CN116178543A

CN116178543A - Anti-procalcitonin Fab antibody and preparation method and application thereof

Info

Publication number: CN116178543A
Application number: CN202211329639.5A
Authority: CN
Inventors: 李小锋; 李晨阳; 覃振俏
Original assignee: Guangzhou Rida Biotech Co ltd
Current assignee: Guangzhou Rida Biotech Co ltd
Priority date: 2022-10-27
Filing date: 2022-10-27
Publication date: 2023-05-30

Abstract

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

Description

Anti-procalcitonin Fab antibody and preparation method and application thereof

Technical Field

The invention belongs to the field of antibodies, and particularly relates to an anti-procalcitonin Fab antibody, and a preparation method and application thereof.

Background

Procalcitonin (PCT), a hormone-free glycoprotein, is a precursor of Calcitonin (CT), and clinically PCT is used as a highly sensitive and specific index for diagnosis of severe systemic bacteria, fungi, parasites, acute malaria infection, systemic Inflammatory Response Syndrome (SIRS), multiple organ failure syndrome (MDS), etc. PCT is generally present in low levels in human blood and is elevated in plasma upon stimulation by endotoxin and inflammatory cytokines in severe bacterial, fungal, parasitic infections, as well as sepsis and multiple organ failure, but is not generally elevated in non-infectious inflammatory states, locally limited bacterial infections, mild infections, or chronic inflammation. PCT can thus be used as an index not only for diagnosing early bacterial and non-bacterial infections and inflammations, meanwhile, the method has important reference value for diagnosing bacterial infection or sepsis of patients with high infection risk or intensive care, prognosis evaluation, treatment monitoring and the like of severe inflammatory diseases.

The commonly used procalcitonin determination method comprises fluorescence immunochromatography, ELISA, chemiluminescence immunoassay and turbidimetry. The detection sample is usually human whole blood, serum or plasma sample, and in the detection process, the detection method, sample treatment, reagent components and other factors often influence, such as poor specificity of the coated antibody, nonspecific binding of interfering substances in the sample, and finally false positive or over-high background conditions occur in the detection result. To reduce interference, a great deal of time and effort is typically required to screen for highly specific and interference-resistant antibodies; or the detection sample and the antibody raw material are pretreated before detection, and when large-scale clinical examination is carried out, the pretreatment loop of the sample can increase the consumption of reagents, increase the workload, further increase the detection time, and are unfavorable for rapid diagnosis.

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 construction of the Fab includes a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a first heavy chain constant region (CH 1). The Fc segment determines the effector function of antibodies and can be combined with Fc receptors (such as FcRn) on the surfaces of cells such as macrophages and NK cells and complement in serum. In clinical detection, the Fc segment can be combined with human anti-mouse antibodies (HAMAS), rheumatoid Factors (RF), staphylococcus A protein (SPA) and other interfering substances in a non-specific manner, so that the specificity and accuracy of antibody detection are affected. The Fab fragment contains an antigen binding region and a partial constant region, so that the Fab fragment not only has the same antibody-antigen affinity as the original whole antibody, but also has smaller molecular volume and stronger tissue penetrating power; the lack of an Fc region reduces nonspecific binding to an interfering substance such as an Fc receptor, and thus has a great effect on clinical diagnosis.

The Fab antibody can be obtained by purifying after degrading the whole monoclonal antibody by papain, the method is rapid and simple, but the required monoclonal antibody has higher cost, and meanwhile, the Fab antibody obtained by the procedures of enzyme degradation, purification and the like can lose certain immunoreactivity. Meanwhile, the Fab antibody does not contain an Fc end, so that post-translational modification and glycosylation are not needed, and the Fab antibody can be expressed in a mammalian system and a prokaryotic system. The escherichia coli expression system has the advantages of low production cost, high production speed and the like, but the antibody expression level is low, inclusion bodies are easy to form, and the activity after renaturation is difficult to ensure. The expression in mammalian cells can be correctly folded, the structure is closer to a natural Fab structure, the activity is higher, and the expression quantity can meet the requirement. The conventional Fab purification method mainly comprises ion exchange chromatography, hydrophobic chromatography and the like, but the problems of antibody recovery rate, antibody purity, mass production, purification time, cost and the like cannot be simultaneously considered, so that the method has certain limitations.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. For this purpose, the invention proposes an anti-procalcitonin Fab antibody.

The invention also provides a nucleic acid molecule for encoding the antibody.

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

The invention also provides a detection kit containing the Fab antibody, the nucleic acid molecule or the expression vector or the transgenic cell line.

The invention also provides a production method of the Fab antibody.

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

In one aspect of the present invention, an anti-procalcitonin Fab antibody is provided, which is composed of a light chain and a heavy chain, wherein the heavy chain variable region of the heavy chain has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown as SEQ ID No.1, the amino acid sequence of CDR2 is shown as SEQ ID No.2, and the amino acid sequence of CDR3 is shown as SEQ ID No. 3; the light chain variable region of the light chain is provided with 3 complementarity determining regions CDR1', CDR2' and CDR3', wherein the amino acid sequence of the CDR1' is shown as SEQ ID NO.4, the amino acid sequence of the CDR2 'is shown as SEQ ID NO.5, and the amino acid sequence of the CDR3' is shown as SEQ ID NO. 6.

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

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

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

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

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

In some embodiments of the invention, the C-terminal of the heavy chain is further fused to two tag sequences.

In some embodiments of the invention, the tag sequences include His, HA, FLAG and Twin-Strep.

In some embodiments of the invention, the tag sequences are linked by a linking peptide.

In some embodiments of the invention, the linker peptide is [ GGGGS ] n, wherein n is an integer from 1 to 3.

In some embodiments of the invention, the linking peptide is [ GGGGS ].

In a second aspect of the invention, nucleic acid molecules encoding the above antibodies are provided.

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

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

In some embodiments of the invention, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID NO.17 and the nucleotide sequence encoding the light chain of the antibody is shown in SEQ ID NO. 18.

In a third aspect of the invention, a nucleic acid molecule, an expression cassette, an expression vector or a transgenic cell line comprising the above is presented.

In some embodiments of the invention, the transgenic cell lines include transgenic cell lines of E.coli cells, yeast cells, insect cells, and mammalian cells.

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

In some embodiments of the invention, the kit further comprises a detection antibody that is IgG monoclonal antibody.

In some embodiments of the invention, the IgG mab comprises a murine or human PCT IgG mab.

In some embodiments of the invention, the IgG monoclonal antibody is an antibody produced by hybridoma cell line mIgG-6A2, wherein the hybridoma cell line mIgG-6A2 is preserved in China Center for Type Culture Collection (CCTCC) NO: C2017143.

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

In some embodiments of the invention, the transgenic cell line is a eukaryotic cell. Eukaryotic-expressed Fab antibodies are more sensitive than prokaryotic-expressed Fab antibodies.

In some embodiments of the invention, the method further comprises a purification step, wherein after the transgenic cell line expressing the procalcitonin Fab antibody is filtered, two tag sequences are added at the C end of the heavy chain of the antibody, the tag sequences are connected through a connecting peptide to obtain a fusion antibody, and the fusion antibody is subjected to Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography respectively to obtain the purified procalcitonin Fab antibody. The invention uses Ni-NTA and Strep-Tactin double-label two-step affinity chromatography to purify, thereby obtaining the Fab antibody with high recovery rate and high purity, the whole purification process is simple and convenient to operate, the output is high, the purification time is short, and the invention is suitable for mass production.

In some embodiments of the invention, the tag sequences include His, HA, FLAG and Strep.

In some embodiments of the invention, the tag sequences are His and Strep.

In some embodiments of the invention, the linker peptide comprises [ GGGGS ] n, wherein n is an integer from 1 to 3.

In some embodiments of the invention, the linking peptide is [ GGGGS ].

In some embodiments of the invention, the means of attachment of the fusion antibody comprises Fab antibody-His-Strep, fab antibody-linker-His-Strep, fab antibody-His-linker-Strep, fab antibody-linker-His-linker-Strep.

In a sixth aspect of the invention, the use of the above-mentioned anti-procalcitonin Fab antibodies, nucleic acid molecules encoding the above-mentioned antibodies, the above-mentioned nucleic acid molecule expression cassettes, expression vectors or transgenic cell lines, detection kits is presented, said use being in an immunoassay method.

In some embodiments of the invention, the immunoassay method comprises flow cytometry, ELISA immunoassay, immunochromatographic assay, immunocytochemical staining assay, and immunohistochemical staining assay.

In some embodiments of the invention, the use is in the preparation of a procalcitonin detection reagent.

According to an embodiment of the invention, at least the following advantages are achieved: the Fab antibody of procalcitonin provided by the scheme of the invention is a fully human genetic engineering antibody, can be effectively used for detecting procalcitonin, can be used as a matched antibody formed by a labeled antibody and other mouse PCT IgG monoclonal antibodies for clinical detection, and has the characteristics of strong specificity, high sensitivity, low cost, strong anti-interference capability and the like. The antibody obtained by the scheme of the invention has simple and efficient preparation, provides important specific raw materials for diagnosis methods such as ELISA, chemiluminescence, fluorescence immunochromatography and the like, and lays a good foundation for the development of clinical diagnosis kits.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a map of pCom3XTT plasmid in example 1 of the present invention;

FIG. 2 is a map of pComb3X Lambda plasmid in example 1 of the present invention;

FIG. 3 is a plasmid map of the eukaryotic expression vector pCDH-puro in example 5 of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

Example 1

This example provides an anti-PCT genetically engineered Fab antibody.

1. Preparation of humanized anti-PCT genetic engineering Fab antibody

(1) Construction of fully human Fab phage antibody library

1) 50mL of whole blood of healthy people is collected, lymphocyte separation liquid is used for separating lymphocyte, and the cell number is 1 x 10 ⁶ Total RNA was extracted from lymphocytes of (a) using Trizol (Invitrogen), and the RNA was reverse transcribed into cDNA (kit: primeScript) using a 2-step method ^TM RT-PCR Kit)。

2) Using the human antibody Fab primer (shown in Table 1), using the cDNA as template, first using different light chain variable region upstream and downstream primers (including kappa (kappa) chain and lambda (lambda) chain) to pair, and amplifying the corresponding light chain variable region (VL) with the primer sequence shown in Table 1; then, respectively pairing the upstream and downstream primers of different heavy chain variable regions, and amplifying corresponding heavy chain variable regions (VH); the light chain constant region (CL) and the first heavy chain constant region (CH 1) (kit: full gold 2X TransTaq High Fidelity (HiFi) PCR Supermix II), CL (including kappa (kappa) and Lambda (Lambda) chains) and CH1 templates were amplified from pCom3XTT (FIG. 1), pComb3X Lambda (FIG. 2) plasmid (adedge), respectively, using primers upstream and downstream of the light/heavy chain constant region. Equal amounts of each light chain variable region fragment (kappa chain and lambda chain separated) were mixed and then synthesized separately with CL using overlay PCR to synthesize the light chain genome; similarly, the VH fragments of the heavy chains are mixed in equal quantity, then the mixed VH fragments and CH1 are synthesized into a heavy chain Fd genome by adopting an overlay PCR, and finally the light chain gene and the heavy chain Fd gene are synthesized into a human Fab genome by adopting the overlay PCR.

3) Digestion of expression vectors Using SfiI (purchased from NEB) endonucleases, respectivelypComb3XTT, pComb3X Lambda and Fab genomes (including His and HA tags after SfiI cleavage site of pComb3XTT, pComb3X Lambda vector). The Fab genomes of the kappa and Lambda chains were then ligated to linearized pComb3XTT, pComb3 Xlambda expression vectors, respectively, using T4 DNA Ligase (purchased from NEB). The ligation product was purified using QIAGEN gel recovery kit to remove salt ions, using 25. Mu.L ddH ₂ O eluted DNA, stored at-20 ℃.

4) Taking 3 mu L of connection products to electrically transform 50 mu L of competent escherichia coli XLl-Blue according to each part, electrically transforming all the connection products, setting 2.5KV by using a 2mm electric shock cup, immediately adding 950 mu L of SOC culture medium after electric shock is finished, and performing 160rmp/min at 37 ℃ for 1h; then all the culture medium is transferred into a 50mL centrifuge tube and supplemented to 10mL at 37 ℃ at 160rmp/min,1hour; centrifuge 12000rmp/min,1min, discard supernatant and leave only 500. Mu.L of plate, 100. Mu.L/plate, incubator overnight at 37 ℃. 2mL of LB liquid culture medium is added into each plate, bacteria are gently scraped by a scraper, bacterial liquid is sucked into a 50mL centrifuge tube at 4000rpm/min for 10min, the supernatant is discarded, 6mL of bacterial liquid is left, 6mL of 60% glycerol is added, and the mixture is resuspended and packaged and stored in a refrigerator at the temperature of minus 80 ℃.

5) 1mL of glycerol bacteria was taken to 200mL of pre-warmed 2TY (1% glucose, 100. Mu.g/mL ampicillin, 10. Mu.g/mL tetracycline).

Shaking at 37℃to OD ₆₀₀ About 0.4. Every 50mL of bacterial liquid is added with 2 multiplied by 10 ¹¹ M13KO7 helper phage (NEB), water bath at 37℃for 30min.3000g, centrifuged for 10min, the supernatant was discarded, 100mL of 2TY (1% glucose, 100. Mu.g/mL ampicillin, 10. Mu.g/mL tetracycline, 50. Mu.g/mL kana antibiotic) was added, and the mixture was shaken overnight at 30 ℃. After overnight incubation 3300g, centrifuged at 4℃for 30min, 20mL PEG/NaCl (20% PEG,2.5M NaCl) was added to 80mL supernatant, mixed well and placed on ice for 1h.3300g,4 ℃, centrifuge for 30min, pour off PEG/NaCl, re-centrifuge, and suck off PEG/NaCl as much as possible. The pellet was resuspended in 4mL PBS, centrifuged at 11600g 4℃for 10min, and the bacterial debris removed. The phage antibody library obtained was stored in sub-packs at-20 ℃.

Table 1: humanized antibody Fab primers

(2) Enrichment screening of phage antibody libraries

Preparation of PCT antigen: PCT protein sequence (NCBI sequence No. ACCESSION NP 001029124) was obtained from NCBI and synthesized, ligated into the true expression vector pcDNA3.1 (+) with the His tag sequence fused at the C-terminus, CHOS suspension cells were transfected with the above plasmid using transfection reagent ExpiFectamineTMCHO reagent (gibco), protein expression was increased by adding ExpiFectamineTMCHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents, and the supernatant was collected by centrifugation at 7000RPM for 20min after 8-10 days of cell culture, filtered with a 0.8 μm filter membrane, filtered with a 0.22 μm filter membrane, and finally purified by a nickel affinity column to obtain PCT antigen.

2) Enrichment screening: taking PCT mouse monoclonal antibody mIgG-6A2 (the preservation number of a corresponding monoclonal hybridoma cell strain screened by the company is CCTCC NO. C2017143) with high affinity and specificity, coating an ELISA plate with 500 ng/hole, and coating overnight at 4 ℃;3% nonfat milk powder PBS was blocked and washed 3 times with 0.05% PBST; PCT antigen was added and incubated at 37 ℃ for 2h and then washed 3 times with 0.05% PBST; 100. Mu.L of the prepared phage Fab antibody library was added and incubated at 37℃for 2h. Discarding unbound phage, and washing off the unbound phage with PBST; 100. Mu.L of Gly-HCI (pH 2.2) eluate was added to each well, and the phage was recovered by incubation at room temperature for 10 minutes. Adding a proper amount of Tris buffer solution to neutralize the eluted phage, adding the eluted phage into 2mL of XL1-Blue bacterial liquid in logarithmic phase, transferring into a triangular flask, adding 10mL of SOC culture medium (20 mg/L ampicillin, 10mg/L tetracycline), and applying 10 mu L of the culture medium to an ampicillin plate for titration of phage. The rest was cultured at 37℃for 1hour, 100mL of SOC was added thereto, and after 1hour of shaking culture at 37℃the helper phage Ml3K07 was added thereto, and shaking culture at 37℃was performed to activate the antibody library. The "adsorption-elution-amplification" procedure was screened 5 times.

3) Screening of positive clones: the phage obtained by the last round of screening is diluted according to a certain ratio and spread on an ampicillin culture plate, 50 single colonies are randomly picked and cultured in an SOC culture medium containing ampicillin, and the culture is carried out by shaking overnight. A small amount of each bacterial night is used for preservation, the rest is added with auxiliary phage VCSM13, shake culture is performed, antibody expression is induced, and phage antibody supernatant is obtained. ELISA plates are coated by PCT, antigen-antibody reaction is detected, positive monoclonal phage strains are screened, and secondary antibodies are HRP marked mouse anti-His-TAG. It was finally found that 12 phage antibodies were able to bind specifically to PCT, and the results are shown in table 2.

4) The positive monoclonal phage strains are amplified, plasmid DNA is extracted according to a conventional method, the sequences are respectively sequenced and then are compared with the sequences of the human antibodies on NCBI, the repeated sequences and incorrect sequences are removed, and 4 Fab sequences with correct sequences and anti-PCT human IgG are obtained and are respectively named as hFab-1B, hFab-3C, hFab-6C and hFab-8C.

TABLE 2

Sequence number	1A	1B	2E	3A	3C	5E	6C	6D	8C	8E	9B	9C	Blank space
														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

Example 2: specificity and sensitivity analysis of anti-PCT-Positive Fab antibodies

1) 4 positive monoclonal phage strains containing 4 kinds of anti-PCT humanized IgG Fab sequences (hFab-1B, hFab-3C, hFab-6C and hFab-8C) with correct sequences and different sequences are inoculated in SOC culture medium containing ampicillin for expansion culture, shaking culture for overnight, adding auxiliary phage VCSM13, shaking culture, inducing expression of antibody, and obtaining phage antibody supernatant. The antibodies in the supernatant were purified using a nickel affinity column.

2) And (3) specificity detection: PCT and CT (calcitonin) antigens are respectively coated, and the cross reaction of the 4 antibodies to CT is detected by ELISA method, so that the specificity of the antibodies is detected. Antigen was coated in 96-well plates at 100/50ng per well, overnight at 4℃and 3 times after overnight washing with PBST. The concentration of the 4 positive antibodies is adjusted to 1mg/mL, the corresponding antibodies are added, the incubation is carried out for 40min at 37 ℃, and the blank control is antibody diluent. After incubation, the plates were washed 5 times with PBST. The secondary antibody was HRP-labeled murine anti-His-TAG, 100. Mu.L per well, incubated at 37℃for 30min. After incubation, the plates were washed 5 times with PBST. Adding a color development solution, and incubating for 15min at 37 ℃ with 100 mu L of each hole; the chromogenic reaction was stopped by adding 50. Mu.L/well of stop solution, and the absorbance was measured as shown in Table 3, and it was found that the three hFab-1B, hFab-3C, hFab-6C antibodies did not cross-react with CT and were specific for PCT antigen only. Therefore, the antibodies can avoid the reaction of the antibodies and CT in clinical application, and false positive results are reduced.

3) Sensitivity detection: the concentration of 3 antibodies (hFab-1B, hFab-3C, hFab-6C) which do not cross react with CT was adjusted to 1mg/mL, ELISA detection sensitivity was performed on antigens with different dilution concentrations, the antigens were coated on 96-well plates at 100/50/25/12.5ng per well, respectively, coated overnight at 4℃and washed 3 times with PBST after overnight. The corresponding antibody was added and incubated at 37℃for 40min with blank as antibody dilution. After incubation, the plates were washed 5 times with PBST. The corresponding HRP-labeled secondary antibody was added and incubated at 37℃for 30min at 100. Mu.L per well. After incubation, the plates were washed 5 times with PBST. Adding a color development solution, and incubating for 15min at 37 ℃ with 100 mu L of each hole; the detection value is still higher when the antigen is detected by using three antibodies such as hFab-1B and the like as primary antibodies, which show that the sensitivity of the three antibodies is high and can meet the diagnosis requirement.

TABLE 3 Table 3

TABLE 4 Table 4

Example 4: screening of optimal PCT paired antibodies by double-antibody sandwich method

And (3) carrying out ELISA detection on the hFab-1B, hFab-3C and hFab-6C monoclonal antibodies obtained after expression and purification, taking part to carry out HRP labeling, respectively pairing with the mIgG-6A2 coated antibodies, and screening out the optimal pairing antibody pair. First, 100 ng/well of mIgG-6A2 antibody was coated separately, and the plates were washed 3 times with PBST after overnight coating at 4 ℃. PCT antigen was added at gradient 200/100/50/25 ng/well, blank wells were set and incubated at 37℃for 40min. After incubation, the plates were washed 5 times with PBST. HRP-labeled antibody diluted 1000-fold with enzyme-labeled diluent was added, 100. Mu.L/well incubated at 37℃for 30min, and after incubation, PBST was washed 5 times. Adding a color development solution, and incubating for 15min at 37 ℃ with 100 mu L of each hole; the reaction was stopped by adding 50. Mu.L/well of stop solution, and the absorbance was measured, and the results are shown in Table 5. As can be seen from the results, the pairing PCT antigen consisting of mIgG-6A2/hFab-1B had higher sensitivity under the same conditions, so that the hFab-1B monoclonal antibody was selected in the subsequent experiments.

TABLE 5

Coating/labelling antigens	200 ng/well	100 ng/well	50 ng/well	25 ng/well	Blank space
						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

Example 5: sensitivity comparison of eukaryotic and prokaryotic expressed anti-PCT recombinant Fab antibodies

1) Construction of eukaryotic expression vectors for anti-PCT recombinant Fab antibodies: the hFab-1B positive phage antibody plasmid prepared in the above example 2 is used as a template, the hFab-1B full-length antibody gene sequence (comprising a light chain leader sequence, a C-terminal His and HA tag sequences) is amplified, and then the amplified hFab-1B full-length antibody gene sequence is inserted between XbaI and BamHI cleavage sites in a eukaryotic expression vector pCDH-puro (shown in figure 3) by a seamless cloning method to construct a corresponding eukaryotic expression plasmid named pCDH-hFab-1B.

2) Expression of anti-PCT recombinant Fab antibodies: inoculating well-grown CHOS suspension cells into a 10cm dish, and growing cells to 5×10 ⁶ When the density is about, subculturing the strain into 250mL conical flasks in a shake flask for continuous culture, wherein the volume is 50mL, and the culture conditions are as follows: 130RPM, 5% carbon dioxide, 37 ℃. Cells are grown to 5 to 7 x 10 ⁶ At about density, 25. Mu.g of the above recombinant plasmids (eukaryotic expression plasmid pCDH-hFab-1B and prokaryotic expression plasmid hFab-1B positive phage antibody plasmid) were transfected with a transfection reagent ExpiFectamineTMCHO reagent (gibco) according to the manufacturer's instructions. After 24h of transfection, the antibody expression was increased by the addition of ExpiFectamineTMCHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents according to the manufacturer's instructions. After 8-10 days of culture, the supernatant was collected by centrifugation at 7000RPM for 20min, filtered with a 0.8 μm filter membrane and then with a 0.22 μm filter membrane, and the antibody in the supernatant was purified using a nickel affinity column.

3) Sensitivity comparison of eukaryotic and prokaryotic expressed anti-PCT recombinant Fab antibodies: the concentrations of eukaryotic and prokaryotic expressed hFab-1B antibodies were adjusted to 1mg/mL, ELISA detection sensitivity was performed on PCT antigens of different dilution concentrations, the antigens were coated in 96-well plates at 100/50/25/12.5ng per well, respectively, overnight at 4℃and after overnight the plates were washed 3 times with PBST. The corresponding antibody was added and incubated at 37℃for 40min with blank as antibody dilution. After incubation, the plates were washed 5 times with PBST. The corresponding HRP-labeled murine anti-His-TAG was added and incubated at 37℃for 30min at 100. Mu.L per well. After incubation, the plates were washed 5 times with PBST. Adding a color development solution, and incubating for 15min at 37 ℃ with 100 mu L of each hole; the chromogenic reaction was stopped by adding a stop solution at 50. Mu.L/well, and the absorbance was measured, and the results are shown in Table 6, and it can be seen from the results that the sensitivity of the eukaryotic-expressed hFab-1B antibody was higher than that of the prokaryotic-expressed hFab-1B antibody.

TABLE 6

Example 6: rapid affinity purification of anti-PCT recombinant Fab antibodies

1) Construction of eukaryotic expression vectors for anti-PCT recombinant Fab antibodies: the following 4 recombinant hFab-1B antibody sequences were obtained by adding different sequences to the C-terminal of the hFab-1B full-length antibody gene sequence (comprising the light chain leader sequence) by a multiple PCR method using the hFab-1B positive phage antibody plasmid as a template: 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 HHHHH, linker represents the flexible linker peptide GGGGS, strep is the tag sequence WSHPQFEK. Then, the recombinant genes are inserted between XbaI and BamHI cleavage sites in eukaryotic expression vectors pCDH-puro (shown in figure 3) by using a seamless cloning method to respectively construct corresponding eukaryotic expression plasmids which are 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 in sequence.

2) Expression of anti-PCT recombinant Fab antibodies: inoculating well-grown CHOS suspension cells into a 10cm dish, and growing cells to 5×10 ⁶ When the density is about, subculturing the strain into 250mL conical flasks in a shake flask for continuous culture, wherein the volume is 50mL, and the culture conditions are as follows: 130RPM, 5% carbon dioxide, 37 ℃. Cells are grown to 5 to 7 x 10 ⁶ At about density, 25. Mu.g of each of the 4 recombinant Fab antibody positive plasmids was transfected with a transfection reagent ExpiFectamineTMCHO reagent (gibco), and the transfection procedure was carried out according to the manufacturer's instructions. After 24h of transfection, the antibody expression was increased by the addition of ExpiFectamineTMCHO Enhancer (gibco) and ExpiCHOTMFeed (gibco) reagents according to the manufacturer's instructions. After 8-10 days of culture, the supernatant was collected by centrifugation at 7000RPM for 20min, filtered with a 0.8 μm filter membrane and then with a 0.22 μm filter membrane, and a small amount of the supernatant was collected and stored for detection of the target antibody content, and the remainder was used for antibody purification.

3) Purification of anti-PCT recombinant Fab antibodies: performing double-tag two-step affinity chromatography purification on the obtained cell expression supernatant, and respectively performing Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography in sequence, wherein the specific steps are as follows:

1. Ni-NTA affinity chromatography:

(1) preparing Ni column: filling Ni column resin nitrilotriacetic acid (NTA) into the column, flushing with 5V pure water, resuspending the resin, and naturally flowing out the pure water; adding 2% nickel chloride into the column, adding 2% nickel sulfate again after the liquid naturally flows out, soaking for 10min, and then flowing out the liquid, wherein the column turns green; washing with 5V pure water to remove excess Ni; equilibrated with 5V equilibration solution (20 mM Tris-HCl pH 7.4, 150mM NaCl,10% glycerol, 20mM imidazole).

(2) Adsorption: the sample was carefully added to the Ni column along the wall.

(3) Washing: after the completion of the loading, the column was equilibrated with 5V equilibration solution, and impurities not bound to the column were washed until effluent OD ₂₈₀ < 0.1. And collecting effluent liquid, and preserving the sample.

(4) Eluting: proteins were eluted with an eluent (20 mM Tris-HCl pH 7.4, 150mM NaCl,10% glycerol, 50/100/200/500mM imidazole) containing 50/100/200/500mM imidazole, respectively; collecting the eluate until OD ₂₈₀ <0.1; a small amount of eluent is taken and stored for detecting the recovery rate and the concentration of the antibody; SDS-PAGE detects that most of the target proteins eluted at a concentration of 200mM imidazole.

2. Strep-Tactin affinity chromatography:

(1) equilibration by loading appropriate amount of Strep-Tactin agarose gel into column, washing with 5V pure water to remove preservation solution, and equilibration with 5V equilibration solution (100 mM Tris-HCl,150mM NaCl,1mM EDTA,pH8.0).

(2) Adsorption: a proper amount of samples eluted by a Ni-NTA column under the condition of 200mM imidazole concentration are dialyzed by a balancing solution (100 mM Tris-HCl,150mM NaCl,1mM EDTA,pH8.0), and the samples are added into a Strep-Tactin column along the wall after the dialysis is finished.

(3) Washing: after loading, the column was equilibrated with 10V equilibration solution and the heteroproteins not bound to the column were washed.

(4) Eluting: eluting the protein with 10-15V eluting buffer (balance +2.5mM desthiobiotin); collecting the eluate until OD ₂₈₀ <0.1; a small amount of eluent was taken and stored for detection of antibody recovery and antibody concentration.

(5) Regeneration: the column was washed with equilibration solution containing 1mM HABA for 15 column volumes, then with equilibration solution at 30V, and then the column was stored with 20% ethanol.

4) Antibody recovery and purity detection: using PCT to coat ELISA plate, using ELISA detection method to detect the antibody concentration of sample solution and eluent before and after each affinity chromatography, and then calculating total antibody and recovery rate; and simultaneously, the non-reducing SDS-PAGE electrophoresis gel running electrophoresis result is subjected to scanning analysis after being dyed by coomassie brilliant blue, and the protein purity is calculated according to the gray scale. The results are shown in Table 7, which shows that the purity of the 4 recombinant antibodies is greatly improved and the loss of antibodies in the second affinity purification process is less after the purification by Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography are sequentially used; the total recovery rate and purity of hFab-1B-linker-His-linker-Strep recombinant antibodies containing 2 segments of flexible connecting peptide GGGGS in the 4 recombinant antibodies are the highest.

TABLE 7

Example 6: construction of stable expression cell line of anti-PCT recombinant Fab antibody

1) Construction of a CHOS stable expression cell line against PCT recombinant Fab antibody: the well-grown CHOS suspension cells were inoculated into 6-well plates, and when the cells grew to 70-90% density, the previously constructed pCDH-hFab-1B-linker-His-linker-Strep eukaryotic expression plasmid (2. Mu.g) was transfected using transfection reagent ExpiFectamineTMCHO reagent (gibco), and the transfection procedure was performed with reference to the manufacturer's instructions. After 24h transfection, cells of each 6-well plate are evenly spread into 4 96-well plates for continuous culture, 100 mu L of culture medium is added into each well, 5 mu g/mL of puromycin (puro) is added into the culture medium for screening, cells to be survived form clone groups after 3-4 weeks, a 10 mu L pipette gun is used for picking single cell groups, the single cell groups are added into other 96-well plates, the pressure is maintained by changing to 1 mu g/mL of puromycin screening culture medium with low concentration, and 100 mu L of culture medium is added into each well for continuous culture. After cells grow almost fully, 50 mu L of cell culture supernatant is collected, the antibody expression level in the supernatant is detected by an ELISA method, a hole with a high detection value is selected, the cells are transferred to a 24-hole plate for continuous culture, after the cells grow fully, 50 mu L of cell culture supernatant is collected, the antibody expression level in the supernatant is also detected by the ELISA method, a hole with a high detection value is selected, the cells are transferred to a 6-hole plate for continuous culture, after the cells grow fully, 50 mu L of cell culture supernatant is collected, and the antibody expression level in the supernatant is also detected by the ELISA method. Finally, selecting 2 cells with highest detection value, performing 96-well plate plating by using a limiting dilution method, wherein the cell density is 0.5 cells per well, cells grow almost fully after about 2 weeks, collecting 50 mu L of cell culture supernatant, detecting the antibody expression level in the supernatant by using an ELISA method, sequentially selecting cells with high detection value, transferring the cells into a 24/6 well plate, culturing and screening high-expression cell lines, screening monoclonal cell lines according to the limiting dilution method, repeating 3 times, and finally selecting positive stable high-expression cell lines for 3-5 expansion culture, freezing and preserving seeds; the cell line was designated CHOS-hFab-1B.

3) And extracting total RNA from the monoclonal cell strain obtained by screening, amplifying recombinant Fab genes by PCR, comparing target genes, and finally confirming that the sequence of the obtained recombinant Fab genes is consistent with the sequence of genes in eukaryotic expression vectors.

4) Resuscitate the stable cell lines obtained by the above screening in a 10cm dish until cells grow to 5X 10 ⁶ When the density is about, subculturing the strain into 250mL conical flasks in a shake flask, and culturing under the following conditions: 130RPM, 5% carbon dioxide, 37 ℃. Cells were grown to 5X 10 ⁶ When the density is about, the culture is continued in a shaking incubator by inoculating a 2000mL conical flask under the same conditions, and the volume of the cell fluid is not more than 1/4 of the volume of the flask. After 6-7 days of culture, the supernatant was collected by centrifugation at 7000rpm, filtered with a 0.8 μm filter membrane and then with a 0.22 μm filter membrane, and finally the supernatant was purified by Ni-NTA+strep-Tactin double-tag two-step affinity chromatography, and the obtained antibody was designated as hFab-1B-linker-His-linker-Strep.

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

Amino acid sequence of antibody heavy chain variable region:

EVQLVESGGGGVQPGGSLRLSCAASGYTFTEYTMHWVRQAPGKGLEWIGGIWPNTGDTYYADSVKGRFTISRDNAKNSLYLQMNSLKTEDTAVYYCARYPIYYDFSHAMDYWGQGTTVTVSSASTKGP(SEQ ID NO.7)。

amino acid sequence of antibody heavy chain constant region:

SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS(SEQ ID NO.9)。

amino acid sequence of antibody light chain variable region:

EIVLTQSPGTLSLSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASSRDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCHQYLSSYTGQGTKVELKRT(SEQ ID NO.8)。

amino acid sequence of antibody light chain constant region:

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC(SEQ ID NO.10)。

amino acid sequence of antibody light chain:

EIVLTQSPGTLSLSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASSRDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCHQYLSSYTGQGTKVELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC(SEQ ID NO.12)。

amino acid sequence of antibody heavy chain:

EVQLVESGGGGVQPGGSLRLSCAASGYTFTEYTMHWVRQAPGKGLEWIGGIWPNTGDTYYADSVKGRFTISRDNAKNSLYLQMNSLKTEDTAVYYCARYPIYYDFSHAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAGQGGGGSHHHHHHGGGGSWSHPQFEK(SEQ ID NO.11)。

nucleotide sequence of the heavy chain variable region of the antibody:

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGAGGAGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATATACCTTTACCGAGTATACCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGATTGGTGGGATTTGGCCGAATACCGGTGATACCTACTATGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCAAAAAATTCCTTGTATCTGCAAATGAACAGTCTGAAGACCGAGGACACCGCCGTGTATTACTGTGCACGGTATCCGATCTATTATGACTTTAGTCACGCAATGGACTACTGGGGCCAGGGAACCACCGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA(SEQ ID NO.13)。

nucleotide sequence of the light chain variable region of the antibody:

ATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAAATCTAGTCAGAGTGTTCTCTACAGCTCTAACCAGAAAAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACTGGGCATCCAGCAGGGACTCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCATCAGTATCTGTCTTCCTACACGGGCCAAGGGACCAAGGTGGAACTCAAACGAACT(SEQ ID NO.14)。

nucleotide sequence of heavy chain constant region of antibody:

TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT(SEQ ID NO.15)。

nucleotide sequence of the light chain constant region of the antibody:

GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQ ID NO.16)。

nucleotide sequence of heavy chain of antibody:

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGAGGAGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATATACCTTTACCGAGTATACCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGATTGGTGGGATTTGGCCGAATACCGGTGATACCTACTATGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCAAAAAATTCCTTGTATCTGCAAATGAACAGTCTGAAGACCGAGGACACCGCCGTGTATTACTGTGCACGGTATCCGATCTATTATGACTTTAGTCACGCAATGGACTACTGGGGCCAGGGAACCACCGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTAGTGGCCAGGCCGGCCAGGGAGGCGGGGGATCCCACCATCACCATCACCATGGCGGCGGAGGATCCTGGAGCCACCCGCAGTTCGAAAAGTAG(SEQ ID NO.17)。

nucleotide sequence of the light chain of the antibody:

ATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAAATCTAGTCAGAGTGTTCTCTACAGCTCTAACCAGAAAAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACTGGGCATCCAGCAGGGACTCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCATCAGTATCTGTCTTCCTACACGGGCCAAGGGACCAAGGTGGAACTCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQ ID NO.18)。

5) The ELISA plate was coated with PCT and the antigen-antibody reaction was examined, and the results are shown in Table 8. As can be seen from Table 8, the recombinant PCT antibody can react with PCT antigen significantly.

TABLE 8

Example 7: detection of clinical samples by anti-PCT recombinant Fab antibodies and murine PCT IgG monoclonal antibodies

Using mIgG-6A2 antibody as coating antibody, using fluorescent microsphere to mark hFab-1B-linker-His-linker-Strep (hereinafter called hFab-1B) as mark antibody, preparing into fluorescent immunochromatography quantitative detection kit of mIgG/hFab paired antibody; the control group uses mIgG-6A2 antibody as coating antibody, another PCT mouse monoclonal antibody mIgG-9C2 as labeling antibody, and prepares the fluorescent immunochromatography quantitative detection reagent of double mIgG pairing antibody. The 2 kits were then compared for detection of clinical samples in several areas, non-specificity, minimum detection limit and linear range.

1) Nonspecific and minimum detection limit

Taking 5 parts of normal human whole blood, plasma and serum reference products without procalcitonin, respectively taking 30 mu L of the reference products, adding the reference products into 200 mu L of sample treatment liquid, blowing and mixing uniformly, respectively sucking 80 mu L of mixed liquid, adding the mixed liquid into sample adding holes of 2 kinds of test cards matched with different antibodies, reacting for 15min, and detecting by using a fluorescence immunoassay analyzer. The results were collected and judged, and the average and minimum detection limits of the reference measurement results were calculated, and the results are shown in Table 9. The results showed that the T/C value of the antibody pair for mIgG-6A2/hFab-1B was lower than that of the antibody pair for mIgG-6A2/mIgG-9C2, indicating that the antibody pair has a lower background and non-specificity in detecting these 3 clinical samples. Regression is carried out according to the fitting curve, and the average minimum detection limit concentration value of the mIgG-6A2/hFab-1B antibody pairing is calculated to be 0.036ng/mL, thereby meeting the technical requirement of procalcitonin fluorescent kit sensitivity.

TABLE 9

2) Linear range

Respectively taking 6 concentrations of reference substances of 0.5ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 50.0ng/mL and 100.0ng/mL, respectively taking 30 mu L of each reference substance, adding the reference substances into 200 mu L of sample treatment liquid, blowing and mixing uniformly, respectively sucking 80 mu L of mixed liquid into sample adding holes of 2 kinds of test cards, reacting for 15min, and detecting by using a fluorescence immunoassay analyzer. Each sample is repeatedly measured for 3 times, the average value of the 3 results is taken, a logarithmic scatter diagram of the theoretical concentration value and the actual detection value is made, a fitting curve is made, a linear equation y=ax+b and a correlation coefficient R are calculated, the experimental result is shown in table 10, from the result, the R value of a kit for pairing the mIgG-6A2/hFab-1B antibody is higher than the R value of a kit for pairing the mIgG-6A2/mIgG-9C2 antibody, the result is closer to 1, the pairing of the antibodies has better linear result, and the kit for preparing the antibodies has good stability.

Table 10

Example 9: anti-PCT recombinant Fab antibody and mouse PCT IgG monoclonal antibody for anti-interference detection of clinical sample

PCT content indicators can be used not only to determine early bacterial and non-bacterial infections and inflammations, but also to diagnose bacterial infections or sepsis in patients at high risk of infection or intensive care. However, staphylococcus aureus cell wall surface antigen exists staphylococcus A protein (SPA), and when bacteria containing SPA protein exist in clinical samples in PCT clinical detection, the protein can be combined with the Fc segment of IgG1, igG2 and IgG4 molecules commonly used in diagnosis and various mammals in a non-specific way, and the combined IgG molecule Fab segment is exposed on the surface of thalli, can still be combined with PCT antigen in a specific way, and generates a synergistic agglutination phenomenon, so that the accuracy and the specificity of detection are finally affected. Meanwhile, human anti-mouse antibodies (HAMAS) and interference substances such as Rheumatoid Factors (RF) existing in blood detection samples can be combined with detection antibodies, and the detection accuracy and specificity are affected. In this example, SPA, HAMAS and RF interference sources were used as examples, respectively, and the anti-interference ability of Fab antibody and murine PCT IgG monoclonal antibody as labeled antibodies in diagnosis was compared.

(1) SPA interferents

Inoculating SPA positive CowanI strain (purchased from ASI 1476) or SPA negative Wood46 strain (purchased from ASI 1477) on an agar slant culture medium, and culturing at 37 ℃ for 18-24 h; washing thallus with small amount of sterile physiological saline, centrifuging at 4000r/min for 15min, and discarding supernatantWashing the precipitated thallus with sterile physiological saline solution for 3 times, preparing 10% (V/V) suspension with 0.01M PBS buffer solution containing 0.5% formalin, and standing at room temperature for 3 hr; the suspension was centrifuged at 4000r/min for 15min in a 56℃water bath for 30min and washed 3 times with PBS. Finally, using NaN containing 0.1% ₃ The PBS buffer solution of (2) is prepared into 10% (V/V) suspension, namely SPA bacteria stabilizing solution, and the solution is placed in a refrigerator at 4 ℃ for standby.

30 mu L of healthy human serum reference without procalcitonin is taken and added into 200 mu L of sample treatment liquid, then positive or negative SPA bacteria stabilizing liquid (40, 8, 2, 0.5 and 0.2 mu L of positive SPA bacteria stabilizing liquid are respectively added into samples 1-5, 40, 8, 2, 0.5 and 0.2 mu L of positive SPA bacteria stabilizing liquid are respectively added into samples 6-10) with different proportions, the mixture is blown and evenly mixed, 80 mu L of mixed liquid is respectively absorbed and added into sample adding holes of the test cards paired with the 2 different antibodies, and the mixture is reacted for 15min and detected by using a fluorescence immunoassay analyzer. The results were collected and judged, and the T/C values were calculated, as shown in Table 11. The results show that the T/C values are substantially identical when the positive or negative SPA bacteria are contained in the detection sample of the kit for pairing the mIgG-6A2/hFab-1B antibodies, and the T/C values are higher when the positive SPA bacteria are contained in the detection sample of the kit for pairing the mIgG-6A2/mIgG-9C2 antibodies than when the negative SPA bacteria are contained in the detection sample, thus generating false positives, and indicating that the Fab antibodies have better capability of being used as anti-SPA interferents in diagnosis.

Samples 1 to 5: 30. Mu.L of serum reference +40/8/2/0.5/0.2. Mu.L of positive SPA bacteria stabilizing solution +200. Mu.L of sample treatment solution; sample 6-10: 30. Mu.L of serum reference +40/8/2/0.5/0.2. Mu.L of negative SPA bacteria stabilizing solution +200. Mu.L of sample treatment solution; the final volume was adjusted to be consistent using PBS buffer. The blank is a sample dilution.

TABLE 11

(2) Hamas interferents

3 parts of HAMAS positive or negative serum (HAMAS positive serum is taken as samples 1-3 and HAMAS negative serum is taken as samples 4-6) which does not contain procalcitonin are added into 200 mu L of sample treatment liquid, blown and evenly mixed, 80 mu L of mixed liquid is respectively sucked and added into the sample adding holes of the mIgG-6A2/hFab-1B or mIgG-6A2/mIgG-9C2 antibody paired test cards, and the mixture is reacted for 15min and detected by using a fluorescence immunoassay analyzer. The results were collected and evaluated, and T/C values were calculated, as shown in Table 12. The results show that the T/C values are substantially identical when the kit for pairing mIgG-6A2/hFab-1B antibodies detects HAMAS positive or negative samples, whereas the T/C values are higher when the kit for pairing mIgG-6A2/mIgG-9C2 antibodies detects HAMAS positive samples than when the kit for pairing HAMAS negative samples detects HAMAS negative samples, and false positives are detected, which indicates that the Fab antibodies have better capacity as labeled antibodies against HAMAS interferents in diagnosis.

Samples 1 to 3: 30. Mu.L of HAMAS positive serum+200. Mu.L of sample treatment solution; samples 4 to 6: 30. Mu.L of HAMAS negative serum+200. Mu.L of sample treatment solution; the blank is a sample dilution.

Table 12

(3) RF interferents

3 parts of RF positive serum and negative serum which do not contain procalcitonin (the RF positive serum is taken as a sample 1-3, the RF negative serum is taken as a sample 4-6) are added into 200 mu L of sample treatment liquid, the mixture is blown and mixed uniformly, 80 mu L of mixed liquid is respectively sucked and added into the sample adding hole of the test card matched with the mIgG-6A2/hFab-1B or the mIgG-6A2/mIgG-9C2 antibody, and the reaction is carried out for 15 minutes, and the detection is carried out by using a fluorescence immunoassay analyzer. The results were collected and judged, and the T/C values were calculated, as shown in Table 13. The results show that the T/C values are basically consistent when the kit for pairing the mIgG-6A2/hFab-1B antibodies detects RF positive or negative samples, and the T/C values are higher when the kit for pairing the mIgG-6A2/mIgG-9C2 antibodies detects RF positive samples than when the kit for pairing the mIgG-6A2/mIgG-9C2 antibodies detects RF negative samples, so that false positives are detected, which indicates that the Fab antibodies have better anti-RF interferents in diagnosis as labeled antibodies.

Samples 1 to 3: 30. Mu.L of RF-positive serum+200. Mu.L of sample treatment solution; samples 4 to 6: 30. Mu.L of RF-negative serum+200. Mu.L of sample treatment solution; the blank is a sample dilution.

TABLE 13

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. An anti-procalcitonin Fab antibody, which is characterized by comprising a light chain and a heavy chain, wherein the heavy chain variable region of the heavy chain is provided with 3 complementarity determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID NO.1, the amino acid sequence of the CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3; the light chain variable region of the light chain is provided with 3 complementarity determining regions CDR1', CDR2' and CDR3', wherein the amino acid sequence of the CDR1' is shown as SEQ ID NO.4, the amino acid sequence of the CDR2 'is shown as SEQ ID NO.5, and the amino acid sequence of the CDR3' is shown as SEQ ID NO. 6.

2. The Fab antibody of claim 1, wherein 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 as SEQ ID NO.9, and the amino acid sequence of the light chain constant region of the Fab antibody is shown as SEQ ID NO. 10.

3. The Fab antibody of claim 1 or 2, wherein the heavy chain of the Fab antibody has the amino acid sequence 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 of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence encoding the heavy chain variable region of said Fab antibody is set forth in SEQ ID No.13 and the nucleotide sequence encoding the light chain variable region of said Fab antibody is set forth in SEQ ID No. 14; the nucleotide sequence of the heavy chain constant region of the Fab antibody is shown as SEQ ID NO.15, and the nucleotide sequence of the light chain constant region of the Fab antibody is shown as SEQ ID NO. 16; the nucleotide sequence of the heavy chain of the Fab antibody is shown as SEQ ID NO.17, and the nucleotide sequence of the light chain of the Fab antibody is shown as SEQ ID NO. 18.

6. An expression cassette, expression vector or transgenic cell line comprising the nucleotide molecule of claim 4 or 5.

7. A test kit comprising the Fab antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, or the expression cassette, expression vector, or transgenic cell line of claim 6; preferably, the kit further comprises a detection antibody, wherein the detection antibody is IgG monoclonal antibody.

8. A method of producing a Fab antibody according to any one of claims 1 to 3, comprising the step of culturing a transgenic cell line according to claim 5 to express the Fab antibody; preferably, the method further comprises a purification step, wherein after filtering the procalcitonin Fab antibody expressed by the transgenic cell line, two sections of tag sequences are added at the C end of the heavy chain of the Fab antibody, the tag sequences are connected through a connecting peptide to obtain a fusion antibody, and the fusion antibody is subjected to Ni-NTA affinity chromatography and Strep-Tactin affinity chromatography respectively to obtain the purified procalcitonin Fab antibody.

9. Use of the Fab antibody of any one of claims 1 to 3, the nucleic acid molecule of claim 4 or 5, the expression cassette of claim 6, an expression vector or a transgenic cell line, or the detection kit of claim 7 in an immunoassay method; preferably, the immunoassay method includes flow cytometry, ELISA immunoassay, immunochromatographic assay, immunocytochemical staining assay, and immunohistochemical staining assay.

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