An anti-procalcitonin Fab antibody and its preparation method and application

Abstract

The present invention discloses an anti-procalcitonin Fab antibody and a preparation method and application thereof, wherein the heavy chain variable region of the heavy chain has three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO.1, 2 and 3 respectively; the light chain variable region of the light chain has three complementary determining regions CDR1', CDR2' and CDR3', and the amino acid sequences thereof are shown in SEQ ID NO.4, 5 and 6. The anti-procalcitonin Fab antibody provided by the scheme of the present invention is a fully human genetic engineering antibody, which can be effectively used to detect procalcitonin, and can also be used as a labeling antibody to be paired with other mouse PCT IgG monoclonal antibodies for clinical detection, and has the characteristics of high specificity, high sensitivity, low cost, strong anti-interference ability, etc.

Description

Technical Field

[0001] The invention belongs to the field of antibodies, and specifically relates to an anti-procalcitonin Fab antibody and a preparation method and application thereof. Background Art

[0002] Procalcitonin (PCT) is a glycoprotein without hormone activity and a precursor of calcitonin (CT). In clinical practice, PCT is used as a highly sensitive and specific indicator for the diagnosis of severe systemic bacterial, fungal, parasitic, acute malaria infection, systemic inflammatory response syndrome (SIRS), multiple organ failure syndrome (MDS), etc. In general, the content of PCT in human blood is very low. When there are severe bacterial, fungal, parasitic infections, sepsis and multiple organ failure, the level of PCT in plasma increases under the stimulation of endotoxins and inflammatory cytokines, while in non-infectious inflammatory states, local limited bacterial infections, mild infections or chronic inflammation, the PCT level generally does not increase. Therefore, PCT can be used as an indicator not only to diagnose early bacterial and non-bacterial infections and inflammations, but also has important reference value for the diagnosis of bacterial infections or sepsis in patients with high infection risk or intensive care, the prognosis evaluation of severe inflammatory diseases and treatment monitoring.

[0003] At present, the commonly used methods for determining procalcitonin include fluorescent immunochromatography, ELISA, chemiluminescent immunoassay, and immunoturbidimetry. The test samples are usually human whole blood, serum, or plasma samples. The test process is often affected by factors such as the test method, sample processing, and reagent components. For example, the specificity of the coated antibody is poor, the interfering substances in the sample are non-specifically bound, and the final test results appear false positive or the background is too high. In order to reduce interference, it usually takes a lot of time and effort to screen antibodies with high specificity and strong anti-interference ability; or the test samples and antibody raw materials are pretreated before testing. When conducting large-scale clinical tests, the sample pretreatment link will increase the consumption of reagents, increase the workload, and then increase the detection time, which is not conducive to rapid diagnosis.

[0004] The antibody IgG commonly used in the detection method can be degraded into two antigen-binding fragments (Fab fragments) and one crystallizable fragment (Fc fragment) under the action of papain. The composition of Fab includes the light chain variable region (VL), the light chain constant region (CL), the heavy chain variable region (VH) and the first heavy chain constant region (CH1). The Fc segment determines the effector function of the antibody and can bind to Fc receptors (such as FcRn) on the surface of macrophages, NK cells and other cells, as well as complement in serum. In clinical testing, the Fc segment can also non-specifically bind to interfering substances such as human anti-mouse antibodies (HAMAS), rheumatoid factors (RF) and Staphylococcus A protein (SPA), affecting the specificity and accuracy of antibody detection. Since the Fab fragment contains both the antigen-binding region and part of the constant region, it not only has the same antibody-antigen affinity as the original whole antibody, but also has a smaller molecular size and stronger tissue penetration; since it does not contain the Fc region, it reduces non-specific binding with interfering substances such as Fc receptors, which plays a huge role in clinical diagnosis.

[0005] Fab antibodies can be purified after the degradation of whole monoclonal antibodies with papain. This method is fast and simple, but the cost of the monoclonal antibody raw materials required is high. At the same time, the Fab antibodies obtained after enzymatic degradation and purification may lose a certain degree of immunoreactivity. At the same time, since Fab antibodies do not contain the Fc end, they do not require post-translational modification and glycosylation, and can be expressed not only in mammalian systems but also in prokaryotic systems. The Escherichia coli expression system has the advantages of low production cost and fast production speed, but the antibody expression level is low, inclusion bodies are easily formed, and the activity after renaturation is difficult to guarantee. Expression in mammalian cells can be correctly folded, closer to the natural Fab structure, higher activity, and the expression level can meet the needs. At present, the commonly used Fab purification methods mainly include ion exchange chromatography, hydrophobic chromatography, etc., but they cannot take into account the antibody recovery rate, antibody purity, large-scale production, purification time, cost and other issues at the same time, so there are certain limitations. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an anti-procalcitonin Fab antibody.

[0007] The present invention also provides a nucleic acid molecule encoding the above antibody.

[0008] The present invention also provides an expression vector and a transgenic cell line containing the nucleotide sequence.

[0009] The present invention also provides a detection kit containing the above-mentioned Fab antibody, nucleic acid molecule or expression vector or transgenic cell line.

[0010] The present invention also provides a method for producing the Fab antibody.

[0011] The present invention also provides an application of the Fab antibody, nucleic acid molecule or expression vector or transgenic cell line.

[0012] In one aspect of the present invention, an anti-procalcitonin Fab antibody is provided, which consists of a light chain and a heavy chain, wherein the heavy chain variable region of the heavy chain has three complementary determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the light chain variable region of the light chain has three complementary determining regions CDR1', CDR2' and CDR3', wherein the amino acid sequence of CDR1' is shown in SEQ ID NO.4, the amino acid sequence of CDR2' is shown in SEQ ID NO.5, and the amino acid sequence of CDR3' is shown in SEQ ID NO.6.

[0013] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.7.

[0014] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.8.

[0015] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of the heavy chain is shown in SEQ ID NO.9.

[0016] In some embodiments of the present invention, the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO.10.

[0017] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.11.

[0018] In some embodiments of the present invention, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.12.

[0019] In some embodiments of the present invention, two tag sequences are further fused to the C-terminus of the heavy chain.

[0020] In some embodiments of the present invention, the tag sequence includes His, HA, FLAG and Twin-Strep.

[0021] In some embodiments of the present invention, the tag sequences are connected via a connecting peptide.

[0022] In some embodiments of the present invention, the connecting peptide is [GGGGS]n, wherein n is an integer of 1-3.

[0023] In some embodiments of the present invention, the connecting peptide is [GGGGS].

[0024] In the second aspect of the present invention, a nucleic acid molecule encoding the above-mentioned antibody is provided.

[0025] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region of the antibody is shown as SEQ ID NO.13, and the nucleotide sequence encoding the light chain variable region of the antibody is shown as SEQ ID NO.14.

[0026] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain constant region of the antibody is shown as SEQ ID NO.15, and the nucleotide sequence encoding the light chain constant region of the antibody is shown as SEQ ID NO.16.

[0027] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain of the antibody is shown as SEQ ID NO.17, and the nucleotide sequence encoding the light chain of the antibody is shown as SEQ ID NO.18.

[0028] In the third aspect of the present invention, a method comprising the above-mentioned nucleic acid molecule, expression cassette, expression vector or transgenic cell line is provided.

[0029] In some embodiments of the present invention, the transgenic cell lines include transgenic cell lines of Escherichia coli cells, yeast cells, insect cells and mammalian cells.

[0030] In the fourth aspect of the present invention, a detection kit is provided, comprising the above-mentioned Fab antibody, nucleic acid molecule or expression vector or transgenic cell line.

[0031] In some embodiments of the present invention, the kit further comprises a detection antibody, and the detection antibody is an IgG monoclonal antibody.

[0032] In some embodiments of the present invention, the IgG monoclonal antibody includes mouse or human PCT IgG monoclonal antibody.

[0033] In some embodiments of the present invention, the IgG monoclonal antibody is an antibody produced by the hybridoma cell line mIgG-6A2, and the hybridoma cell line mIgG-6A2 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2017143.

[0034] In the fifth aspect of the present invention, a method for producing the above-mentioned Fab antibody is proposed, comprising the step of culturing the above-mentioned transgenic cell line to express anti-procalcitonin Fab antibody.

[0035] In some embodiments of the present invention, the transgenic cell line is a eukaryotic cell. Fab antibodies expressed in eukaryotes are more sensitive than Fab antibodies expressed in prokaryotes.

[0036] In some embodiments of the present invention, the method further comprises a purification step, wherein the purification step is to filter the anti-procalcitonin Fab antibody expressed by the transgenic cell line, add two tag sequences to the C-terminus of the heavy chain of the antibody, connect the tag sequences by a connecting peptide, obtain a fusion antibody, and subject the fusion antibody to Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography, respectively, to obtain a purified anti-procalcitonin Fab antibody. The present invention uses Ni-NTA and Strep-Tactin double-label two-step affinity chromatography for purification, and can obtain a Fab antibody with high recovery rate and high purity. The entire purification process is simple to operate, has high output, and short purification time, and is suitable for large-scale production.

[0037] In some embodiments of the present invention, the tag sequence includes His, HA, FLAG and Strep.

[0038] In some embodiments of the present invention, the tag sequences are His and Strep.

[0039] In some embodiments of the present invention, the connecting peptide comprises [GGGGS]n, wherein n is an integer of 1-3.

[0040] In some embodiments of the present invention, the connecting peptide is [GGGGS].

[0041] In some embodiments of the present invention, the connection modes of the fusion antibody include Fab antibody-His-Strep, Fab antibody-connecting peptide-His-Strep, Fab antibody-His-connecting peptide-Strep, and Fab antibody-connecting peptide-His-connecting peptide-Strep.

[0042] In the sixth aspect of the present invention, the use of the above-mentioned anti-procalcitonin Fab antibody, the nucleic acid molecule encoding the above-mentioned antibody, the above-mentioned nucleic acid molecule expression box, expression vector or transgenic cell line, and detection kit is proposed, and the application of the application in an immunoassay method.

[0043] In some embodiments of the present invention, the immunoassay method includes flow cytometry, ELISA immunoassay, immunochromatography assay, immunocytochemical staining assay and immunohistochemical staining assay.

[0044] In some embodiments of the present invention, the application is application in the preparation of a procalcitonin detection reagent.

[0045] According to the embodiments of the present invention, at least the following beneficial effects are achieved: the anti-procalcitonin Fab antibody provided by the scheme of the present invention is a fully human genetically engineered antibody, which can be effectively used to detect procalcitonin, and can also be used as a labeled antibody to form a paired antibody with other mouse PCT IgG monoclonal antibodies for clinical testing, and has the characteristics of strong specificity, high sensitivity, low cost, strong anti-interference ability, etc. The antibody obtained by the scheme of the present invention is simple to prepare and efficient, and provides important specific raw materials for diagnostic methods such as ELISA, chemiluminescence, and fluorescent immunochromatography, laying a good foundation for the development of clinical diagnostic kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 This is the plasmid map of pCom3XTT in Example 1 of the present invention;

[0048] Figure 2 This is the pComb3X Lambda plasmid map in Example 1 of the present invention;

[0049] Figure 3 This is the plasmid map of the eukaryotic expression vector pCDH-puro in Example 5 of the present invention. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] Example 1

[0052] This embodiment provides an anti-PCT genetically engineered Fab antibody.

[0053] 1. Preparation of human anti-PCT genetically engineered Fab antibody

[0054] (1) Construction of a fully human Fab phage antibody library

[0055] 1) Collect 50 mL of whole blood from a healthy person, separate lymphocytes using lymphocyte separation fluid, and take 1*10 cells. 6Total RNA was extracted from lymphocytes using Trizol (Invitrogen), and RNA was reverse transcribed into cDNA using a two-step method (Kit: PrimeScript TM RT-PCR Kit).

[0056] 2) Using human antibody Fab primers (as shown in Table 1), with the above cDNA as template, first use different light chain variable region upstream and downstream primers (including kappa (κ) chain and lambda (λ) chain) to pair respectively, the primer sequences are shown in Table 1, and amplify the corresponding light chain variable regions (VL); then use different heavy chain variable region upstream and downstream primers to pair respectively, and amplify the corresponding heavy chain variable regions (VH); then use light / heavy chain constant region upstream and downstream primers to amplify the light chain constant region (CL) and the first heavy chain constant region (CH1) (kit: All-Gold 2×TransTaq High Fidelity (HiFi) PCR SuperMix II), CL (including kappa (κ) chain and lambda (λ) chain) and CH1 templates are from pCom3XTT (as shown in Table 1). Figure 1 )、pComb3X Lambda(such as Figure 2 ) plasmid (addgen). Each light chain variable region fragment (κ chain and λ chain separately) was mixed in equal amounts, and then respectively mixed with CL by Overlap PCR to synthesize the light chain genome; similarly, each heavy chain VH fragment was mixed in equal amounts, and then mixed with CH1 by Overlap PCR to synthesize the heavy chain Fd genome, and finally the light chain gene and heavy chain Fd gene were combined by Overlap PCR to synthesize the human Fab genome.

[0057] 3) Use SfiI (purchased from NEB) endonuclease to digest the expression vectors pComb3XTT, pComb3X Lambda and Fab genome (the SfiI restriction site of pComb3XTT and pComb3X Lambda vectors contains His and HA tags). Then use T4 DNA Ligase (purchased from NEB) to connect the Fab genome of the kappa chain and lambda chain to the linearized pComb3XTT and pComb3X Lambda expression vectors. The ligation products were purified with QIAGEN gel recovery kit to remove salt ions, and the DNA was eluted with 25 μL ddH2O and stored at -20°C.

[0058] 4) Take 3 μL of the ligation product from each portion and electro-transform 50 μL of competent E. coli XL1-Blue. Electro-transform all ligation products using a 2mm electroporation cup with a voltage set to 2.5KV. Immediately after the electroporation, add 950 μL of SOC medium and incubate at 37°C, 160 rpm, for 1 hour; then transfer all to a 50 mL centrifuge tube and fill the medium to 10 mL, 37°C, 160 rpm, for 1 hour; centrifuge at 12000 rpm, 1 minute, discard the supernatant and leave only 500 μL for coating, 100 μL / plate, and incubate at 37°C overnight. Add 2 mL of LB liquid medium to each plate, gently scrape off the bacteria with a scraper, and pipette the bacterial solution into a 50 mL centrifuge tube at 4000 rpm, 10 minutes, discard the supernatant, leave 6 mL of bacterial solution, add 6 mL of 60% glycerol, resuspend and aliquot, and store in a -80°C refrigerator.

[0059] 5) Take 1 mL of glycerol bacteria and add it to 200 mL of preheated 2TY (1% glucose, 100 μg / mL ampicillin, 10 μg / mL tetracycline).

[0060] Shake at 37℃ until OD 600 About 0.4. Add 2×10 11 M13KO7 helper phage (NEB), 37℃ water bath for 30min. 3000g, centrifuge for 10min, discard the supernatant, add 100mL 2TY (1% glucose, 100μg / mL ampicillin, 10μg / mL tetracycline, 50μg / mL kanamycin), shake overnight at 30℃. After overnight culture, centrifuge at 3300g, 4℃ for 30min, add 20mL PEG / NaCl (20% PEG, 2.5M NaCl) to 80mL supernatant, mix well, and place on ice for 1h. 3300g, 4℃, centrifuge for 30min, pour out PEG / NaCl, centrifuge again, and remove PEG / NaCl as much as possible. Add 4mL PBS to resuspend the precipitate, centrifuge at 11600g, 4℃ for 10min, and remove bacterial residues. The obtained phage antibody library is divided and stored at -20℃.

[0061] Table 1: Human antibody Fab primers

[0062]

[0063]

[0064] (2) Enrichment and screening of phage antibody library

[0065] Preparation of PCT antigen: The protein sequence of PCT was obtained from NCBI (NCBI sequence number: ACCESSIONNP001029124) and the gene sequence was synthesized and connected to the true expression vector pcDNA3.1(+), and the His tag sequence was fused to the C-terminus. The above plasmid was transfected into CHOS suspension cells using the transfection reagent ExpiFectamineTM CHO reagent (gibco), and ExpiFectamineTM CHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents were added to increase protein expression. After 8-10 days of cell culture, the supernatant was collected by centrifugation at 7000RPM for 20 minutes, first filtered with a 0.8μm filter membrane, then filtered with a 0.22μm filter membrane, and finally purified through a nickel affinity column to obtain the PCT antigen.

[0066] 2) Enrichment screening: Take high affinity and specific PCT mouse monoclonal antibody mIgG-6A2 (screened by our company, the corresponding monoclonal hybridoma cell line deposit number is CCTCC NO.C2017143), coat the ELISA plate with 500ng / well, and coat overnight at 4℃; block with 3% skim milk powder·PBS and wash 3 times with 0.05% PBST; add PCT antigen, incubate at 37℃ for 2h, and then wash 3 times with 0.05% PBST; add 100μL of the prepared phage Fab antibody library and incubate at 37℃ for 2h. Discard unbound phages, and fully wash unadsorbed phages with PBST; add 100μL of Gly-HCI (pH2.2) elution solution to each well, and incubate at room temperature for 10 minutes to recover phages. Add an appropriate amount of Tris buffer to neutralize the eluted phage, add the eluted phage to 2mL of XL1-Blue bacterial solution in the logarithmic growth phase, then transfer to a triangular flask, add 10mL of SOC medium (20mg / L ampicillin, 10mg / L tetracycline), take 10μL and apply it on the ampicillin plate to titrate the phage. The rest is cultured at 37℃ for 1 hour, add 100mL SOC and shake culture at 37℃ for 1 hour, then add helper phage Ml3K07 and shake culture at 37℃ to activate the antibody library. This "adsorption-elution-amplification" process is screened 5 times.

[0067] 3) Screening of positive clones: Take the phages obtained in the last round of screening and dilute them at a certain ratio on an ampicillin culture plate. Randomly pick 50 single colonies and culture them in SOC medium containing ampicillin, and shake and culture them overnight. Take a small amount of bacteria for preservation, and add auxiliary phage VCSM13 to the rest, shake and culture, induce antibody expression, and harvest phage antibody supernatant. Coat ELISA plates with PCT to detect antigen-antibody reactions and screen positive monoclonal phage strains. The secondary antibody is HRP-labeled mouse anti-His-TAG. Finally, it was found that 12 phage antibodies can specifically bind to PCT, and the results are shown in Table 2.

[0068] 4) After amplifying the positive monoclonal phage strain, plasmid DNA was extracted according to the conventional method, and the sequences were sequenced and compared with the human antibody sequences on NCBI. The repeated sequences and incorrect sequences were removed to obtain 4 Fab sequences of anti-PCT human IgG with correct sequences, which were named hFab-1B, hFab-3C, hFab-6C and hFab-8C.

[0069] Table 2

[0070] Serial number 1A 1B 2E 3A 3C 5E 6C 6D 8C 8E 9B 9C blank Detection value 0.92 0.82 0.80 0.40 0.76 0.66 0.81 0.35 0.83 0.63 0.53 0.66 0.03

[0071] Example 2: Specificity and sensitivity analysis of anti-PCT positive Fab antibodies

[0072] 1) Four positive monoclonal phage strains with correct sequencing containing four correct anti-PCT human IgG Fab sequences (hFab-1B, hFab-3C, hFab-6C and hFab-8C) were inoculated in SOC medium containing ampicillin for expansion and culture, shaken overnight, and helper phage VCSM13 was added and shaken to induce antibody expression, and the phage antibody supernatant was harvested. The antibody in the supernatant was purified using a nickel affinity column.

[0073] 2) Specificity detection: PCT and CT (calcitonin) antigens were coated separately, and the cross-reaction of the above four antibodies to CT was detected by ELISA to detect the antibody specificity. The antigen was coated on a 96-well plate at 100 / 50ng per well, coated overnight at 4°C, and washed three times with PBST overnight. The concentrations of the four positive antibodies were adjusted to 1mg / mL, and the corresponding antibodies were added. Incubated at 37°C for 40min, and the blank control was the antibody diluent. After incubation, the plate was washed 5 times with PBST. The secondary antibody was HRP-labeled mouse anti-His-TAG, 100μL per well, incubated at 37°C for 30min. After incubation, the plate was washed 5 times with PBST. Add 100 μL of colorimetric solution to each well and incubate at 37°C for 15 min; add 50 μL of stop solution per well to terminate the colorimetric reaction and detect the absorbance. The results are shown in Table 3. It can be seen from the results that hFab-1B, hFab-3C, and hFab-6C antibodies do not cross-react with CT and are only specific for PCT antigens. Therefore, these antibodies can avoid antibody reaction with CT and reduce false positive results in clinical applications.

[0074] 3) Sensitivity test: The concentration of three antibodies (hFab-1B, hFab-3C, hFab-6C) that do not cross-react with CT was adjusted to 1 mg / mL. At the same time, ELISA was performed on the sensitivity of antigens at different dilution concentrations. The antigens were coated on 96-well plates at 100 / 50 / 25 / 12.5 ng per well, respectively, and coated overnight at 4°C. After overnight, the plates were washed three times with PBST. The corresponding antibodies were added and incubated at 37°C for 40 minutes. The blank control was the antibody diluent. After incubation, the plates were washed five times with PBST. The corresponding HRP-labeled secondary antibodies were added, 100 μL per well, and incubated at 37°C for 30 minutes. After incubation, the plates were washed five times with PBST. Add 100 μL of colorimetric solution to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution per well to terminate the colorimetric reaction and detect the absorbance. The results are shown in Table 4. It can be seen from the results that when the antigen is coated with 12.5 ng per well and hFab-1B and other three antibodies are used as primary antibodies for detection, the detection values ​​are still high, indicating that these three antibodies have high sensitivity and can meet the diagnostic requirements.

[0075] Table 3

[0076]

[0077] Table 4

[0078]

[0079] Example 4: Screening of the optimal PCT paired antibody by double antibody sandwich method

[0080] The hFab-1B, hFab-3C and hFab-6C monoclonal antibodies obtained after the above expression and purification were partially labeled with HRP and paired with the mIgG-6A2 coated antibody for ELISA detection to screen out the best paired antibody pair. First, 100 ng / well of mIgG-6A2 antibody was coated at 4°C overnight, and the plate was washed 3 times with PBST overnight. PCT antigen was added according to the gradient of 200 / 100 / 50 / 25 ng / well, a blank control well was set, and incubated at 37°C for 40 minutes. After incubation, the plate was washed 5 times with PBST. HRP-labeled antibody diluted 1000 times with enzyme label diluent was added, 100 μL / well, incubated at 37°C for 30 minutes, and the plate was washed 5 times with PBST after incubation. Add 100 μL of colorimetric solution to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution per well to terminate the colorimetric reaction and detect the absorbance. The results are shown in Table 5. It can be seen from the results that under the same conditions, the paired PCT antigen composed of mIgG-6A2 / hFab-1B has higher sensitivity, so the hFab-1B monoclonal antibody was selected for subsequent experiments.

[0081] Table 5

[0082] Coating / labeling antigen 200ng / well 100ng / well 50ng / well 25ng / well blank mIgG-6A2 / hFab-1B 2.35 1.68 1.12 0.69 0.04 mIgG-6A2 / hFab-3C 2.04 1.44 0.86 0.51 0.04 mIgG-6A2 / hFab-6C 1.79 1.27 0.80 0.44 0.03

[0083] Example 5: Comparison of the sensitivity of eukaryotically expressed and prokaryotically expressed anti-PCT recombinant Fab antibodies

[0084] 1) Construction of eukaryotic expression vector of anti-PCT recombinant Fab antibody: Using the hFab-1B positive phage antibody plasmid prepared in Example 2 as a template, the hFab-1B full-length antibody gene sequence (including the light chain leader sequence, and the His and HA tag sequences at the C-terminus) was amplified and then inserted into the eukaryotic expression vector pCDH-puro (such as Figure 3 ) between the XbaI and BamHI restriction sites, and the corresponding eukaryotic expression plasmid was constructed and named pCDH-hFab-1B.

[0085] 2) Expression of anti-PCT recombinant Fab antibody: Take well-growing CHOS suspension cells and inoculate them into a 10 cm culture dish. When the cells grow to 5*10 6 When the density is about 100 μg / mL, the cells are inoculated into 250 mL conical flasks and cultured in a shaking incubator with a volume of 50 mL. The culture conditions are: 130 RPM, 5% carbon dioxide, and 37°C. When the cells grow to 5-7*10 6When the density is about 1.577 nm, 25 μg of the above recombinant plasmids (eukaryotic expression plasmid pCDH-hFab-1B and prokaryotic expression plasmid hFab-1B positive phage antibody plasmid) were transfected using the transfection reagent ExpiFectamineTMCHO reagent (gibco) according to the manufacturer's instructions. After 24 hours of transfection, add ExpiFectamineTMCHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents according to the manufacturer's instructions to increase antibody expression. After 8-10 days of culture, centrifuge at 7000RPM for 20 minutes to collect the supernatant, first filter with a 0.8μm filter membrane, then filter with a 0.22μm filter membrane, and purify the antibody in the supernatant with a nickel affinity column.

[0086] 3) Comparison of sensitivity of eukaryotic and prokaryotic expressed anti-PCT recombinant Fab antibodies: The concentration of eukaryotic and prokaryotic expressed hFab-1B antibodies was adjusted to 1 mg / mL, and the sensitivity of PCT antigens at different dilution concentrations was tested by ELISA. The antigens were coated on 96-well plates at 100 / 50 / 25 / 12.5 ng per well, respectively, and coated overnight at 4°C. After overnight, the plates were washed 3 times with PBST. The corresponding antibodies were added and incubated at 37°C for 40 minutes. The blank control was the antibody diluent. After incubation, the plates were washed 5 times with PBST. The corresponding HRP-labeled mouse anti-His-TAG was added, 100 μL per well, and incubated at 37°C for 30 minutes. After incubation, the plates were washed 5 times with PBST. Add 100 μL of colorimetric solution to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution per well to terminate the colorimetric reaction and detect the absorbance. The results are shown in Table 6. It can be seen from the results that the hFab-1B antibody expressed in eukaryotes is more sensitive than the hFab-1B antibody expressed in prokaryotes.

[0087] Table 6

[0088]

[0089] Example 6: Rapid affinity purification of anti-PCT recombinant Fab antibodies

[0090] 1) Construction of eukaryotic expression vector of anti-PCT recombinant Fab antibody: Using the above hFab-1B positive phage antibody plasmid as a template, different sequences were added to the C-terminus of the hFab-1B full-length antibody gene sequence (including the light chain leader sequence) by multiple PCR methods to obtain the following four recombinant hFab-1B antibody sequences: hFab-1B-His-Strep, hFab-1B-linker-His-Strep, hFab-1B-His-linker-Strep and hFab-1B-linker-His-linker-Strep; wherein His represents the tag sequence HHHHHH, linker represents the flexible linker peptide GGGGS, and Strep is the tag sequence WSHPQFEK. Then, the above recombinant genes were inserted into the eukaryotic expression vector pCDH-puro (such as Figure 3 ) between the XbaI and BamHI restriction sites, and the corresponding eukaryotic expression plasmids were constructed, and the plasmids were named pCDH-hFab-1B-His-Strep, pCDH-hFab-1B-linker-His-Strep, pCDH-hFab-1B-His-linker-Strep and pCDH-hFab-1B-linker-His-linker-Strep, respectively.

[0091] 2) Expression of anti-PCT recombinant Fab antibody: Take well-growing CHOS suspension cells and inoculate them into a 10 cm culture dish. When the cells grow to 5*10 6 When the density is about 100 μg / mL, the cells are inoculated into 250 mL conical flasks and cultured in a shaking incubator with a volume of 50 mL. The culture conditions are: 130 RPM, 5% carbon dioxide, and 37°C. When the cells grow to 5-7*10 6 When the density is about 1.5, 25 μg of each of the above four recombinant Fab antibody positive plasmids were transfected using the transfection reagent ExpiFectamineTMCHO reagent (gibco). The transfection process was carried out according to the manufacturer's instructions. 24 hours after transfection, ExpiFectamineTMCHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents were added according to the manufacturer's instructions to increase antibody expression. After 8-10 days of culture, the supernatant was collected by centrifugation at 7000RPM for 20 minutes, first filtered with a 0.8μm filter membrane, and then filtered with a 0.22μm filter membrane. A small amount of supernatant was taken and saved for detection of the target antibody content, and the rest was used for antibody purification.

[0092] 3) Purification of anti-PCT recombinant Fab antibody: The obtained cell expression supernatant was subjected to double-label two-step affinity chromatography purification, and Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography were performed in sequence. The specific steps are as follows:

[0093] 1. Ni-NTA affinity chromatography:

[0094] ① Preparation of Ni column: Load Ni column resin nitrilotriacetic acid (NTA) into the column, rinse with 5V pure water, resuspend the resin, and wait for the pure water to flow out naturally; fill the column with 2% nickel chloride, wait for the liquid to flow out naturally, then fill it with 2% nickel sulfate again, soak for 10 minutes and then let the liquid flow out, the column turns green; rinse with 5V pure water to remove excess Ni; balance with 5V balancing solution (20mM Tris-HCl pH 7.4, 150mM NaCl, 10% glycerol, 20mM imidazole).

[0095] ② Adsorption: Carefully add the sample to the Ni column along the wall.

[0096] ③ Washing: After loading, balance the column with 5V balance solution to wash away impurities that are not bound to the column until the OD value of the outflow liquid is 280 <0.1. Collect the outflow liquid and keep the sample.

[0097] ④ Elution: Elute the protein with elution buffer containing 50 / 100 / 200 / 500 mM imidazole (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 50 / 100 / 200 / 500 mM imidazole) respectively; collect the eluate until OD 280 <0.1; a small amount of elution solution was taken and saved for detecting antibody recovery rate and antibody concentration; SDS-PAGE detection showed that most of the target protein was eluted at a concentration of 200mM imidazole.

[0098] 2. Strep-Tactin affinity chromatography:

[0099] ① Balance: Load an appropriate amount of Strep-Tactin agarose gel into the column, wash with 5V pure water to remove the preservation solution, and then balance with 5V balance solution (100mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH8.0).

[0100] ② Adsorption: Take an appropriate amount of sample eluted from the Ni-NTA column under 200mM imidazole concentration conditions, dialyze it using equilibrium solution (100mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH8.0), and then add it to the Strep-Tactin column along the wall after dialysis is completed.

[0101] ③ Washing: After loading, use 10V equilibration solution to equilibrate the column and wash away the impurities that are not bound to the column.

[0102] ④ Elution: Elute the protein with 10-15V elution buffer (balance solution + 2.5mM desthiobiotin); collect the eluate until OD 280 <0.1; save a small amount of eluate for testing antibody recovery rate and antibody concentration.

[0103] ⑤ Regeneration: Wash with 15 column volumes of equilibrium solution containing 1 mM HABA, then wash with 30V equilibrium solution, and then preserve the column with 20% ethanol.

[0104] 4) Antibody recovery rate and purity detection: PCT was coated on the ELISA plate, and the antibody concentration of the sample solution and the eluate before and after each affinity chromatography was detected by ELISA detection method, and then the total antibody amount and recovery rate were calculated; at the same time, the gel electrophoresis results were scanned and analyzed after Coomassie Brilliant Blue staining, and the protein purity was calculated according to the grayscale. The results are shown in Table 7, which show that when Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography were used for purification in sequence, the purity of the four recombinant antibodies was greatly improved, and the loss of antibodies in the second step of affinity purification was less; among the four recombinant antibodies, the hFab-1B-linker-His-linker-Strep recombinant antibody containing two flexible linker peptides GGGGS had the highest total recovery rate and purity.

[0105] Table 7

[0106]

[0107] Example 6: Construction of a stable expression cell line of anti-PCT recombinant Fab antibody

[0108] 1) Construction of CHOS stable expression cell line of anti-PCT recombinant Fab antibody: Take well-growing CHOS suspension cells and inoculate them into 6-well plates. When the cells grow to 70-90% density, use the transfection reagent ExpiFectamineTM CHO reagent (gibco) to transfect 2μg of the previously constructed pCDH-hFab-1B-linker-His-linker-Strep eukaryotic expression plasmid. The transfection process is carried out according to the manufacturer's instructions. 24 hours after transfection, the cells in each 6-well plate are evenly spread into 4 96-well plates for continued culture, 100μL of culture medium is added to each well, and 5μg / mL of puromycin (puro) is added to the culture medium for screening. After 3-4 weeks, the surviving cells form clones, take a 10μL pipette and use the tip to pick up a single cell group, add it to another 96-well plate, and replace it with a low concentration of 1μg / mL puromycin screening culture medium to maintain pressure, add 100μL culture medium to each well, and continue to culture. After the cells are almost full, 50 μL of cell culture supernatant is collected and the antibody expression in the supernatant is detected by ELISA method. The wells with high detection values ​​are selected, and the cells are transferred to 24-well plates for continued culture. After the cells are full, 50 μL of cell culture supernatant is collected and the antibody expression in the supernatant is detected by ELISA method. The wells with high detection values ​​are selected and the cells are transferred to 6-well plates for continued culture. After the cells are full, 50 μL of cell culture supernatant is collected and the antibody expression in the supernatant is detected by ELISA method. Finally, the two cell lines with the highest detection values ​​were selected and plated in 96-well plates using the limiting dilution method. The cell density was 0.5 cells per well. After about 2 weeks, the cells were almost full. 50 μL of cell culture supernatant was collected and the antibody expression in the supernatant was detected by ELISA. According to the above-mentioned operating procedures, the cells in the wells with high detection values ​​were selected in turn and transferred to 24 / 6-well plates for culture and screening of high-expressing cell lines. The process of limiting dilution screening of monoclonal cell lines was repeated 3 times. Finally, 3 to 5 positive stable high-expressing cell lines were selected for expansion culture and cryopreserved for seed preservation. The cell line was named CHOS-hFab-1B.

[0109] 3) Extract total RNA from the monoclonal cell line obtained by the above screening, amplify the recombinant Fab gene by PCR, compare the target gene, and finally confirm that the obtained recombinant Fab gene sequence is consistent with the gene sequence in the eukaryotic expression vector.

[0110] 4) Resuscitate the stable cell lines obtained by the above screening into 10 cm culture dishes and grow the cells to 5×10 6 When the density was about 5 × 10 cells / mL, the cells were inoculated into 250 mL conical flasks and continued to be cultured in a shaking incubator under the following conditions: 130 RPM, 5% carbon dioxide, and 37°C. 6When the density is about 1.5, the cells are inoculated into 2000mL conical flasks and cultured in a shaking incubator under the same conditions. The volume of the cell fluid does not exceed 1 / 4 of the flask capacity. After 6-7 days of culture, the supernatant is collected by centrifugation at 7000rpm, first filtered with a 0.8μm filter membrane, and then filtered with a 0.22μm filter membrane. The supernatant is finally purified by Ni-NTA+Strep-Tactin double-label two-step affinity chromatography. The obtained antibody is named hFab-1B-linker-His-linker-Strep.

[0111] The hFab-1B-linker-His-linker-Strep antibody of the present embodiment is composed of a light chain and a heavy chain. The heavy chain variable region of the heavy chain has three complementary determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is: GYTFTEYT (SEQ ID NO.1), the amino acid sequence of CDR2 is: WPNTGD (SEQ ID NO.2), and the amino acid sequence of CDR3 is: YPIYYDFSHAMDY (SEQ ID NO.3); the light chain variable region of the light chain has three complementary determining regions CDR1', CDR2' and CDR3', wherein the amino acid sequence of CDR1' is: KSSQSVLYSSNQKNYLA (SEQ ID NO.4), the amino acid sequence of CDR2' is: WASSRDS (SEQ ID NO.5), and the amino acid sequence of CDR3' is HQYLSSYT (SEQ ID NO.6).

[0112] Amino acid sequence of the antibody heavy chain variable region:

[0113] EVQLVESGGGGVQPGGSLRLSCAASGYTFTEYTMHWVRQAPGKGLEWIGGIWPNTGDTYYADSVKGRFTISSRDNAKNSLYLQMNSLKTEDTAVYYCARYPIYYDFSHAMDYWGQGTTVTVSSASTKGP (SEQ ID NO. 7).

[0114] Amino acid sequence of the antibody heavy chain constant region:

[0115] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS (SEQ ID NO. 9).

[0116] The amino acid sequence of the antibody light chain variable region:

[0117] EIVLTQSPGTLSLSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASSRDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCHQYLSSYTGQGTKVELKRT (SEQ ID NO. 8).

[0118] The amino acid sequence of the antibody light chain constant region:

[0119] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSLLPVTKSFNRGEC (SEQ ID NO. 10).

[0120] Amino acid sequence of antibody light chain:

[0121] EIVLTQSPGTLSLSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASSRDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCHQYLSSYTGQGTKVELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC(SEQ ID NO.12).

[0122] Amino acid sequence of antibody heavy chain:

[0123] EVQLVESGGGGVQPGGSLRLSCAASGYTFTEYTMHWVRQAPGKGLEWIGGIWPNTGDTYYADSVKGRFTISRDNAKNSLYLQMNSLKTEDTAVYYCARYPIYYDFSHAMDYWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAGQGGGGSHHHHHHGGGGSWSHPQFEK(SEQ ID NO.11).

[0124] Nucleotide sequence of the heavy chain variable region of the antibody:

[0125] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGAGGAGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATATACCTTTACCGAGTATACCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGATTGGTGGGATTTGGCCGAATACCGGTGATACCTACTATGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCAAAAAATTCCTTGTATCTGCAAATGAACAGTCTGAAGACCGAGGACACCGCCGTGTATTACTGTGCACGGTATCCGATCTATTATGACTTTAGTCACGCAATGGACTACTGGGGCCAGGGAACCACCGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA(SEQ ID NO.13).

[0126] Nucleotide sequence of the variable region of the light chain of the antibody:

[0127] ATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAAATCTAGTCAGAGTGTTCTCTACAGCTCTAACCAGAAAAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACTGGGCATCCAGCAGGGACTCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCATCAGTATCTGTCTTCCTACACGGGCCAAGGGACCAAGGTGGAACTCAAACGAACT(SEQ ID NO.14).

[0128] Nucleotide sequence of the constant region of the heavy chain of the antibody:

[0129] TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT(SEQ ID NO.15).

[0130] Nucleotide sequence of the constant region of the light chain of the antibody:

[0131] GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQ ID NO.16).

[0132] Nucleotide sequence of the heavy chain of the antibody:

[0133] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGAGGAGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATATACCTTTACCGAGTATACCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGATTGGTGGGATTTGGCCGAATACCGGTGATACCTACTATGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCAAAAAATTCCTTGTATCTGCAAATGAACAGTCTGAAGACCGAGGACACCGCCGTGTATTACTGTGCACGGTATCCGATCTATTATGACTTTAGTCACGCAATGGACTACTGGGGCCAGGGAACCACCGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTAGTGGCCAGGCCGGCCAGGGAGGCGGGGGATCCCACCATCACCATCACCATGGCGGCGGAGGATCCTGGAGCCACCCGCAGTTCGAAAAGTAG(SEQ ID NO.17).

[0134] Nucleotide sequence of the light chain of the antibody:

[0135] (SEQ ID NO.18).

[0136] 5) ELISA plates were coated with PCT to detect antigen-antibody reactions. The results are shown in Table 8. As can be seen from Table 8, the recombinant PCT antibody can react significantly with the PCT antigen.

[0137] Table 8

[0138]

[0139] Example 7: Detection of clinical samples with anti-PCT recombinant Fab antibody and mouse PCT IgG monoclonal antibody

[0140] The mIgG-6A2 antibody was used as the coating antibody, and the fluorescent microsphere-labeled hFab-1B-linker-His-linker-Strep (hereinafter referred to as hFab-1B) was used as the labeling antibody to prepare a fluorescent immunochromatographic quantitative detection kit for mIgG / hFab paired antibodies; the control group used the mIgG-6A2 antibody as the coating antibody, and another PCT mouse monoclonal antibody mIgG-9C2 as the labeling antibody to prepare a fluorescent immunochromatographic quantitative detection reagent for double mIgG paired antibodies. Then the two kits were compared in terms of non-specificity, minimum detection limit and linear range for the detection of clinical samples.

[0141] 1) Nonspecificity and minimum detection limit

[0142] Take 5 normal human whole blood, plasma and serum reference materials without procalcitonin, take 30 μL of each, add to 200 μL sample treatment solution, blow and mix, and take 80 μL of the mixture and add it to the sample wells of the test cards with two different antibody pairs, react for 15 minutes, and detect using a fluorescent immunoassay analyzer. Collect and determine the results, calculate the mean and minimum detection limit of the reference product determination results, and the results are shown in Table 9. The results show that the T / C value of the antibody pairing of mIgG-6A2 / hFab-1B is lower than that of the antibody pairing of mIgG-6A2 / mIgG-9C2, indicating that the antibody pairing has lower background and non-specificity when detecting these three clinical samples. According to the regression of the fitting curve, the average minimum detection limit concentration value of the mIgG-6A2 / hFab-1B antibody pairing is calculated to be 0.036 ng / mL, which meets the technical requirements of the sensitivity of the procalcitonin fluorescence kit.

[0143] Table 9

[0144]

[0145]

[0146] 2) Linear range

[0147] Take 6 kinds of reference samples of 0.5ng / mL, 2.0ng / mL, 5.0ng / mL, 10.0ng / mL, 50.0ng / mL, and 100.0ng / mL, take 30μL of each, add it to 200μL sample treatment solution, blow and mix, take 80μL of the mixture and add it to the sample wells of 2 test cards, react for 15min, and use fluorescent immunoassay to detect. Each sample is measured 3 times, the average of the 3 results is taken, and the logarithmic scatter plot of the theoretical concentration value and the actual detection value is made, and the fitting curve is made to calculate the linear equation y=ax+b and the correlation coefficient R. The experimental results are shown in Table 10. It can be seen from the results that the R value of the kit paired with mIgG-6A2 / hFab-1B antibody is higher than that of the kit paired with mIgG-6A2 / mIgG-9C2 antibody, and is closer to 1, indicating that the antibody pairing has a better linear result, indicating that the kit prepared by the antibody has good stability.

[0148] Table 10

[0149]

[0150]

[0151] Example 9: Anti-interference detection of clinical samples using anti-PCT recombinant Fab antibody and mouse PCT IgG monoclonal antibody

[0152] The PCT content index can not only be used to judge early bacterial and non-bacterial infections and inflammations, but also to diagnose bacterial infections or sepsis in patients with high infection risks or intensive care. However, Staphylococcus aureus cell wall surface antigens exist in Staphylococcus A protein (SPA). In the clinical detection of PCT, when bacteria containing SPA protein are present in clinical samples, the protein can non-specifically bind to the Fc segments of IgG1, IgG2 and IgG4 molecules commonly used in diagnosis in humans and various mammals. The Fab segments of the bound IgG molecules are exposed to the surface of the bacteria and can still specifically bind to the PCT antigen, resulting in a synergistic agglutination phenomenon, which ultimately affects the accuracy and specificity of the detection. At the same time, interfering substances such as human anti-mouse antibodies (HAMAS) and rheumatoid factors (RF) present in blood test samples can also bind to the detection antibodies, which also affects the accuracy and specificity of the detection. This embodiment takes SPA, HAMAS and RF interference sources as examples to compare the anti-interference ability of Fab antibodies and mouse PCT IgG monoclonal antibodies as marker antibodies in diagnosis.

[0153] (1) SPA disruptors

[0154] Take SPA-positive CowanI strain (purchased from ASI 1476) or SPA-negative Wood46 strain (purchased from ASI 1477) and inoculate it on agar slant medium, and culture it at 37℃ for 18-24h; wash the bacteria with a small amount of sterile saline, centrifuge at 4000r / min for 15min, discard the supernatant, wash the precipitated bacteria with sterile saline for 3 times, and then make a 10% (V / V) suspension with 0.01M PBS buffer containing 0.5% formalin, and place it at room temperature for 3h; place the suspension in a 56℃ water bath for 30min, centrifuge at 4000r / min for 15min, and then wash it with PBS for 3 times. Finally, make a 10% (V / V) suspension with PBS buffer containing 0.1% NaN3, which is the SPA bacteria stabilization solution, and place it in a 4℃ refrigerator for use.

[0155] Take 30 μL of healthy human serum reference without procalcitonin, add it to 200 μL sample treatment solution, then add different proportions of positive or negative SPA bacteria stabilizing solution (samples 1-5 are added with 40, 8, 2, 0.5, 0.2 μL positive SPA bacteria stabilizing solution respectively; samples 6-10 are added with 40, 8, 2, 0.5, 0.2 μL positive SPA bacteria stabilizing solution respectively), pipette and mix, and take 80 μL of the mixture and add it to the sample wells of the above two different antibody paired test cards, react for 15 minutes, and use a fluorescent immunoassay analyzer for detection. Collect and determine the results, calculate the T / C value, and the results are shown in Table 11. The results showed that the T / C values ​​of the mIgG-6A2 / hFab-1B antibody paired kit were basically the same when the samples contained positive or negative SPA bacteria, while the T / C value of the mIgG-6A2 / mIgG-9C2 antibody paired kit was higher when the samples contained positive SPA bacteria than when the samples contained negative SPA bacteria, resulting in false positives, indicating that Fab antibodies have better anti-SPA interference ability as labeled antibodies in diagnosis.

[0156] Samples 1-5: 30μL serum reference + 40 / 8 / 2 / 0.5 / 0.2μL positive SPA bacteria stabilization solution + 200μL sample treatment solution; Samples 6-10: 30μL serum reference + 40 / 8 / 2 / 0.5 / 0.2μL negative SPA bacteria stabilization solution + 200μL sample treatment solution; Use PBS buffer to adjust the final volume to the same. The blank control is the sample diluent.

[0157] Table 11

[0158]

[0159] (2) HAMAS Interferors

[0160] Take 30 μL of each of the three HAMAS positive or negative sera without procalcitonin (samples 1 to 3 are HAMAS positive sera, and samples 4 to 6 are HAMAS negative sera), add them to 200 μL of sample treatment solution, blow and mix, and take 80 μL of the mixture and add them to the sample wells of the test card paired with the above mIgG-6A2 / hFab-1B or mIgG-6A2 / mIgG-9C2 antibodies, react for 15 minutes, and use a fluorescent immunoassay analyzer for detection. Collect and determine the results, calculate the T / C value, and the results are shown in Table 12. The results show that the T / C values ​​of the mIgG-6A2 / hFab-1B antibody paired kit are basically the same when detecting HAMAS positive or negative samples, while the T / C value of the mIgG-6A2 / mIgG-9C2 antibody paired kit when detecting HAMAS positive samples is higher than the T / C value of the HAMAS negative sample, and a false positive is detected, indicating that the Fab antibody has a better ability to resist HAMAS interference as a labeled antibody in diagnosis.

[0161] Samples 1-3: 30 μL HAMAS positive serum + 200 μL sample treatment solution; samples 4-6: 30 μL HAMAS negative serum + 200 μL sample treatment solution; blank control is sample diluent.

[0162] Table 12

[0163]

[0164]

[0165] (3) RF interference

[0166] Take 30 μL of each of the RF positive and negative sera without procalcitonin (samples 1 to 3 are RF positive sera, and samples 4 to 6 are RF negative sera), add them to 200 μL of sample treatment solution, blow and mix, and take 80 μL of the mixture and add them to the sample wells of the test card paired with the above mIgG-6A2 / hFab-1B or mIgG-6A2 / mIgG-9C2 antibodies, react for 15 minutes, and use a fluorescent immunoassay analyzer to detect. Collect and determine the results, calculate the T / C value, and the results are shown in Table 13. The results show that the T / C values ​​of the mIgG-6A2 / hFab-1B antibody paired kit are basically the same when detecting RF positive or negative samples, while the T / C value of the mIgG-6A2 / mIgG-9C2 antibody paired kit when detecting RF positive samples is higher than the T / C value of the RF negative sample, and a false positive is detected, indicating that the Fab antibody as a labeled antibody has better anti-RF interference ability in diagnosis.

[0167] Samples 1-3: 30 μL RF-positive serum + 200 μL sample treatment solution; Samples 4-6: 30 μL RF-negative serum + 200 μL sample treatment solution; blank control is sample diluent.

[0168] Table 13

[0169]

[0170] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-procalcitonin Fab antibody, characterized in that The Fab antibody is composed of a light chain and a heavy chain, and the heavy chain variable region of the heavy chain has three complementary determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the light chain variable region of the light chain has three complementary determining regions CDR1', CDR2' and CDR3', wherein the amino acid sequence of CDR1' is shown in SEQ ID NO.4, the amino acid sequence of CDR2' is shown in SEQ ID NO.5, and the amino acid sequence of CDR3' is shown in SEQ ID NO.

6.

2. The Fab antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the heavy chain is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the light chain is shown in SEQ ID NO.8; the amino acid sequence of the heavy chain constant region of the Fab antibody is shown in SEQ ID NO.9, and the amino acid sequence of the light chain constant region of the Fab antibody is shown in SEQ ID NO.

10.

3. The Fab antibody according to claim 1 or 2, characterized in that The amino acid sequence of the heavy chain of the Fab antibody is shown in SEQ ID NO.11; the amino acid sequence of the light chain of the Fab antibody is shown in SEQ ID NO.

12.

4. A nucleic acid molecule encoding the Fab antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region of the Fab antibody is shown in SEQ ID NO.13, and the nucleotide sequence encoding the light chain variable region of the Fab antibody is shown in SEQ ID NO.14; the nucleotide sequence encoding the heavy chain constant region of the Fab antibody is shown in SEQ ID NO.15, and the nucleotide sequence encoding the light chain constant region of the Fab antibody is shown in SEQ ID NO.16; the nucleotide sequence encoding the heavy chain of the Fab antibody is shown in SEQ ID NO.17, and the nucleotide sequence encoding the light chain of the Fab antibody is shown in SEQ ID NO.

18.

6. An expression cassette, expression vector or transgenic cell line containing the nucleotide molecule according to claim 4 or 5.

7. A detection kit, characterized in that: It comprises the Fab antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, or the expression cassette, expression vector or transgenic cell line according to claim 6.

8. The detection kit according to claim 7, characterized in that The kit also includes a detection antibody, which is an IgG monoclonal antibody; the IgG monoclonal antibody is an antibody produced by the hybridoma cell line mIgG-6A2, and the hybridoma cell line mIgG-6A2 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2017143.

9. A method for producing a Fab antibody according to any one of claims 1 to 3, characterized in that: The method comprises the step of culturing the transgenic cell line as claimed in claim 6 to express Fab antibodies.

10. The production method according to claim 9, characterized in that: The production method also includes a purification step, wherein the purification step is to filter the anti-procalcitonin Fab antibody expressed by the transgenic cell line, add two tag sequences to the C-terminus of the heavy chain of the Fab antibody, connect the tag sequences by connecting peptides to obtain a fusion antibody, and respectively subject the fusion antibody to Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography to obtain a purified anti-procalcitonin Fab antibody.

11. Use of the Fab antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the expression cassette, expression vector or transgenic cell line according to claim 6 or the detection kit according to claim 7 or 8 in the preparation of a procalcitonin detection reagent.

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