CN114106175B

CN114106175B - PD-L1 monoclonal antibody, kit, preparation method and application thereof

Info

Publication number: CN114106175B
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: 2023-06-23
Anticipated expiration: 2040-12-30
Also published as: CN114106175A

Abstract

The invention is suitable for the field of biotechnology, and provides a PD-L1 monoclonal antibody, a kit, a preparation method and application thereof. 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; 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. 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, high 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 targeting 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 B7 family member belonging to the class I transmembrane protein having a molecular weight of about 65kDa and a full length of 290 amino acids and comprising 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 Keystuda of PD-L1 approved first in 2017 is used for treating non-small cell lung cancer which fails to be treated by chemotherapy or targeted therapy, and research shows that the concentration of PD-L1 in serum of a patient with non-small cell lung cancer is related to aspects such as tumor disease progression, treatment effect evaluation, disease prognosis and the like, and has great application value in aspects such as disease diagnosis, disease prediction and the like.

In recent years, the research and application of the marker immunoassay technology are rapid, and the marker immunoassay technology is widely applied to various fields of biomedical basic theory research and clinical disease diagnosis. Methods for detecting serological indicators mainly include radioisotopic immunoassays, enzyme-linked immunosorbent assays, and chemiluminescent immunoassays. The methods can be used as a preliminary 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, the products for monitoring and prognosis of PD-1/PD-L1 pathway targeted drugs are blank in China at present, and the similar products have the possibility of further improving the specificity, the affinity and the like, 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, and aims to solve the problems in the prior art pointed out in the background art.

The embodiment of the invention is realized by 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 as 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 as SEQ ID NO. 9.

As another preferred scheme of the embodiment of the invention, the amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2, the heavy chain hypervariable region CDR-H3, the heavy chain framework region FR-H1, the heavy chain framework region FR-H2 and the heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively amino acid sequences shown as 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 amino acid sequences shown as 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, which are formed by replacing, deleting and/or adding one or more amino acid residues and have the same functions.

As another preferable scheme of the embodiment of the invention, the nucleic acid sequence of 387bp (5 '-3') 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 equivalent functions 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 scheme of the embodiment of the invention, the amino acid sequences of 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 of the PD-L1 monoclonal antibody are respectively amino acid sequences shown as 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 amino acid sequences shown as 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, which are formed by replacing, deleting and/or adding one or more amino acid residues and have the same functions.

As another preferable scheme of the embodiment of the invention, the nucleic acid sequence of 360bp (5 '-3') 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 equivalent functions formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 16.

Another object of the embodiment of the present invention is to provide a method for preparing the PD-L1 monoclonal antibody, which includes the following steps:

1. preparing PD-L1 protein antigen;

2. preparing PD-L1 polyclonal antibody;

3. preparing PD-L1 monoclonal antibody;

4. monoclonal antibody screening: establishing a dose response curve, and screening an optimal monoclonal antibody;

5. modulation of monoclonal antibody genes in hybridomas:

extracting total RNA of the hybridoma cells;

performing reverse transcription synthesis by using oligo dT as a primer to obtain cDNA;

performing PCR amplification by taking the synthesized cDNA as a template;

obtaining a gene sequence encoding a heavy chain variable region and a gene sequence encoding a light chain variable region;

the heavy chain variable region gene sequence, the coding light chain variable region gene sequence and the constant region of the antibody are subjected to codon optimization and synthesis to respectively obtain the heavy chain and the light chain of the complete antibody gene;

cloning the obtained heavy chain gene and light chain gene into a pMD18-T expression vector respectively;

transfecting the obtained plasmid into competent cells;

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

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

Another object of the embodiment of the invention is to provide a PD-L1 detection kit, which contains the PD-L1 monoclonal antibody.

Another object of the embodiment of the present invention is to provide a method for preparing the PD-L1 detection kit, which includes the following steps:

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

washing buffer solution;

adding PBS sealing liquid containing fetal calf serum, magnetically separating after reaction, and removing supernatant;

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

diluting the PD-L1 protein antigen by a plurality of gradients in proportion, and subpackaging to prepare a PD-L1 calibrator;

adding the PD-L1 polyclonal antibody into an acridinium ester solution, and reacting in a dark place;

taking out after the reaction is finished, adding a lysine salt solution, and continuing the light-shielding reaction;

purifying to obtain PD-L1 acridine ester antibody;

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

respectively adding HNO ₃ 、H ₂ O ₂ The luminescence excitation liquid A, naOH, triton-100 luminescence excitation liquid B is immediately placed into a chemiluminescence immunoassay instrument to detect the luminescence intensity of each hole;

and calculating the content of PD-L1 in the sample according to the reaction curve.

The embodiment of the invention also aims to provide an application of the PD-L1 detection kit in preparing a detection kit for diagnosing clinical assistance of non-small cell lung cancer and evaluating the curative effect of PD-L1 targeted drugs.

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, high 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 targeting drugs.

Drawings

Fig. 1: PD-L1 assay dose-response scheme.

Fig. 2: regression analysis plots of the evaluation reagent measurements versus the comparison reagent measurements.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Specific implementations of the invention are described in detail below in connection with 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 as SEQ ID NO. 1; or the amino acid sequence shown in SEQ ID NO.1 has the same function by replacing, deleting and/or adding one or more amino acid residues.

The amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2, the heavy chain hypervariable region CDR-H3, the heavy chain framework region FR-H1, the heavy chain framework region FR-H2 and the heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively shown as 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 replaced, deleted and/or added with one or more amino acid residues to form the amino acid sequence with the same function.

The nucleic acid sequence of 387bp (5 '-3') 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 shown in SEQ ID NO.8 has the same function by replacing, deleting and/or adding one or more amino acid residues.

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 the amino acid sequence shown in SEQ ID NO.9 has the same function by replacing, deleting and/or adding one or more amino acid residues.

The amino acid sequences of 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 of the PD-L1 monoclonal antibody are respectively the amino acid sequences shown as 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 as 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 replaced, deleted and/or added with one or more amino acid residues to form the amino acid sequence with the same function.

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

1. preparation of PD-L1 protein antigen:

(1) A pair of primers was designed based on the sequence of human PD-L1 (GQ 904196.1) 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 matched with corresponding multiple cloning sites on a mammalian cell high-efficiency expression plasmid vector pSec/WG;

the cloning plasmid containing the human PD-L1 gene fragment is synthesized through total genes, the plasmid is used as a template, and the Pyrobest DNA polymerase is used for carrying out PD-L1 gene specific amplification, and the PCR amplification conditions are as follows: denaturation at 95℃for 5min;94 ℃ for 1min;50 ℃ for 2min;72 ℃,1min,35 cycles; extending at 72deg.C for 10min;

performing gel recovery, chloroform extraction of the amplified PCR product, ethanol precipitation and TE dissolution 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 digestion, and recovered by a gel electrophoresis method;

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

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

(2) Transfecting recombinant PD-L1/pSec/WG plasmid into eukaryotic expression cell line Flp-In CHO by adopting liposome reagent Lipofectamine 2000, culturing the transfected cells In hygromycin-containing culture medium, and screening;

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

culturing Flp-In CHO cells transfected with PD-L1/pSec/WG recombinant plasmid In an ultraCHO serum-free medium, and collecting cell culture supernatant 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 supernatant ₃ And 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; washing the column with 20-30 volumes of PBS (pH 7.2); 28. Mu.L of 1.25M Tris-HCl (pH 8.0) was previously added to each of the collection vials; eluting with 100mM Glycine-HCl (pH 3.0) eluent, and collecting in the collecting small tube at 1.0rnl per tube to allow the eluent to be immediately neutralized in the collecting small tube; typically proteins elute at tubes 3-10, with peaks at about 3, 4, 5; collecting all the eluate with A280 > 0.01, and concentrating with centrifugal filtration 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 the protein by a 0.22 mu m filter membrane, and storing the protein at the temperature of minus 20 ℃ for later use;

2. preparation of PD-L1 polyclonal antibody

(1) Using male big ear rabbits as immunized animals, firstly using 10mg BCG vaccine to inject and stimulate the animals, and starting immunization after one week by using PD-L1 protein as an immune antigen; subcutaneous injection at 4-6 points under the feet, and four times of immunization of animals are performed by using Freund's complete adjuvant as an immune adjuvant; taking 1mg of PD-L1 protein each time, respectively sucking equivalent Freund complete adjuvant and antigen solution into two syringes, fully emulsifying for 1 hour, and performing subcutaneous injection every two weeks;

taking the detection titer of the ear vein blood ELISA method to reach 1:40000, carrying out carotid blood discharge, centrifuging at 5000rpm to obtain serum, and purifying by DEAE ion exchange to obtain a polyclonal antibody of crude serum for later use;

(2) Diluting serum crude polyclonal antibody to 1mg/mL with 0.5mol/L PBS (pH 7.5), preparing 3mL agarose gel of CNBr-Sephaose 4B, coupling 9mg PD-L1 protein antigen with coupling agent, reacting at room temperature for 4 hours to obtain PD-L1 antibody affinity chromatography column, purifying serum crude polyclonal antibody, eluting, collecting, measuring antibody OD value at 280nm by ultraviolet-visible spectrophotometer, dividing the obtained OD value by 1.35 to obtain concentration of the measured antibody, adding 40-50% glycerol, and standing at-20deg.C for long term storage;

3. preparation of PD-L1 monoclonal antibodies

(1) Culturing the above-obtained stably expressed PD-L1/pSec/WG recombinant plasmid Flp-In CHO cells to immunize 5 female BALA/c mice, each BALB/c mouse was subcutaneously injected 1X 10 ⁷ Cells were immunized 4 times consecutively, 2 weeks apart; blood is taken after 7 days of immunization, serum titer is detected by CLIA method, mice with highest titer are selected, and 1X 10 is injected into spleen ⁶ Individual cells are boosted, the spleen of the mouse is taken after 3 days, ground and spleen cells are counted for standby;

(2) Fusing spleen cells 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 culture, half-volume liquid exchange by 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 strain of hybridoma cells (B7-H1) with continuous secretion positive rate of PD-L1Ab of more than 98%, performing expansion culture, and preparing a mice by intraperitoneal injection, wherein 500 mu L of liquid paraffin is injected into the abdominal cavity of the mice 1 week ago;

collecting hybridoma cells by centrifugation, suspending with incomplete culture solution, mixing, and adjusting cell number to 1×10 ⁹ Starting inoculating mice, injecting 500 mu L of each mouse into the abdominal cavity, extracting ascites from the mice with obvious abdominal swelling after 1 week, centrifuging the obtained ascites at 3000r/min for 3 minutes, and collecting supernatant;

(3) purifying ascites by using a protein G purification column; balancing the purification column with 0.02mol/L PB buffer solution, adding ascites to sample, 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 freeze-preserving at-20deg.C;

4. monoclonal antibody screening

(1) Coating the prepared PD-L1 monoclonal antibody, and diluting the antibody to 2-5 mug/mL by 0.5mol/L PBS; 100 mu L/hole is added into an ELISA plate for coating, after 2-8 ℃ overnight, 0.9% NaCl is washed 3 times, a blocking solution containing 1% BSA is added, 150 mu L/hole is blocked, and after 2-8 ℃ overnight, the mixture is dried for standby;

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

a dose response curve was established with the PD-L1 concentration as the X-value on the abscissa and the OD value as the Y-value on the ordinate (see fig. 1). As can be seen from FIG. 1, the linear coefficient R is more than 0.99, the detection range can reach 0-800pg/ml, and the analysis performance of the PD-L1 detection kit is excellent; final selection of the PD-L1 monoclonal antibody with the best evaluation index; the monoclonal antibody protuberance antibody is used as an evaluation standard of a sandwich ELISA detection method, the evaluation standard simultaneously meets the conditions that the S0 OD value is less than 0.1, the S7/S1 (P/N) is maximum, the phase relation number of a dose response curve is more than 0.99, and the detection rate of 30 quality control serum is more than 90%;

5. modulation of monoclonal antibody genes in hybridomas

(1) Extraction with Trizol reagent 5X 10 ⁶ Total RNA of hybridoma cells (B7-H1);

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

using synthesized cDNA as a template, and performing 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℃for 5min;94 ℃ for 1min;50 ℃ for 2min;72 ℃,1min,35 cycles; extending at 72deg.C for 10min;

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

codon optimization and synthesis are carried out on the obtained heavy chain and light chain variable region gene sequences and the constant region of the antibody, the heavy chain and the light chain of the complete antibody gene are respectively obtained, and the obtained heavy chain and light chain genes are respectively cloned into a pMD18-T expression vector; the plasmids were separately extracted in an amount of 150. Mu.g and endotoxin removed (< 1 EU/mg), and the 2 plasmids obtained were 1:1 co-transfected into 50 mL-volume of suspended competent TG1 cells, with PEI being used as the transfection reagent, and the ratio of plasmid to PEI being 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 detection kit comprising the PD-L1 monoclonal antibody of example 1.

Example 4

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

(1) Taking streptavidin magnetic beads as an immunoreaction carrier, diluting a PD-L1 monoclonal antibody to 2.5 mug/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 wash buffer solution for about 3 times;

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

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

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

the biotinylated PD-L1 polyclonal antibody was added to 50. Mu.l of 0.5mM acridinium ester (4- (2-succinimidyl carboxyl) phenyl-10-methylacridine-9-carboxylate fluorosulfonate) solution and reacted in the shaking table at 25℃and 180rpm in the absence of light for 20min;

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

purifying by using a G-25 desalting column to obtain a PD-L1 acridine ester-labeled antibody;

(3) Adding 50 mu L/hole serum samples and calibrator into the PD-L1 coated magnetic beads, adding 50 mu L/hole PD-L1 acridine ester labeled antibody, incubating for 1h at 37 ℃, and washing for 5 times at 0.01mol/LPBS to remove unbound PD-L1 polyclonal antibody and acridine ester;

adding 0.1mol/L HNO respectively ₃ ，0.1％H ₂ O ₂ 50 μl of luminescence excitation solution A, 0.25mol/L NaOH, 50 μl of 2% Triton-100 luminescence excitation solution B, and immediately adding chemiluminescence immunityIn the epidemic detector, detecting the luminous intensity (RLU) of each hole with the accumulated time of 1.5 s;

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

Experimental example detection of performance of PD-L1 detection kit

(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%; the variation Coefficient (CV) between batches is not more than 15%;

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

assay specificity: the detection result of the specific substances (PD-1 and PD-L2) with a certain concentration is not more than 6pg/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 cases of detection, 650 cases of normal people, 348 cases of serum PD-L1 detection of non-small cell lung cancer patients, and the normal reference value of the kit is 0-150pg/mL, and the detection result of the kit is as follows:

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

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

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

2.2 detection consistency analysis of PD-L1 detection kit

The humanized IgG4-kappa antibody of PD-L1 of Keystuda is used as a coating antibody, the preparation method of the kit is used for preparing a preparation contrast kit for detecting PD-L1, and the consistency of the capability of detecting PD-L1 in serum with the kit is evaluated.

56 gastric cancer patient samples and 59 normal person samples are selected to form 115 test samples.

The compliance and confidence intervals are shown in the following table.

Table 3 coincidence rate and confidence interval thereof

Table 4 symmetrical measurements

a. No null hypothesis is used.

b. Asymptotic standard errors with no assumption of false are being used.

The consistency factor Kappa (K) value is: 1.000.

2.3 regression analysis

a. Regression analysis was performed on each sample measurement of the assessment system with the corresponding comparison system measurement on a scatter plot (Y-axis for the assessment system-assessment kit versus X-axis for the comparable kit).

b. Correlation analysis

TABLE 5 correlation analysis

* *. correlation is significant on the 0.01 layer (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 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 two reagent detection results have strong correlation.

c. Regression equation for two reagents and 95% confidence interval for equation slope b

95% confidence interval of Table 6b

Coefficients of ^a

a. Strain number \contrast agent measuring value

From the upper graph of the SPSS output, it can be seen that the regression equation between the clinical trial kit test results and the contrast agent test results is y=1.008X-0.923, with equation slope b within the 95% confidence interval.

The detection capability of the programmed death ligand 1 (PD-L1) chemiluminescent detection kit disclosed by the invention is consistent with that of a control kit, so that the capability of the PD-L1 monoclonal antibody disclosed by the invention for combining free PD-L1 protein in serum with the humanized IgG4-kappa antibody of PD-L1 of Keystuda is consistent, and the PD-L1 detection kit can be used as a monitoring tool of a PD-1/PD-L1 pathway targeted drug.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

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Sequence listing

<110> Beijing Prime vitamin technology Co., ltd

<120> a 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 is characterized in that,

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;

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.

2. The PD-L1 monoclonal antibody according to claim 1,

the amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2, the heavy chain hypervariable region CDR-H3, the heavy chain framework region FR-H1, the heavy chain framework region FR-H2 and the heavy chain framework region FR-H3 of the PD-L1 monoclonal antibody are respectively shown as 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 according to claim 1,

the nucleic acid sequence of 387bp (5 '-3') of the 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 according to claim 1,

the amino acid sequences of 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 and the light chain framework region FR-L2 of the PD-L1 monoclonal antibody are respectively shown as 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 according to claim 1,

the nucleic acid sequence of 360bp (5 '-3') 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 PD-L1 detection kit, comprising the PD-L1 monoclonal antibody of any one of claims 1-5.

7. The method for preparing the PD-L1 detection kit according to claim 6, comprising the steps of:

washing buffer solution;

purifying to obtain PD-L1 acridine ester antibody; the PD-L1 coated magnetic beads, the PD-L1 calibrator and the PD-L1 acridinium ester antibody jointly form a PD-L1 detection kit.

8. The use of the PD-L1 detection kit of claim 6 in the preparation of a detection kit for diagnosis of non-small cell lung cancer in clinical assistance, evaluation of the efficacy of a PD-L1 targeted drug.