CN116284382A - Procalcitonin-resistant antibodies and uses thereof - Google Patents

Procalcitonin-resistant antibodies and uses thereof Download PDF

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CN116284382A
CN116284382A CN202211667710.0A CN202211667710A CN116284382A CN 116284382 A CN116284382 A CN 116284382A CN 202211667710 A CN202211667710 A CN 202211667710A CN 116284382 A CN116284382 A CN 116284382A
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antigen
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amino acid
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罗海峰
奚建红
王文峰
鲍勇刚
滕宏
陈超
周昊
万欢
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Cnpair Biotech Co ltd
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Abstract

The invention relates to the technical field of antibodies, in particular to an anti-procalcitonin antibody and application thereof. The amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO.1-3, and the amino acid sequences of light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6. The antibody can be specifically combined with procalcitonin and has higher affinity. The immune detection reagent developed by 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 in the field of procalcitonin immune detection.

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 (PCT) is a hormone-free glycoprotein of 116 amino acids with a molecular weight of 13KD, which is a precursor of procalcitonin (CT), and is produced by coding with 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. Other inflammatory factors such as IL-6, IL-10, TNF- α peak at 2 hours post infection and decrease rapidly and are not easily detected; c-reactive protein (CRP) peaks 24-48 hours after infection and remains at a high level after inflammatory control, which is detrimental to early diagnosis and therapeutic evaluation. 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 electrochemical luminescence method of Swiss Roche company, enzyme-linked immunofluorescence method of French Mei Liai company, colloidal gold colorimetry method and chemiluminescence method of Siemens Fedder, U.S.A. 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 set forth in SEQ id No. 9; 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 herein can 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 label or protein; 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, and 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; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, 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 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 the above (3) is a sample not derived from a living human or animal, for example: cells cultured in vitro, cell culture fluid, procalcitonin standard, etc.
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 a specific targeting procalcitonin antibody which has higher affinity to procalcitonin protein, can be specifically combined with 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 the synthesized polypeptide protein kata-KLH and Freund's complete adjuvant are uniformly mixed according to the ratio of 1:1 and emulsified, 4 mice of female Balb/c with the age of 6-8 weeks are subcutaneously injected in multiple points, the antigen inoculation dosage of each mouse is 50 mug, after three weeks, the antigen and Freund's incomplete adjuvant are uniformly mixed according to the ratio of 1:1 and emulsified according to the ratio of 500 mug, the subcutaneous multiple point injection is carried out, the antigen inoculation dosage of each mouse is 50 mug, and after three weeks, the intraperitoneal injection is carried out by 50 mug of antigen without adjuvant, and the total immunization is carried out for 4 times.
3. Immune serum potency assay
Immune serum titers were determined by indirect ELISA. 50 μg of the synthetic peptide kata-KLH was dissolved in 10ml of 0.05M phosphate buffer pH 9.6 and coated on a polystyrene 96-well plate, 100 μl/well, overnight at 4deg.C. The plate was washed three times with PBS (containing 0.05% (V/V) Tween-20), 100. Mu.L/well with 1% BSA blocking solution in 10mM PBS, blocked at 37℃for 2h, three times with PBS (containing 0.05% (V/V) Tween-20), 10 days post-third immunization, blood was collected from the tail vein of the mice, and murine immune serum was subjected to 10 with 10mM PBS containing 1% BSA -2 ～10 -8 Multiple dilutions were added to 96-well plates, incubated at 37℃for 1h at 100. Mu.L/well, and after three plate washes with PBS (containing 0.05% (V/V) Tween-20), 1: after washing the plates according to the above procedure with 100. Mu.L/well of 10000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) incubated at 37℃for 30min, TMB developed, 100. Mu.L/well was protected from light at room temperature for 10min, and 50. Mu.L/well 2M H was added 2 SO 4 Stopping the reaction, measuring the 450nm absorption value, taking the serum of the mice before immunization as a negative control, and taking the ratio of the measured value to the control value as a positive judgment value which is more than or equal to 2.1 to determine the immune serum titer. The results are shown in Table 1.
TABLE 1
Figure BDA0004015264280000071
Figure BDA0004015264280000081
4. Preparation of hybridomas
Mice with serum titers greater than 1:160000 were taken, 3 days prior to fusion, and synthetic peptide kata-KLH was mixed with an equal volume of PBS and injected intraperitoneally with BALB/c at a dose of 50 μg/500 μl for booster immunization. Taking mouse spleen aseptically, preparing spleen cell suspension and mouse myeloma cell strain SP2/0 in logarithmic growth phase according to the ratio of 1:1, 1000g room temperature centrifugation for 5min, discarding supernatant, flicking the bottom of the centrifuge tube with a finger, placing the centrifuge tube in a 37 ℃ water bath, adding 50% polyethylene glycol (PEG, MW4000, sigma) incubated in the 37 ℃ water bath dropwise into the centrifuge tube with a dropper, shaking the centrifuge tube while dripping over 1min, standing for 2min after dripping over, adding 1mL, 2mL, 3mL, 4mL, 5mL and 10mL of preheated serum-free 1640 medium at 37 ℃ every 1min to terminate the action of polyethylene glycol, centrifuging 1000g of the cell mixture for 5min at room temperature, discarding supernatant, adding HAT culture (hypoxanthine (H), aminopterin (a) and thymidine (T) (HAT, sigma)) to gently resuspend cells into 96 well plates, 200 μl per well. 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 the cell positive rate reaches 100%, and finally, a cell strain which stably secretes anti-PCT-kata monoclonal antibodies 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 cell C2F9 was grown at 1X 10 6 The abdominal cavity of 8-10 week old BALB/c female mice pretreated with liquid paraffin is injected into the amount of the liquid paraffin, and after 10-14 days of feeding and observation, the abdominal cavity of the mice is enlarged, and ascites is extracted. Purifying by affinity chromatography Protein G Sepharose Fast Flow to obtain monoclonal antibody C2F9, determining purity of monoclonal antibody by SDS-PAGEThe degree reaches more than 90 percent.
Example 2 Performance test of anti-procalcitonin monoclonal antibodies
1. Determination of antibody concentration
The ascites fluid prepared by hybridoma cell C2F9 is purified to obtain anti-PCT-kata protein monoclonal antibody C2F9, and the anti-PCT-kata protein monoclonal antibody C2F9 is measured by using a Nanodrop nucleic acid protein measuring instrument manufactured by Thermofisher company, and the concentration of the anti-PCT-kata protein monoclonal antibody C2F9 is more than 1mg/mL.
2. Antibody subtype identification
Subtype of hybridoma cell strain is identified by using a mouse monoclonal antibody subtype identification kit of Thermofisher, subtype of C2F9 secretion antibody is IgG1 type, and 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 4deg.C. PBS (containing 0.05% (V/V) Tween-20) plates were washed three times, 1% BSA blocking solution was used with 10mM PBS 150. Mu.L/well, blocking was performed at 37℃for 2h, PBS (containing 0.05% (V/V) Tween-20) plates were used three times, 100. Mu.L of gradient diluted purified antibody was added to each well, incubated at 37℃for 1h, PBS (containing 0.05% (V/V) Tween-20) plates were used three times, horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was used as secondary antibody, incubated at 37℃for 30min, PBS (containing 0.05% (V/V) Tween-20) plates were used three times, 100. Mu.L of LTMB color development solution was added to each well, and after incubation at 37℃for 15min 2M H was added 2 SO 4 The reaction was stopped and the microplate reader was tested at an absorbance of 450 nm. Multiple results showed that the antibody titer could reach 1X 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 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 at 4 ℃ overnight. Before use, taking out the closed microplate from the temperature of 4 ℃, spin-drying, and adding a cleaning solution (1 XPBS-T) to moisten the ELISA plate; monoclonal antibody C2F9 was pre-diluted to 30. Mu.g/mL with 1 XPBS and the fold m of pre-dilution was recorded, with the 10-fold dilution, i.e., 3. Mu.g/mL, as the highest concentration (S1), followed by 1:3 gradient dilutions (dilutions in 96 deep well plates) for a total of 8 dilution gradients (S1-S8).
Adding 100 mu L of diluted monoclonal antibody C2F9 into a 96-well microplate which is cleanly shot on absorbent paper, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating on absorbent paper, cleaning the ELISA plate 3 times with a plate washer, and adding 100 μl of 1×PBS into each hole of 1-4 columns; 200 mu L of urea treatment solution is added into each of the 5 columns and the 6 columns, and the mixture is incubated for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating the ELISA plate on absorbent paper, cleaning the ELISA plate 3 times by using a plate washer, adding 100 mu L of GAM-HRP ELISA secondary antibody which is diluted 10000 times by secondary antibody diluent in advance into each hole, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating the ELISA plate on absorbent paper, cleaning the ELISA plate 3 times by using a plate washer, taking TMB color development liquid, adding 100 mu L color development liquid into each hole, and incubating for 5-10min at 37 ℃; after development, 50. Mu.L of stop solution was added to each well. 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 absorbance value OD, corresponding polynomial curves are obtained through fitting, the corresponding binding can see obvious gradient along with the variation of 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 concentration corresponding to the binding force of the antibody reaches 1.45E-9mol/L respectively (under the condition of 0.5 mug/mL antigen coating, the highest OD reading is 50 percent, namely, when the concentration of the antigen-antibody complex is half of the total concentration, the concentration value K corresponding to the antibody is shown by the concentration value K 0.5 ) And 9.08E-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 corresponding concentration value of the antibody, K) 1 )。
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 detection is carried out by using the N-terminal recombinant protein, the CT recombinant protein and the CRP recombinant protein of PCT, and the result shows that the result is negative, and the PCT-kata protein is positive, so that the specificity of the monoclonal antibody C2F9 is good.
EXAMPLE 3 sequencing of anti-procalcitonin monoclonal antibodies
Sequencing a light chain variable region and a heavy chain variable region of an anti-PCT-kata protein monoclonal antibody C2F9 prepared by purified hybridoma cell C2F9, wherein the sequencing result shows that the amino acid sequences of a heavy chain complementarity determining region CDR1, a CDR2 and a CDR3 are shown as 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. 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 to be used 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
Binding the coated antibody D1B6 to the magnetic particles by covalent bonds: the carboxyl magnetic beads of 1 micrometer are activated by EDC and NHS, washed by MES buffer with pH of 5.0, added with 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 azdining amide are covalently bound: antibody C2F9 was first dialyzed to PBS buffer at pH 8.5, and the NHS group-containing azdining amide was added at 1:10, and dialyzing to remove the azing ester after the reaction is finished.
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
Figure BDA0004015264280000121
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
Figure BDA0004015264280000131
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 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 (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
Figure BDA0004015264280000132
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
Figure BDA0004015264280000141
5. Precision of
Experiment requirements: the variation coefficient of the detection result should be not more than 15%.
The experimental method comprises the following steps: the samples were each repeated 5 times a day for 5 consecutive days, each sample yielding 25 data. The total inaccuracy was calculated.
The results are shown in tables 6 and 7.
TABLE 6
Figure BDA0004015264280000142
Figure BDA0004015264280000151
TABLE 7
Figure BDA0004015264280000152
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
Figure BDA0004015264280000153
7. Clinical consistency
At least 40 samples were taken, both high and low values were distributed and analyzed for identity with the reference reagent (cobas, rochanter). 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.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. An anti-procalcitonin antibody or an antigen binding fragment thereof, which is characterized in that the amino acid sequences of a heavy chain complementarity determining region CDR1, CDR2 and CDR3 of the antibody or the antigen binding fragment thereof are shown in SEQ ID NO.1-3, and the amino acid sequences of a light chain complementarity determining region CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID No.7 or at least 80% similarity to the amino acid sequence as shown in SEQ ID No.7, and the light chain variable region has an amino acid sequence as shown in SEQ ID No.8 or 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, wherein the antibody or antigen-binding fragment thereof is selected from any one of monoclonal antibodies, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies.
4. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3;
preferably, 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.
6. A biological material comprising the nucleic acid molecule of claim 5, wherein the biological material is an expression cassette, a vector or a host cell.
7. An antibody conjugate, which is obtained by coupling the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 3 or the bispecific antibody or the multispecific antibody according to claim 4 with a label or a protein;
the marker is one or more selected from chemiluminescent dye markers, enzyme markers, biotin markers, fluorescent dye markers, colloidal gold markers and radioactive markers.
8. An antibody composition comprising a first antibody that is the antibody or antigen-binding fragment thereof of any one of claims 1-3 and a second antibody having the amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2, CDR3 of SEQ ID nos. 14-16; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR3 are shown in SEQ ID NO. 17-19.
9. Use 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 biological material of claim 6 or the antibody conjugate of claim 7 or the antibody composition of claim 8 for any one of the following:
(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.
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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