CN114106175A

CN114106175A - PD-L1 monoclonal antibody, kit, preparation method and application thereof

Info

Publication number: CN114106175A
Application number: CN202011614762.2A
Authority: CN
Inventors: 吕鹏辉
Original assignee: Jiangsu Proway Biotechnology Co ltd
Current assignee: Jiangsu Proway Biotechnology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-03-01
Anticipated expiration: 2040-12-30
Also published as: CN114106175B

Abstract

The invention is applicable to the technical field of biology, and provides a PD-L1 monoclonal antibody, a kit, a preparation method and application thereof. The heavy chain variable region amino acid sequence of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 9. The PD-L1 monoclonal antibody has higher affinity, strong specificity and good biological activity in vitro; the detection kit containing the PD-L1 monoclonal antibody has the advantages of simple detection method, strong accuracy and low cost. The detection kit can be used as a means for auxiliary diagnosis of non-small cell lung cancer and a monitoring tool of PD-1/PD-L1 pathway tumor-targeted drugs.

Description

PD-L1 monoclonal antibody, kit, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PD-L1 monoclonal antibody, a kit, a preparation method and application thereof.

Background

Programmed death ligand 1(PD-L1) is a ligand for the PD-1 protein receptor that is widely expressed on lymphoid and non-lymphoid tissues as well as on tumor cells and virus-infected cells (Dong et al, 1999, Nature Med.). PD-L1 is a member of the B7 family, belongs to a type I transmembrane protein, has a molecular weight of about 65kDa, has a full length of 290 amino acids, and comprises 1 IgV region, 1 IgC region, 1 transmembrane hydrophobic region and 1 intracellular region consisting of 30 amino acids. PD-L1 is expressed primarily in activated T cells, B cells, various tumor cells, and some non-lymphoid tissues.

The humanized IgG4-kappa antibody Keytruda of PD-L1 approved in 2017 is used for treating non-small cell lung cancer which fails in previous chemotherapy or targeted therapy and is on the market, and researches show that the concentration of PD-L1 in serum of a patient with the non-small cell lung cancer has important relation with the aspects of tumor disease progression, treatment effect evaluation, disease outcome and the like, and has great application value in the aspects of disease diagnosis, disease treatment prediction and the like.

In recent years, research and application of a labeling immunoassay technology are rapidly developed, and the labeling immunoassay technology is widely applied to various fields of biomedical basic theory research and clinical disease diagnosis. The method for detecting serological indexes mainly comprises radioactive isotope immunoassay, enzyme-linked immunosorbent assay and chemiluminescence immunoassay. The methods can be used as a primary screening test and a confirmation test, wherein the chemiluminescence method has the advantages of wide detection linear range, simple detection instrument, convenient operation and the like.

In the prior art, products aiming at PD-1/PD-L1 pathway targeted drug monitoring and prognosis are blank at present in China, and the specificity, the affinity and other aspects of similar products are possible to be further improved, so that the invention provides a PD-L1 monoclonal antibody with high affinity and strong specificity, a kit, a preparation method and application thereof.

Disclosure of Invention

The embodiment of the invention aims to provide a PD-L1 monoclonal antibody, a kit, a preparation method and application thereof, aiming at solving the problems in the prior art pointed out in the background technology.

The embodiment of the invention is realized by that, a PD-L1 monoclonal antibody,

the heavy chain variable region amino acid sequence of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

The light chain variable region amino acid sequence of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO.9, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 9.

As another preferred embodiment of the present invention, the amino acid sequences of the heavy chain hypervariable region CDR-H1, heavy chain hypervariable region CDR-H2, heavy chain hypervariable region CDR-H3, heavy chain framework region FR-H1, heavy chain framework region FR-H2 and heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, or the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are the amino acid sequences having equivalent functions formed by replacing, deleting and/or adding one or several amino acid residues.

As another preferred embodiment of the present invention, the 387bp (5 '-3') nucleic acid sequence of the heavy chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO.8, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 8.

As another preferred embodiment of the present invention, the light chain hypervariable region CDR-L1, light chain hypervariable region CDR-L2, light chain hypervariable region CDR-L3, light chain framework region FR-L1, light chain framework region FR-L2 and light chain framework region FR-L3 amino acid sequences of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, or the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 are the amino acid sequences having equivalent functions formed by replacing, deleting and/or adding one or several amino acid residues.

As another preferred embodiment of the present invention, the 360bp (5 '-3') nucleic acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO.16, or the amino acid sequence shown in SEQ ID NO.16 is an amino acid sequence with equivalent functions formed by replacing, deleting and/or adding one or more amino acid residues.

Another objective of the embodiments of the present invention is to provide a method for preparing the PD-L1 monoclonal antibody, comprising the following steps:

firstly, preparing a PD-L1 protein antigen;

secondly, preparing a PD-L1 polyclonal antibody;

thirdly, preparing a PD-L1 monoclonal antibody;

fourthly, screening monoclonal antibodies: establishing a dose response curve, and screening an optimal monoclonal antibody;

fifthly, the monoclonal antibody gene in the hybridoma is regulated:

extracting total RNA of the hybridoma cells;

carrying out reverse transcription synthesis by taking OligodT as a primer to obtain cDNA;

carrying out PCR amplification by taking the synthesized cDNA as a template;

obtaining a gene sequence for coding a heavy chain variable region and a gene sequence for coding a light chain variable region;

carrying out codon optimization and synthesis on the heavy chain variable region gene sequence, the encoding light chain variable region gene sequence and the constant region of the antibody to respectively obtain a heavy chain and a light chain of the complete antibody gene;

cloning the obtained heavy chain and light chain genes into pMD18-T expression vectors respectively;

transfecting the obtained plasmid into a competent cell;

collecting cell supernatant, purifying and concentrating to obtain the PD-L1 monoclonal antibody.

As another preferred embodiment of the present invention, the PCR amplification conditions are: denaturation at 95 deg.C for 5 min; 94 ℃ for 1 min; at 50 ℃ for 2 min; 72 ℃, 1min, 35 cycles; extension at 72 ℃ for 10min

The embodiment of the invention also aims to provide a PD-L1 detection kit, which contains the PD-L1 monoclonal antibody.

Another objective of the embodiments of the present invention is to provide a method for preparing the PD-L1 detection kit, comprising the following steps:

diluting the PD-L1 monoclonal antibody with a buffer solution, and adding streptavidin magnetic beads for reaction;

washing a buffer solution;

adding PBS confining liquid containing fetal calf serum, performing magnetic separation after reaction, and removing supernatant;

after being washed by buffer solution, the magnetic particles are suspended in the preservation buffer solution of PBS containing bovine serum albumin to prepare PD-L1 coated magnetic beads;

diluting multiple gradients with PD-L1 protein antigen in proportion, and subpackaging to obtain PD-L1 calibrator;

adding the PD-L1 polyclonal antibody into the acridine ester solution, and reacting in the dark;

taking out after the reaction is finished, adding a lysine salt solution, and continuing the reaction in a dark place;

purifying to obtain a PD-L1 acridinium ester standard antibody;

adding a serum sample and a calibrator into the PD-L1-coated magnetic beads, adding a PD-L1 acridinium ester standard antibody, and incubating to form an antibody-antigen-labeled antibody complex;

adding HNO respectively₃、H₂O₂Immediately putting the luminescence excitation liquid A, NaOH and Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity of each hole;

the content of PD-L1 in the sample is calculated according to the reaction curve.

The embodiment of the invention also aims to provide application of the PD-L1 detection kit in preparation of a detection kit for diagnosing the curative effect of a non-small cell lung cancer clinically-assisted and PD-L1-targeted drug.

The PD-L1 monoclonal antibody has higher affinity, strong specificity and good biological activity in vitro; the detection kit containing the PD-L1 monoclonal antibody has the advantages of simple detection method, strong accuracy and low cost. The PD-L1 protein detection kit established by the PD-L1 monoclonal antibody and the PD-L1 polyclonal antibody can be used as a means for auxiliary diagnosis of non-small cell lung cancer and a monitoring tool of PD-1/PD-L1 pathway tumor targeted drugs.

Drawings

FIG. 1: PD-L1 check dose-response system diagrams.

FIG. 2: a regression analysis plot of the assessed reagent measurement and the compared reagent measurement.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Specific implementations of the present invention are described in detail below with reference to specific embodiments.

Example 1

The embodiment provides a PD-L1 monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; or the amino acid sequence shown in SEQ ID NO.1 is formed by replacing, deleting and/or adding one or more amino acid residues, and has the same function.

The amino acid sequences of a heavy chain hypervariable region CDR-H1, a heavy chain hypervariable region CDR-H2, a heavy chain hypervariable region CDR-H3, a heavy chain framework region FR-H1, a heavy chain framework region FR-H2 and a heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7; or the amino acid sequence shown by SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 is formed by replacing, deleting and/or adding one or more amino acid residues to form the amino acid sequence with the same function.

The nucleic acid sequence of the 387bp (5 '-3') heavy chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 8; or the amino acid sequence shown in SEQ ID NO.8 is formed by replacing, deleting and/or adding one or more amino acid residues, and has the same function.

The amino acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 9; or an amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 9.

The light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2, the light chain hypervariable region CDR-L3, the light chain framework region FR-L1, the light chain framework region FR-L2 and the light chain framework region FR-L3 amino acid sequences of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, or the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 are subjected to substitution, deletion and/or addition of one or more amino acid residues to form the amino acid sequences with the same functions.

The 360bp (5 '-3') nucleic acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO.16, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 16.

SEQ ID NO.1：

SEQ ID NO.2：

SEQ ID NO.3：

SEQ ID NO.4：

SEQ ID NO.5：

SEQ ID NO.6：

SEQ ID NO.7：

SEQ ID NO.8：

SEQ ID NO.9：

SEQ ID NO.10：

SEQ ID NO.11：

Thr Val Lys

1

SEQ ID NO.12：

SEQ ID NO.13：

SEQ ID NO.14：

SEQ ID NO.15：

SEQ ID NO.16：

Example 2

This example provides a method for preparing a PD-L1 monoclonal antibody, comprising the steps of:

firstly, preparing a PD-L1 protein antigen:

(1) a pair of primers is designed according to the sequence of human PD-L1(GQ904196.1) in GenBank database: the upstream primer is vF: 5' -cgacatgtGCTABCATGCTGCTCCTGG-3’；

The downstream primer is vR: cc (cc)ctcgagGCGGCCGCCTAGATCCTCTTTC-3’；

The underlined parts of the upstream primer and the downstream primer are enzyme cutting sites with sequences of Pci I and Xho I respectively, and the two sites are coincided with corresponding multiple cloning sites on the mammalian cell high-efficiency expression plasmid vector pSec/WG;

the cloning plasmid containing the human PD-L1 gene fragment is synthesized by the whole gene, the PD-L1 gene specific amplification is carried out by Pyrobest DNA polymerase by taking the plasmid as a template, and the PCR amplification condition is as follows: denaturation at 95 deg.C for 5 min; 94 ℃ for 1 min; at 50 ℃ for 2 min; 72 ℃, 1min, 35 cycles; extending for 10min at 72 ℃;

carrying out gel recovery, chloroform extraction, ethanol precipitation and TE dissolution on the amplified PCR product to obtain pSec/WG plasmid for later use;

the recovered PD-L1 gene and pSec/WG plasmid are respectively subjected to Pci I and Xho I double enzyme digestion, and are recovered by a gel electrophoresis method;

respectively purifying the PCR enzyme digestion product and the recovered product of the vector again, mixing PD-L1 and pSec/WG according to the molar ratio of 1:1, dissolving with TE, reacting for 12 hours at 16 ℃, and placing at 70 ℃ for 10min to terminate the reaction;

connecting the reaction product with DH-5 alpha competent cells, screening out positive clones by an ampicillin (1mg/mL) antibody, amplifying, extracting recombinant PD-L1/pSec/WG plasmid, carrying out enzyme digestion and sequencing identification for later use;

(2) transfecting a recombinant PD-L1/pSec/WG plasmid into a eukaryotic expression cell line Flp-In CHO by using a liposome reagent Lipofectamine 2000, and culturing and screening cells In a hygromycin-containing culture medium after transfection;

untransfected Flp-In CHO cell lines were cultured In complete Hams F12 medium (containing 10% FBS, 2 mM L-glutamine) supplemented with 1% cyan/streptomycin and 100. mu.g/mL Zeocin transfected Flp-In CHO cells of recombinant plasmid PD-L1/pSec/WG because the insertion of the PD-L1 gene inactivated the Zeocin resistance gene In the host cell genome but at the same time brought In the hygromycin resistance gene (Hyg +), so that host cells transfected with recombinant plasmid could be cultured In hygromycin-containing medium, whereas untransfected host cells could not survive; culturing in complete Hams F12 culture medium containing 800 μ g/mL hygromycin, and screening out corresponding recombinant gene expression clone in 6-7 days;

culturing Flp-In CHO cells transfected with the PD-L1/pSec/WG recombinant plasmid In an UltraCHO serum-free culture medium, and collecting cell culture supernatant for 1 time every 3-4 days;

centrifuging the collected culture supernatant at 10000r/min for 5 minutes, and removing the precipitate; adding 0.02% NaN into the supernatant₃And all possible cell debris was removed by filtration through a 0.22 μm filter;

the column was packed with l.0mL of resin and equilibrated with approximately 10 volumes of PBS (pH 7.2); passing the supernatant containing the target Protein through a Protein G-Sepharose 4B column for 2 times; 20-30 volumes of PBS (pH 7.2) column wash; 28 μ L of 1.25M Tris-HCl (pH 8.0) was added to each collection vial beforehand; eluting with 100mM Glycine-HCl (pH 3.0) eluent, and collecting in the collection tubules at a rate of 1.0rnl per tube to neutralize the eluent in the collection tubules immediately; usually the protein is eluted in the 3 rd to 10 th tube, with peaks around 3, 4, 5; collecting all the eluate with A280 > 0.01, and concentrating with centrifugal filter column of Ultrafree 15(MWCO 10000, Millipore); the expression level is 50mg/L, SDS-PAGE electrophoresis identification is carried out, and the purity of the purified PD-L1 protein is more than 98 percent; after the protein is quantified, filtering and sterilizing through a 0.22 mu m filter membrane, and storing at-20 ℃ for later use;

II, preparation of PD-L1 polyclonal antibody

(1) Using male big ear rabbit as immune animal, firstly injecting 10mg BCG vaccine to stimulate animal, using PD-L1 protein as immune antigen, and starting immune after one week; 4-6 points of subcutaneous injection are injected below feet, and Freund complete adjuvant is used as an immunologic adjuvant to immunize animals for four times; 1mg of PD-L1 protein is taken each time, equivalent Freund's complete adjuvant and antigen solution are respectively sucked into two syringes for full emulsification for 1 hour, and foot subcutaneous injection is carried out, and two weeks are separated each time;

taking auricular venous blood to detect titer by an ELISA method, performing carotid bleeding when the ratio reaches 1:40000, centrifuging at 5000rpm to take serum, and purifying by DEAE ion exchange to obtain crude serum polyclonal antibody for later use;

(2) diluting the crude serum polyclonal antibody to 1mg/mL by using 0.5mol/L PBS (pH 7.5), preparing 3mL of CNBr-Sepharose 4B agarose gel, coupling 9mg of PD-L1 protein antigen by using a coupling agent, reacting for 4 hours at room temperature to prepare a PD-L1 antibody affinity chromatographic column, purifying the crude serum polyclonal antibody on the column, eluting and collecting, measuring the OD value of the antibody at the wavelength of 280nm by using an ultraviolet visible spectrophotometer, dividing the OD value by 1.35 to obtain the concentration of the measured antibody, adding 40-50% of glycerol, and placing at-20 ℃ for long-term storage;

preparation of PD-L1 monoclonal antibody

(1) Culturing the stably expressed PD-L1/pSec/WG recombinant plasmid Flp-In CHO cells obtained above, immunizing 5 female BALA/c mice, injecting 1 × 10 subcutaneously into each BALB/c mouse⁷Cells, immunised 4 times consecutively, each time with 2 weeks intervals; collecting blood 7 days after immunization, detecting serum titer by CLIA method, selecting mice with highest titer, and injecting into spleen for 1 × 10⁶Boosting the immunity of each cell, taking the spleen of the mouse 3 days later, grinding the spleen, and counting the spleen cells for later use;

(2) fusing splenocytes and bone marrow cells Sp2/0 according to the ratio of 5:1 of cell count, inducing by PEG1200, adding the fused cells into a 96-well plate containing a feeder layer for culturing, changing the culture medium by half by using HAT culture medium after one week, and observing the cell state after fusion;

screening positive hybridoma cell strains by an indirect CLIA method, selecting 1 hybridoma cell (B7-H1) with PD-L1Ab continuous secretion positive rate of more than 98% for amplification culture to prepare an intraperitoneal injection mouse, and performing intraperitoneal injection on the mouse by 500 mu L of liquid paraffin 1 week before;

collecting hybridoma cells by centrifugation, suspending with incomplete culture medium, mixing, and adjusting cell number to 1 × 10⁹Performing inoculation on mice at the beginning, performing intraperitoneal injection on each mouse at 500 mu L, extracting ascites from mice with obvious abdominal swelling after 1 week, centrifuging the obtained ascites at 3000r/min for 3 minutes, and collecting supernatant;

(3) purifying ascites with protein G purifying column; balancing the purification column with 0.02mol/L PB buffer solution, adding ascites for sampling, eluting with 0.1mol/L glycine hydrochloric acid buffer solution (pH 2.7), collecting with an EP tube, dialyzing with 0.05mol/LPB, concentrating to obtain PD-L1 monoclonal antibody, and freezing at-20 deg.C;

fourth, monoclonal antibody screening

(1) Coating the prepared PD-L1 monoclonal antibody, and diluting the antibody to 2-5 mu g/mL by 0.5mol/L PBS; adding 100 mu L/hole into an enzyme label plate for coating, after overnight at 2-8 ℃, washing with 0.9% NaCl for 3 times, adding a blocking solution containing 1% BSA, blocking at 150 mu L/hole, and airing for later use after overnight at 2-8 ℃;

(2) screening the optimal monoclonal antibody: diluting the prepared PD-L1 protein antigen, diluting 8 gradients (0, 10, 50, 100, 200, 400, 1000 and 2000pg/mL) according to a proportion, sequentially adding the diluted PD-L1 protein antigen into the prepared enzyme label, adding 50 mu L of the diluted PD-L1 protein antigen into each hole, adding a biotinylated polyclonal antibody and streptavidin marked with horseradish peroxidase, incubating for 1 hour at 37 ℃, washing for 5 times by PBST, adding a chromogenic substrate solution, and detecting an OD value by using an enzyme label instrument;

dose-response curves were constructed with the concentration of PD-L1 as X-axis and the OD as Y-axis (see FIG. 1). As can be seen from FIG. 1, the dose-response curve established by the PD-L1 detection kit has a linear coefficient R of >0.99, a detection range of 0-800pg/ml and excellent analysis performance; finally, selecting the PD-L1 monoclonal antibody with the best evaluation index; the monoclonal antibody used as an evaluation standard of a sandwich ELISA detection method simultaneously meets the requirements that an OD value of S0 is less than 0.1, an OD value of S7/S1(P/N) is the largest, a related coefficient of a dose-response curve is more than 0.99, and the detection rate of 30 cases of quality control serum is more than 90%;

fifth, the retrieval of monoclonal antibody genes in hybridomas

(1) Extraction with Trizol reagent 5X 10⁶Total RNA of selected hybridoma cells of (B7-H1);

then using OligodT as a primer, carrying out reverse transcription by AMV reverse transcriptase at the temperature of 37 ℃ for 15 minutes, and synthesizing to obtain cDNA;

taking the synthesized cDNA as a template, and carrying out nested PCR amplification aiming at a primer of an RNA sequence and a gene specific primer GSP, wherein the PCR conditions are as follows: denaturation at 95 deg.C for 5 min; 94 ℃ for 1 min; at 50 ℃ for 2 min; 72 ℃, 1min, 35 cycles; extending for 10min at 72 ℃;

(2) identifying the PCR product by 1% agarose gel electrophoresis, cutting and recovering a target gel strip, calling a target gene, connecting the target gene to a pGEM-T vector, carrying out EcoRI enzyme digestion to identify a positive recombinant plasmid, and determining a DNA sequence to respectively obtain a coding heavy chain variable region gene sequence and a coding light chain variable region gene sequence;

carrying out codon optimization and synthesis on the obtained heavy chain and light chain variable region gene sequences and the constant region of the antibody to respectively obtain a heavy chain and a light chain of a complete antibody gene, and respectively cloning the obtained heavy chain and light chain genes into a pMD18-T expression vector; respectively extracting 150 mu g of plasmids, removing endotoxin (less than 1EU/mg), co-transfecting the obtained 2 plasmids 1:1 into suspension competent TG1 cells with the volume of 50mL, wherein PEI is adopted as a transfection reagent, and the ratio of the plasmids to the PEI is 1: 2;

after 3-7 days of transfection, collecting cell supernatant, detecting whether the antibody expression is correct by SDS-PAGE, purifying by a ProteinA column, and concentrating to obtain qualified PD-L1 monoclonal antibody with the purity of more than 98%.

Example 3

This example provides a PD-L1 assay kit containing the PD-L1 monoclonal antibody described in example 1.

Example 4

The embodiment provides a preparation method of a PD-L1 detection kit, which comprises the following steps:

(1) using streptavidin magnetic beads as an immunoreaction carrier, diluting the PD-L1 monoclonal antibody to 2.5 mu g/mL by using 0.02mol/L Tris-HCl coupling buffer solution, adding the streptavidin magnetic beads, and reacting for 30min in a shaking table at 37 ℃;

washing with 0.01mol/L PBS and 0.05% tween-20 washing buffer solution for about 3 times;

adding 1ml of 0.01mol/L PBS blocking solution containing 2% fetal calf serum, reacting for 2h at 37 ℃ and 180rpm in a shaking bed, performing magnetic separation, and removing supernatant;

washing with 1ml of 0.01mol/L PBS buffer solution containing 0.1% tween-20 and pH7.4 for 3 times, suspending the magnetic particles in 1ml of 0.01mol/L PBS buffer solution containing 1% bovine serum albumin and pH7.4 to obtain PD-L1-coated magnetic beads, and keeping at 4 deg.C;

(2) diluting the prepared PD-L1 protein antigen with 1% BSA/0.5mol/L PBS, and subpackaging by 6 gradients (10, 50, 100, 200, 400, 800pg/mL) according to the proportion to prepare a PD-L1 calibrator;

adding 50 μ L of 0.5mM acridinium ester (4- (2-succinimidylcarboxyl) phenyl-10-methylacridine-9-carboxylate fluorosulfonate) solution to biotinylated PD-L1 polyclonal antibody, and reacting in a shaker at 25 deg.C and 180rpm in the absence of light for 20 min;

taking out after the reaction is finished, adding 100 mu l of 10% lysine salt solution, keeping out of the sun at 25 ℃, reacting for 30min at 180rpm in a shaking table;

purifying with G-25 desalting column to obtain PD-L1 acridinium ester standard antibody;

(3) adding 50 mu L/hole of serum sample and calibrator into the magnetic beads coated with PD-L1, adding 50 mu L/hole of PD-L1 acridinium ester standard antibody, incubating for 1h at 37 ℃, forming a solid phase antibody-antigen-labeled antibody complex after incubation, washing for 5 times at 0.01mol/LPBS, and removing unbound PD-L1 polyclonal antibody and acridinium ester;

respectively adding 0.1mol/L HNO₃，0.1％H₂O₂Immediately putting 50 mul of luminescence excitation liquid A, 0.25mol/L NaOH and 50 mul of 2% Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity (RLU) of each hole within the accumulated time of 1.5 s;

the RLU value of the sample increases along with the increase of the concentration of PD-L1, and the content of PD-L1 in the sample can be calculated according to the reaction curve.

Experimental example Performance of detection kit for detecting PD-L1

(1) Performance analysis of PD-L1 detection kit

Repeatability: the intra-batch Coefficient of Variation (CV) of the PD-L1 detection kit is not more than 10 percent; the inter-batch variation Coefficient (CV) is not more than 15%;

analysis sensitivity: the lowest detection limit of the kit is not more than 6 pg/mL;

analysis of specificity: the detection result of specific substances (PD-1 and PD-L2) with certain concentration is not more than 6 pg/mL;

linear range: in the concentration range of 0-800pg/mL, the linear correlation coefficient r is not less than 0.9900.

(2) Clinical performance of PD-L1 detection kit

2.1, 998 tests, 650 normal persons, 348 non-small cell lung cancer patient serum PD-L1 tests are carried out, the normal reference value of the kit is 0-150pg/mL, and the test results of the kit are as follows:

TABLE 1 detection results of PD-L1 and CYFRA21-1 kit for non-small cell lung cancer

TABLE 2 differential diagnosis effect of PD-L1 and CYFRA21-1 kit on non-small cell lung cancer

The sensitivity of the chemiluminescence detection kit for programmed death ligand 1(PD-L1) to non-small cell lung cancer is 82.76%, and the specificity is 94.92%; is superior to similar products in the current market, and can play a role in clinical auxiliary diagnosis of the non-small cell lung cancer.

2.2 assay consistency analysis of PD-L1 assay kit

A preparation comparison kit for detecting PD-L1 is prepared by using a humanized IgG4-kappa antibody of PD-L1 of Keytruda as a coating antibody by using the preparation method of the reagent kit, and the consistency of the capability of detecting PD-L1 in serum of the reagent kit is evaluated with the reagent kit.

The test samples 115 were composed of 56 gastric cancer patient samples and 59 normal human samples.

The percent-of-compliance and its confidence interval are shown in the table below.

TABLE 3 percent of agreement and confidence intervals

TABLE 4 measurement of symmetry

a. Null hypotheses are not used.

b. Asymptotic standard errors with null hypotheses are being used.

The coefficient of conformity kappa (K) has the value: 1.000.

2.3 regression analysis

a. And (4) performing regression analysis on each sample measured value and the corresponding measured value of the comparison system of the assessment system by using a scatter diagram (the assessment system and the evaluation kit are used as Y axes, and the comparison kit is used as an X axis).

b. Correlation analysis

TABLE 5 correlation analysis

Correlation was significant on 0.01 layers (double tail).

As can be seen from FIG. 2 and the SPSS output table, the correlation coefficient of the two reagent detection results is 0.997, and the result of the hypothesis test performed on the correlation coefficient is P < 0.05, which indicates that there is a linear correlation between the two reagent detection results; the correlation coefficient r is close to the maximum possible value 1, which indicates that the detection results of the two reagents have strong correlation.

c. Regression equations for both reagents and 95% confidence intervals for equation slope b

95% confidence interval of Table 6b

Coefficient of performance^a

a. Strain number/contrast reagent measurement

From the upper graph outputted by SPSS, it can be seen that the regression equation between the test results of the clinical test kit and the test results of the contrast agent is Y ═ 1.008X-0.923, and the slope b of the equation is within the 95% confidence interval.

The detection capability of the programmed death ligand 1(PD-L1) chemiluminescence detection kit is consistent with that of a control kit, which shows that the binding capability of the PD-L1 monoclonal antibody of the invention and the humanized IgG4-kappa antibody of PD-L1 of Keytruda to free PD-L1 protein in serum is consistent, and the PD-L1 detection kit can be used as a monitoring tool of PD-1/PD-L1 pathway targeted drugs.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Peking Puff vitamin technology Co., Ltd

<120> PD-L1 monoclonal antibody, kit, preparation method and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 132

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn

20 25 30

Leu Gly Gly Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr

35 40 45

Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Ser Leu Ile

50 55 60

Tyr Asp Asn Thr Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly

65 70 75 80

Ala Leu Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala

85 90 95

Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Arg Asp Ser Ser Asn Ala

100 105 110

Gly Ser Val Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser

115 120 125

Ser Arg Ser Ser

130

<210> 2

<211> 95

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Tyr

65 70 75 80

Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Ser Leu Ile Tyr Asp

85 90 95

<210> 3

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ser Ser Val Ser Ala Asn Leu Gly Gly Lys Asn Asp Asp Ser

1 5 10

<210> 4

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asn Thr Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser

1 5 10

<210> 5

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Gly Thr Thr Leu

1 5

<210> 6

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala Ala Leu Thr Gln Pro

20 25

<210> 7

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Tyr

1 5 10 15

Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Ser Leu Ile Tyr

20 25 30

<210> 8

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gly Ala Leu Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln

1 5 10 15

Ala Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Arg Asp Ser Ser Asn

20 25 30

Ala Gly Ser Val Phe Gly

35

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

1 5 10

<210> 10

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Thr Val Lys

1

<210> 11

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val

1 5 10

<210> 12

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Glu Val Gln Leu Leu Glu

1 5

<210> 13

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ile Met

1 5 10 15

Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

20 25 30

<210> 14

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ile Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp

1 5 10

<210> 15

<211> 387

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtggcccag 60

gcggccctga ctcagccgtc ctcggtgtca gcaaacctgg gaggaaccgt caagatcacc 120

tgctccgggg gtagtggcag ctacggctgg tatcagcaga aggcacctgg cagtgcccct 180

gtcagtctga tctatgacaa caccaacaga ccctcggaca tcccttcacg attctccggt 240

gccctatccg gctccacagc cacattaacc atcactgggg tccaagccga ggacgaggct 300

gtctattact gtgggagcag ggacagcagt aatgctggtt ctgtatttgg ggccgggaca 360

accctgaccg tcctaggtca gtcctct 387

<210> 16

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatatca tgatgtgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttatccta gtggtggtat cacattctac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gcgaatcaag 300

cttggtaccg tcacgacagt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360

<210> 17

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgacatgtgc tabcatgctg ctcctgg 27

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccctcgaggc ggccgcctag atcctctttc 30

Claims

1. A PD-L1 monoclonal antibody, characterized in that,

the heavy chain variable region amino acid sequence of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 9.

2. The PD-L1 monoclonal antibody of claim 1,

the amino acid sequences of a heavy chain hypervariable region CDR-H1, a heavy chain hypervariable region CDR-H2, a heavy chain hypervariable region CDR-H3, a heavy chain framework region FR-H1, a heavy chain framework region FR-H2 and a heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

3. The PD-L1 monoclonal antibody of claim 1,

the nucleic acid sequence of the 387bp (5 '-3') heavy chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 8.

4. The PD-L1 monoclonal antibody of claim 1,

the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2, the light chain hypervariable region CDR-L3, the light chain framework region FR-L1, the light chain framework region FR-L2 and the light chain framework region FR-L3 amino acid sequences of the PD-L1 monoclonal antibody are respectively amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15.

5. The PD-L1 monoclonal antibody of claim 1,

the 360bp (5 '-3') nucleic acid sequence of the light chain variable region of the PD-L1 monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 16.

6. A method for preparing the PD-L1 monoclonal antibody of any one of claims 1 to 5, characterized by comprising the following steps:

firstly, preparing a PD-L1 protein antigen;

secondly, preparing a PD-L1 polyclonal antibody;

thirdly, preparing a PD-L1 monoclonal antibody;

fifthly, the monoclonal antibody gene in the hybridoma is regulated:

extracting total RNA of the hybridoma cells;

carrying out PCR amplification by taking the synthesized cDNA as a template;

transfecting the obtained plasmid into a competent cell;

7. The method for producing the PD-L1 monoclonal antibody according to claim 6, characterized in that the PCR amplification conditions are: denaturation at 95 deg.C for 5 min; 94 ℃ for 1 min; at 50 ℃ for 2 min; 72 ℃, 1min, 35 cycles; extension at 72 ℃ for 10 min.

8. A PD-L1 detection kit, characterized by comprising the PD-L1 monoclonal antibody of any one of claims 1 to 5.

9. A method of making the PD-L1 test kit of claim 8, comprising the steps of:

washing a buffer solution;

purifying to obtain a PD-L1 acridinium ester standard antibody;

10. The use of the PD-L1 detection kit of claim 8 in the preparation of a detection kit for the clinical assistance of diagnosing non-small cell lung cancer and assessing the efficacy of a PD-L1-targeted drug.