NO.7:

EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYAMNWVRQSPEKGLEWVAEITL ESNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTSMINMDWGQG TTLMVSS.

SEQ ID NO.8:

DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQENYLAWYQQKLGQSPKLLI YWASTHESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYYTYPDTFGGGTKLE IK.

In some embodiments of the 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.

In the case of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and light chain complementarity determining regions CDR1, CDR2, CDR3 described above, the amino acid sequence of the heavy chain variable region is 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 shown as SEQ ID No.7, and the amino acid sequence of the light chain variable region is an antibody or antigen binding fragment thereof 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 99.5% sequence similarity to the amino acid sequence shown as SEQ ID No. 8.

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 that retains the ability to specifically bind procalcitonin or that competes 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 monoclonal antibodies, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies.

Wherein the monoclonal antibody comprises an animal-derived antibody, a chimeric antibody, a humanized antibody or 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, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO. 11.

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

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

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

The present invention provides bispecific or multispecific antibodies comprising an antibody or antigen-binding fragment thereof described above.

The present invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.

Based on the amino acid sequence and codon regularity of the above-described antibodies or antigen-binding fragments thereof, one skilled in the art can obtain the nucleotide sequence of a nucleic acid molecule encoding the above-described antibodies or antigen-binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.

In some embodiments of the invention, 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.

The present invention provides a biological material comprising the nucleic acid molecule described above, which biological material is an expression cassette, a vector or a host cell.

The nucleic acid molecules described above are operably linked to promoters and/or terminators to provide expression cassettes.

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

The host cells described above include microbial cells or animal cells. Wherein the microbial cells include, but are not limited to, E.coli, yeast, etc., and the animal cells include, but are not limited to, insect cells, CHO cells, 293T cells, etc.

The antibodies or antigen-binding fragments thereof provided by the present invention may be prepared by methods conventional in the art, including chemical synthesis, host expression, and the like.

In some embodiments of the invention, the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are introduced into host cells to obtain recombinant cells, the recombinant cells are cultured, and the antibodies or antigen binding fragments thereof are isolated and purified.

The invention provides an antibody conjugate, which is obtained by coupling the antibody or antigen binding fragment thereof or the bispecific antibody or multispecific antibody with a marker or protein, wherein the marker is one or more selected from chemiluminescent dye markers, enzyme markers, biotin markers, fluorescent dye markers, colloidal gold markers and radioactive markers.

The 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, the amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the second antibody are shown as SEQ ID NO.14-16, and the amino acid sequences of light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 17-19.

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.

Further preferably, the amino acid sequence of the full length of the heavy chain of the second antibody 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.

The present invention provides the use of any of the antibodies or antigen binding fragments thereof or the bispecific or multispecific antibodies or the nucleic acid molecules or the biological material or the antibody conjugates or the antibody compositions described above:

(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 a product for diagnosing a bacterial infection;

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

(4) Use in the manufacture of a product for assessing the development of inflammation and/or prognosis of treatment.

In the applications described in the above (1) and (2), the product may be a detection reagent or a kit. (1) The sample may be a sample derived from a living human or animal (including blood, etc.), or may be a sample derived from a non-living human or animal such as cells or cell culture fluid cultured in vitro.

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

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

The present invention provides a detection reagent comprising the above-described antibody or antigen-binding fragment thereof, or comprising the bispecific or multispecific antibody, or comprising the antibody conjugate, or comprising the antibody composition.

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

The above-mentioned detection reagent may contain, in addition to the antibody or antigen-binding fragment thereof or the bispecific antibody or multispecific antibody or the antibody conjugate, a secondary antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention. Among them, detectable labels include, but are not limited to, chemiluminescent labels, enzymes, colloidal gold, radioisotopes, fluorescent dyes, and the like.

The detection reagent may be any immunodiagnostic reagent including, but not limited to, chemiluminescent detection reagents, enzyme linked immunosorbent detection reagents, lateral flow immunochromatographic detection reagents, immunofluorescent detection reagents, and the like.

In some embodiments of the invention there is provided a chemiluminescent kit for procalcitonin detection comprising a chemiluminescent dye-labeled antibody or antigen binding fragment thereof.

The invention has the beneficial effects that the invention provides the antibody for specifically targeting procalcitonin, which has higher affinity to procalcitonin, can specifically bind procalcitonin and has higher sensitivity. The immune detection reagent developed by the antibody has higher specificity and sensitivity when being used for detecting procalcitonin, has higher relativity 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 procalcitonin immune detection.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the binding of antibodies of example 2 of the present invention to PCT antigen at gradient concentrations, respectively. Wherein 0.5. Mu.g (/ mL) is the binding curve of 50ng (100. Mu.L per well) of the coated antigen per well, and 1. Mu.g (/ mL) is the binding curve of 100ng (100. Mu.L per well) of the coated antigen per well.

FIG. 2 shows the results of a clinical uniformity analysis of PCT detection reagent in example 5 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 acquisition of anti-procalcitonin monoclonal antibodies

1. Design, preparation and carrier coupling of PCT antigen

PCT protein sequences were obtained from the procalcitonin protein sequence on GenBank (accession number: NP-001029124). Through immunogenicity, hydrophilicity and hydrophobicity and surface accessibility analysis, the kata region sequence of PCT protein is finally determined as antigen (SEQ ID NO. 13) through screening, and the peptide and the synthetic peptide (kata-KLH) antigen of the C-terminal coupled keyhole limpet hemocyanin carrier protein KLH are synthesized by the division of biological engineering (Shanghai).

2. Immunized mice

After mixing and emulsifying the synthetic polypeptide protein kata-KLH and Freund's complete adjuvant according to a ratio of 1:1, 4 mice of female Balb/c with 6-8 weeks old are subjected to subcutaneous multipoint injection, the antigen inoculation dosage of each mouse is 50 mug, after three weeks, the antigen and Freund's incomplete adjuvant are mixed and emulsified according to a ratio of 1:1, 500 mug of the antigen is subjected to subcutaneous multipoint injection, the antigen inoculation dosage of each mouse is 50 mug, and after three weeks, the total immunization is 4 times by intraperitoneal injection with 50 mug of antigen without adjuvant.

3. Immune serum potency assay

Immune serum titers were determined by indirect ELISA. 50 μg of synthetic peptide kata-KLH was dissolved in 10ml of 0.05M phosphate buffer pH 9.6 and coated on polystyrene 96-well plates, 100 μl/well, at 4℃overnight. The plate was washed three times with PBS (containing 0.05% (V/V) Tween-20), 100. Mu.L/well with 10mM PBS containing 1% BSA blocking solution, blocked for 2h at 37 ℃, washed three times with PBS (containing 0.05% (V/V) Tween-20), the mice were bled from the tail vein 10 days after the third immunization, the mouse immune serum was 10 ^-2～10^-8 -fold diluted with 10mM PBS containing 1% BSA, 96-well plates were added, 100. Mu.L/well incubated for 1h at 37 ℃, PBS (containing 0.05% (V/V) Tween-20) washed three times, horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) was added, 100. Mu.L/well incubated for 30min at 37 ℃, after washing the plate according to the above method, TMB color development was 100. Mu.L/well, room temperature was 10min, and the reaction was stopped at 2. 2M H ₂SO₄ with 50. Mu.L/well, the absorbance at 450nm was measured, and the positive serum ratio of the mouse serum as a negative control was not less than 2.1 was determined. The results are shown in Table 1.

TABLE 1

4. Preparation of hybridomas

Mice with serum titers greater than 1:160000 were taken, 3 days prior to fusion, synthetic peptide kata-KLH was mixed with an equal volume of PBS, and BALB/c was injected intraperitoneally at a dose of 50 μg/500 μl for booster immunization of the mice to be fused. The spleen of the mice is aseptically taken, a spleen cell suspension is prepared, the spleen cell suspension is mixed with a mouse myeloma cell strain SP2/0 in a logarithmic phase at a ratio of 1:1, 1000g of the mixture is centrifuged at room temperature for 5min, the supernatant is discarded, the bottom of a centrifuge tube is flicked by a finger, the sediment is loosened, the centrifuge tube is placed in a 37 ℃ water bath, 50% polyethylene glycol (PEG, MW4000, sigma) which is insulated in the 37 ℃ water bath is added into the centrifuge tube by a dropper dropwise while shaking the centrifuge tube, the centrifuge tube is completely dripped in 1min, the mixture is left to stand for 2min after dripping, the action of polyethylene glycol is stopped by adding 1mL, 2mL, 3mL, 4mL, 5mL and 10mL of a serum-free 1640 medium preheated at 37 ℃ every 1min, the cell mixture is centrifuged at room temperature for 5min, the supernatant is discarded, HAT culture solution (hypoxanthine (H), aminopterin (A) and thymidine (HAT, sigma)) is added to light-resuspend the cells into a 96-well plate, and 200 mu L of cells are separated into each well plate. After three days of culture, the cell fusion was observed, half of HAT medium was changed, and the culture was continued for several days until clone formation, and HT medium (hypoxanthine (H) and thymidine (T) (HT, sigma)) was changed seven days after fusion.

5. Screening of hybridoma cells secreting monoclonal antibodies against PCT-kata protein

The cell culture supernatant is screened by an indirect ELISA method, positive clone hybridoma cells with higher titers are selected for subcloning, and cloning is carried out continuously for 2-3 times by a limiting dilution method until 100% of cell positive rate is reached, and finally, a cell strain which stably secretes anti-PCT-kata monoclonal antibody and is marked as C2F9 is obtained. And (5) performing liquid nitrogen freezing after amplification culture on the cells with the positive rate reaching 100% after cloning.

6. Preparation and purification of ascites

Hybridoma cells C2F9 were injected into the abdominal cavity of liquid paraffin-pretreated 8-10 week-old BALB/C female mice in an amount of 1X 10 ⁶/mouse, and after 10-14 days of feeding observation, ascites were withdrawn when the abdomen of the mice swelled. Purifying by affinity chromatography Protein G Sepharose Fast Flow to obtain monoclonal antibody C2F9, and determining purity of monoclonal antibody by SDS-PAGE to reach purity above 90%.

Example 2 Performance test of anti-procalcitonin monoclonal antibodies

1. Determination of antibody concentration

The ascites fluid prepared by hybridoma cell C2F9 was purified to obtain anti-PCT-kata protein monoclonal antibody C2F9, which was measured using a Nanodrop nucleic acid protein meter manufactured by Thermofisher, and the concentration was >1mg/mL.

2. Antibody subtype identification

The subtype of the hybridoma cell strain is identified by adopting Thermofisher mouse monoclonal antibody subtype identification kit, the subtype of the C2F9 secretion antibody is IgG1 type, and the light chain is kappa chain.

3. Potency identification of purified antibodies

50 Μg of the synthetic PCT-kata protein peptide was dissolved in 10mL of 0.05M carbonate coating buffer pH 9.6, and added to a 96-well plate at 100 μl per well overnight at 4 ℃. PBS (containing 0.05% (V/V) Tween-20) was used to wash plates three times, 10mM PBS containing 1% BSA blocking solution 150. Mu.L/well, 37℃was used to block plates three times, PBS (containing 0.05% (V/V) Tween-20) was used to wash plates three times, 100. Mu.L of gradient diluted purified antibody was added to each well, 37℃was incubated for 1h, PBS (containing 0.05% (V/V) Tween-20) was used to wash plates three times, horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was used as secondary antibody, 37℃was incubated for 30min, PBS (containing 0.05% (V/V) Tween-20) was used to wash plates three times, 100. Mu.L of LTMB color development solution was added to each well, after 15min incubation at 37℃was added to stop the reaction by adding 2M H ₂SO₄ solution, and the microplate reader was detected at absorbance value of 450 nm. Multiple results showed that the antibody titers could reach 1 x 10 ⁶.

4. Affinity test:

PCT-kata antigen protein was diluted to 0.5. Mu.g/mL and 1. Mu.g/mL with 1 XCB, added to the wells of the ELISA plate at a rate of 100. Mu.L/well, and multiplexed, and adsorbed at 4℃overnight or 37℃for 2 hours. The coated microplates were spin-dried, washed once according to the procedure set by the plate washer, and blocking solution was added in an amount of 200 μl/well, placed in a 37 ℃ incubator for 2 hours, and then placed overnight at 4 ℃. Immediately before use, the blocked microwell plate was removed from 4 ℃, dried, and wash solution (1 XPBS-T) was added to wet the ELISA plate, monoclonal antibody C2F9 was pre-diluted to 30 μg/mL with 1 XPBS, the pre-dilution factor m was recorded, 10-fold dilution, i.e., 3 μg/mL, was taken as the highest concentration (S1), and then diluted with a 1:3 gradient (diluted in 96 deep well plates), for a total of 8 dilution gradients (S1-S8).

Adding 100 mu L of diluted monoclonal antibody C2F9 into 96-well micro-pore plates which are well beaten on absorbent paper, incubating for 30min at 37 ℃, spin-drying the ELISA plates after incubation, beating dry on absorbent paper, washing the ELISA plates 3 times by using a plate washer, adding 100 mu L of 1 XPBS into each hole of 1-4 columns, adding 200 mu L of urea treatment liquid into each hole of 5 columns and 6 columns, incubating for 30min at 37 ℃, spin-drying the ELISA plates after incubation, beating dry on absorbent paper, washing the ELISA plates 3 times by using a plate washer, adding 100 mu L of GAM-HRP (gamma-HRP) which is pre-diluted 10000 times by using a secondary anti-diluent into each hole, incubating for 30min at 37 ℃, spin-drying the ELISA plates after incubation, beating dry on absorbent paper, washing the ELISA plates 3 times by using a plate washer, taking TMB color development liquid, adding 100 mu L of color development liquid into each hole, incubating for 5-10min at 37 ℃, and adding 50 mu L of stop liquid into each hole after color development. The read at 450nm/630nm was set up on a microplate reader.

ELISA antigen-antibody binding force experiments are tested under the conditions of 0.5 mug/mL and 1 mug/mL antigen coating respectively, data (figure 1) are obtained through testing the absorbance OD, corresponding polynomial curves are obtained through fitting, the corresponding binding can see obvious gradient along with the variation of the dilution ratio of the antibody, meanwhile, the affinity binding reflected by the two curves under the conditions of 0.5 mug/mL and 1 mug/mL antigen coating shows the binding specificity, and the corresponding concentration of the antibody binding force respectively reaches 1.45E-9mol/L (under the condition of 0.5 mug/mL antigen coating, the highest OD reading is 50 percent, namely, the concentration value K _0.5 corresponding to the antibody is half the total concentration) and 9.08E-10mol/L (under the condition of 1 mug/mL antigen coating, namely, the concentration value K ₁ corresponding to the highest OD reading value is half the total concentration of the antigen-antibody).

5. Specific detection

The specificity detection of the monoclonal antibody C2F9 is carried out by referring to an ELISA antibody titer detection method, and the N-terminal recombinant protein, the CT recombinant protein and the CRP recombinant protein of PCT are used for detection, so that the result shows that the result is negative, and the PCT-kata protein is positive, thus indicating that the specificity of the monoclonal antibody C2F9 is good.

EXAMPLE 3 sequencing of anti-procalcitonin monoclonal antibodies

Sequencing the light chain variable region and the heavy chain variable region of the monoclonal antibody C2F9 of the anti-PCT-kata protein prepared by the purified hybridoma cell C2F9, wherein the sequencing result shows that the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain complementarity determining regions are shown as SEQ ID NO.1-3, and the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the light chain complementarity determining regions are shown as 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 whole heavy chain is shown as SEQ ID NO.11, and the amino acid sequence of the whole 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 developing recombinant antibodies, single-chain antibodies and bispecific antibodies and related products for diagnostic use.

EXAMPLE 4 development of chemiluminescent kit Using anti-procalcitonin monoclonal antibody C2F9

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

1. Coating of magnetic particles

The coated antibody D1B6 and the magnetic particles are combined by covalent bonds, namely 1 micrometer carboxyl magnetic beads are activated by EDC and NHS, washed by MES buffer with pH of 5.0, added with the coated antibody which is dialyzed in advance into the MES buffer with pH of 5.0 for reaction, washed by the MES buffer with pH of 5.0, and blocked by BSA for later use.

2. Labeling of az dines

The labeled antibody C2F9 and the azulenamide are combined by covalent bond, namely, the antibody C2F9 is dialyzed to PBS buffer solution with pH of 8.5, the azulenamide containing NHS groups is added for reaction according to the proportion of 1:10, and the azulene ester is removed by dialysis after the reaction is finished for standby.

3. Optimization of the reaction System

The concentration of the magnetic particles and the azlactone labels were screened using orthogonal experiments to determine the reaction concentration, and the reaction system was optimized to determine the specific formulation and pH.

4. Tracing of PCT standard

And tracing the magnitude relation of the standard substance to the measured value of the Roche kit to form the unit magnitude 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 loading amount of the sample is 15 mu L, the sample loading amount of the detection reagent is 50 mu L respectively, reacting for 10 minutes, and adding the excitation liquid and the pre-excitation liquid to perform luminescence test. The luminescence signal and the PCT content of the sample to be detected form a positive correlation.

Example 5 evaluation of Performance of chemiluminescent kits

1. Detection limit

S0 zero value samples are continuously detected for 20 times, and LOB of the kit is calculated. The results are shown in Table 2, and the sensitivity was 0.0035ng/mL.

TABLE 2

2. Accuracy (recovery experiment)

And adding the high-value sample (A liquid) into the low-value sample (B liquid), repeatedly detecting for 3 times, taking an average value, and calculating the recovery rate according to the formula (1).

R= (c× (v0+vs) -c0×v0)/(vs×cs) ×100% formula (1)

Wherein:

R is the recovery rate;

c, adding the average value of the detection concentration of the solution A into the solution B;

V0-volume of liquid B;

vs—volume of liquid a;

C0-average value of concentration of B solution;

CS-concentration of A solution.

The results are shown in Table 3, and the recovery rate R was 93.13%.

TABLE 3 Table 3

3. Linearity of

High value samples near the upper line of the linear interval are diluted in proportion by 5 concentrations, wherein the low value sample concentration is near the lower limit of the linear interval, and each sample concentration is repeatedly measured for 2 times. The 7 sample concentrations were L, 0.833L+0.167H, 0.667L+0.333H, 0.5L+0.5H, 0.333L+0.667H, 0.167L+0.833H, H. And (5) performing linear fitting on the average value of the measured concentration and the theoretical concentration, and obtaining a linear correlation coefficient r.

The results are shown in Table 4, where the linear correlation coefficient r is 0.9967.

TABLE 4 Table 4

4. Repeatability of

The samples were each repeated 10 times and the CV values of the respective results were calculated.

The results are shown in Table 5.

TABLE 5

5. Precision of

The experiment requires that the variation coefficient of the detection result should be not more than 15%.

The test method is that the samples are repeatedly tested for 5 times each day for 5 days continuously, and 25 data are obtained for each 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) sample was repeatedly tested 3 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.

TABLE 8

7. Clinical consistency

At least 40 samples were taken, both high and low values were distributed and analyzed for consistency with a reference reagent (roche cobas). The results are shown in FIG. 2, which shows that the detection reagent prepared by the antibody C2F9 of the application has higher serum consistency with the reference reagent.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution 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 the antigen-binding fragment thereof are as shown in SEQ ID NO.1-3, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO.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 as shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8 or has at least 80% similarity to the amino acid sequence as 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 biomaterial comprises the nucleic acid molecule according to claim 5 or 6, and the biomaterial 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, 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 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.

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:

(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.