CN112661816B - Artificial antigen and kit for detecting blood concentration of rituximab - Google Patents
Artificial antigen and kit for detecting blood concentration of rituximab Download PDFInfo
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Abstract
The invention relates to the detection of blood concentration of a monoclonal antibody, in particular to an artificial antigen and a kit for detecting the blood concentration of rituximab. The artificial antigen comprises an amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. The kit comprises the artificial antigen. The artificial antigen or the kit provided by the invention can sensitively and accurately detect the concentration of the rituximab in a serum sample, and can be used for providing accurate monitoring data of a therapeutic drug, so that the accurate treatment of the rituximab drug is realized.
Description
Technical Field
The invention relates to blood concentration detection of a monoclonal antibody, in particular to an artificial antigen and a kit for blood concentration detection of rituximab.
Background
With the intensive research and development and clinical use of monoclonal antibody drugs, clinical problems are gradually revealed, different individuals have significant differences in response degrees to the monoclonal antibody drugs, the rate of initial treatment response is 40-90%, and a part of patients who respond to initial treatment also have response loss during treatment, so that the disease progresses. The current detailed mechanisms that are not responsive to the initial treatment are still poorly understood and have more influence factors, such as epigenetics, pathological mechanisms, immunogenicity, etc. The individual differences in treatment response and the loss of response are thought to be related to factors such as the concentration of the monoclonal antibody in serum and immunogenicity after drug use, and therefore, dose optimization based on Therapeutic Drug Monitoring (TDM) can provide an effective means for precise treatment of the monoclonal antibody drug. The detection of drug concentration is an important link in TDM practice, and for monoclonal antibody drugs, immunological platform detection is usually adopted, and the most adopted method at present is an enzyme-linked immunosorbent assay. The method is used for a reaction mode of monoclonal antibody medicine TDM, recombinant protein of a monoclonal antibody in-vivo acting target spot is coated on an enzyme label plate, the monoclonal antibody medicine is added to be combined with antigen, enzyme-labeled anti-human IgG antibody is added to be combined with the antigen, enzyme catalysis substrate TMB is added, stop solution is added to stop reaction, and the concentration of the monoclonal antibody medicine can be detected according to absorbance measurement.
Rituximab injection (trade name: rituximab) was widely used for CD20 positive lymphoma patients as the first clinically used CD20 monoclonal antibody drug. As early as the clinical development of rituximab, differences in drug concentration were observed in patients with low-grade lymphoma whose response varied and in patients who achieved clinical remission after initial administration (71.3. Mu.g.mL) -1 ) Higher than non-responders (57.9. Mu.g.mL) -1 ) The concentration of drug in the patient was clinically alleviated after 4 doses (206.2. Mu.g.mL) -1 ) Much higher than the non-remission population (129.1. Mu.g.mL) -1 ). Therefore, accurate Therapeutic Drug Monitoring (TDM) of rituximab is very important for the therapeutic effect of rituximab.
Rituximab (rituximab) is a human-mouse chimeric antibody, is composed of a variable region Fab of mouse anti-CD 20 monoclonal antibody and a human IgG1 antibody stable region Fc fragment, contains 1328 amino acid residues, and has a relative molecular weight of about 144 multiplied by 10 3 Da. The widely used treatment for B-cell lymphoma, developed by IDEC corporation, was the first monoclonal antibody approved for clinical treatment of non-hodgkin lymphoma (NHL). Among the approved indications are rheumatoid arthritis, secondary symptoms of rheumatoid arthritis, chronic lymphocytic leukemia, wegener's granulomatosis and microscopic polyangiitis. Rituximab is sold year by year after approved by the U.S. FDA to market in 1997, the sales amount is $ 67.52 billion in 2018, and the sales amount of famous global antitumor drugs is prosperous. The target of action of rituximab is CD20, CD20 is a specific mark on the surface of human B lymphocytes, and consists of 297 amino acid residues, and the relative molecular mass is about 33 multiplied by 10 3 Da, belonging to non-glycosylated phosphoproteins, has a regulatory effect on the proliferation and differentiation of B lymphocytes. CD20 is ubiquitously expressed on circulating B cells, has four transmembrane helices with two extracellular loops ECL1 andECL2, second longer, and contains disulfide bonds.
Although rituximab has been successfully used in the clinic, little is known about the specific antigenic site of rituximab on CD20 and how rituximab activates complement to kill B cells. It is clear that rituximab does not act on a single linear epitope of the CD20 antigen, but on multiple epitopes of the transmembrane region of CD 20. The three-dimensional structure of the full-length CD20 protein and the complex of CD20 protein and rituximab was resolved in a breakthrough in Genentech, a paper published in Science at 2.2020. The article proves that rituximab can recognize not only the primary epitope ECL2 but also the secondary epitope ECL1/ECL2 of CD 20. Other in vitro studies have shown that the CD20 full-length protein expressed by prokaryotic or eukaryotic cells as an antigen is unable to react with rituximab in vitro and therefore cannot be used for drug concentration monitoring (TDM) of rituximab.
Disclosure of Invention
In order to accurately monitor the blood concentration of rituximab, the invention constructs an artificial antigen by using peptide mimic epitope. The codon of the coded artificial antigen is optimized and then is subjected to whole gene synthesis, and then the recombinant protein is obtained through prokaryotic expression. The artificial antigen contains a plurality of peptide mimic sequences, and has higher capability of combining rituximab and strong specificity.
In one aspect, the invention provides an artificial antigen for detecting blood concentration of rituximab, which is characterized in that the artificial antigen comprises an amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO.
In some embodiments of the invention, the artificial antigen further comprises a tag sequence. The tag sequence may be a GST tag sequence.
In some embodiments of the invention, the amino acid sequence of the artificial antigen is as set forth in SEQ ID NO 10, SEQ ID NO.
The invention also claims a gene for coding the artificial antigen for detecting the blood concentration of the rituximab.
In some embodiments of the invention, the gene comprises the nucleotide sequence set forth in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.
In another aspect, the invention further provides a kit, which is characterized by comprising the artificial antigen for blood concentration detection of rituximab.
In some embodiments of the kits of the invention, the artificial antigen is coated on an elisa plate at a concentration of 0.05-0.25 μ g/mL.
In some embodiments of the invention, the kit further comprises an enzyme-labeled secondary antibody, a sample diluent, a standard, a substrate developing solution, a stop solution, and/or a concentrated washing solution. The concentrated washings were diluted before use.
In some embodiments of the kits of the invention, the enzyme-labeled secondary antibody is HRP-labeled mouse anti-human IgG.
In some embodiments of kits of the invention, the standard is a rituximab solution at 30ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 3000ng/mL, and 10000 ng/mL.
In some embodiments of the kits of the invention, the sample diluent is 50mM phosphate buffer containing 0.3M NaCl; the stop solution is 2mol/L sulfuric acid; the concentrated washing solution is 0.02mol/L phosphate buffer solution with pH 7.4 and containing 0.05% (v/v) Proclin300 and 2.0% (v/v) Tween-20.
In still another aspect, the present invention provides a method for preparing an artificial antigen for blood concentration detection of rituximab, which is characterized by comprising the following steps: synthesizing the coding gene of the artificial antigen, constructing an expression vector, and inducing protein expression after transforming host bacteria.
In some embodiments of the preparation method of the invention, a gene with a nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 is obtained by an artificial synthesis technology, the synthesized gene is introduced into a pGEX-6p-1 vector and transformed into Escherichia coli BL-21 (DE 3), recombinant Escherichia coli is cultured and protein expression is induced by IPTG, and purified recombinant protein is obtained by an affinity chromatography method, thus obtaining the artificial antigen of the invention.
Within the scope of legal permission, the invention also claims a method for detecting the blood concentration of rituximab, which adopts the kit of the invention to carry out detection and comprises the following steps:
(1) Adding 90 mu L of sample diluent into the ELISA plate coated with the artificial antigen, adding 10 mu L of sample into each hole, adding 100 mu L of rituximab standard substance solution into each reserved hole, sealing the plate by using a cover plate film, and reacting in a 37 ℃ thermostat for 60 minutes;
(2) Spin-drying the liquid in the holes, washing the plate for 5 times, and patting to dry;
(3) Adding 100 mu L of mouse anti-human IgG solution marked by HRP into each hole, sealing the plate by a cover plate membrane, and reacting in a constant temperature box at 37 ℃ for 30 minutes;
(4) Spin-drying the liquid in the holes, washing the plate for 5 times, and patting dry;
(5) Adding 50 mu L of substrate color development liquid A and 50 mu L of substrate color development liquid B into each hole, lightly oscillating and uniformly mixing, and developing for 10 minutes in a constant temperature box at 37 ℃ in a dark place;
(6) Adding 50 mu L of 2mol/L sulfuric acid stop solution into each hole, and lightly shaking and uniformly mixing;
(7) Setting the wavelength of the microplate reader at 450/630nm, and measuring the absorbance value of each hole;
(8) And drawing a four-parameter fitting curve according to the absorbance value and the concentration value of the standard substance, substituting the absorbance value of the sample into the curve, and calculating the concentration of the rituximab in each sample.
The artificial antigen provided by the invention contains a plurality of CD20 peptide mimotopes (polypeptide 1, polypeptide 2 and polypeptide 3) in the same protein, overcomes the defect that the CD20 full-length protein can not react with rituximab in vitro, improves the capacity of combining with the rituximab in vitro compared with a single polypeptide, is not influenced by synthesis conditions, and can be prepared in a large number of stable ways by an in vitro expression method. The rituximab standard solution is used for carrying out performance verification on the artificial antigen and the polypeptide, and the result shows that the detection limits of the polypeptide 1, the polypeptide 2 and the polypeptide 3 are all larger than 30ng/mL, the detection sensitivity of the artificial antigen is below 30ng/mL, and the linear range of the artificial antigen with the amino acid sequence shown as SEQ ID NO. 2 is the widest. The kit for detecting the blood concentration of the rituximab, which is prepared by the invention, adopts the artificial antigen-coated ELISA plate to detect the blood concentration of the rituximab in a serum sample by an enzyme-linked immunosorbent assay, and the specificity is as high as 99%. Clinical test results show that the kit provided by the invention is used for detecting a patient serum sample taking rituximab drugs, and the detection result has high consistency with that of a commercial kit.
In conclusion, the artificial antigen and the kit provided by the invention can sensitively and accurately detect the concentration of rituximab in a serum sample, can be used for providing accurate monitoring data of therapeutic drugs and realizing accurate treatment of rituximab drugs.
Drawings
FIG. 1 is SDS-PAGE electrophoresis of purified recombinant proteins Rp1, rp2, rp3.
FIG. 2 Western Blot detection scheme of purified recombinant proteins Rp1, rp2, rp3.
FIG. 3 comparison of the detection performance of different antigens ( polypeptide 1,2,3, proteins Rp1, rp2, rp 3) on rituximab; wherein the abscissa is rituximab standard substance concentration (ng/ml), and the ordinate is the absorbance value of the antigen-antibody reactant at 450/630 nm.
FIG. 4 is a comparison of the results of the rituximab blood levels measured using the kit of the present invention and a commercial kit; wherein, the abscissa is the plasma concentration (mu g/ml) of rituximab detected by the kit P1, the kit P2 and the kit P3, and the ordinate is the plasma concentration (mu g/ml) of rituximab detected by the commercial kit.
Detailed Description
The present invention is further illustrated below by reference to examples, which are to be understood as being merely illustrative and explanatory of the invention and not limiting the scope of the invention in any way.
Main reagent and consumable
Vector pGEX-6p-1: purchased from the biotechnology limited of hunan fenghui, cat # goods: YH005.
T-Vector pMD TM 19 (Simple): purchased from baozi physician technology (beijing) limited, cat #: 3271.
restriction enzymes: both BamH I and Not I are available from NEB (Beijing) Inc. under the # R0136S and # R0189S designations.
T4 DNA ligase: baobaozhi limited (da lian), cat #: 2011A.
The general agarose gel recovery kit (cat # DP 209) and the plasmid miniprep kit (cat # DP 103-02) were purchased from Tiangen Biotechnology (Beijing) Ltd.
Trans 5. Alpha. Competent cells (cat # CD 201-01), E.coli BL-21 (DE 3) competent cells (cat # CD 601), and goat anti-mouse secondary antibody (cat # HS 201-01) were purchased from Beijing Quanjin Biotechnology Ltd.
Sepharose 4B: thermo Fisher, available from beijing jinshi baiyou technologies ltd, cat #: 16100.
anti-GST antibody: purchased from Shanghai Biyuntian Biotechnology Co., ltd, cat #: AG768.
Red-PAGE color quick gel preparation kit 12% (Sevenbio, cat # SW 145-02), 5 × protein loading buffer solution (Sevenbio, cat # SW 163-01), blocking solution (Sevenbio, cat # SW 162-02-02), 10 × TBST (Sevenbio, cat # SW 111-02) were purchased from Severn Innovation (Beijing) science and technology Co., ltd.
SuperSignal chemiluminescent substrate: purchased from siemer feishier technologies, inc, cat # s: 7JU6896.
BCA-100 protein quantification kit: purchased from beijing seich biotechnology limited, cat #: 300001-B.
Rituximab: roche USA under the batch number H0278.
The two-component TMB developing solution (Solebao, cat # PR 1210) and the HRP-labeled mouse-anti-human IgG (Solebao, cat # K0001M-HRP) were purchased from Beijing Solebao scientific Co., ltd.
An enzyme label plate: corning, cargo number: 42592.
enzyme-linked dilution: manufactured by Biopanda diagnostic reagents, inc., under the trade name EL0004.
The gene synthesis in the following examples was carried out by the Beijing Nonosel genome research center, inc. The normal human serum samples and patient serum samples used in the examples described below were provided by the national hospital of Jiangxi province.
Unless otherwise specified, the experimental reagents used in the following examples are conventional in the art, and may be prepared according to conventional methods in the art or commercially available. Unless otherwise specified, the experimental procedures used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, such as Molecular cloning, A laboratory Manual (Sambrook J & Russell DW, molecular cloning: a laboratory Manual, 2001), or the manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1 expression and purification of Artificial antigen for Rituximab blood concentration detection
1. Sequence design of recombinant proteins
We sought peptide mimetic sequences found using phage peptide library technology, polypeptide 1: CALMIANSC, polypeptide 2: CWWEWTIGC, polypeptide 3: WPTWLE. Three polypeptide sequences of polypeptide 1, polypeptide 2 and polypeptide 3 are connected by flexible peptide GGGGS, and different sequence combinations are carried out to obtain the following recombinant protein sequences:
P1：CALMIANSCGGGGSCWWEWTIGCGGGGSWPTWLE(SEQ ID NO:1)；
P2：CWWEWTIGCGGGGSCALMIANSCGGGGSWPTWLE(SEQ ID NO:2)；
P3：CALMIANSCGGGGSWPTWLEGGGGSCWWEWTIGC(SEQ ID NO:3)。
2. gene synthesis and vector construction
Coli strain BL-21 (DE 3) for prokaryotic expression has a certain preference for codons, so we first performed codon optimization of the gene coding sequences of recombinant proteins P1, P2 and P3, the optimized gene coding sequences are as follows:
rP1 sequence (5 '-3'):
tgcgcgctgatgattgcgaacagctgcggcggcggcggcagctgctggtgggaatggaccattggctgcggcggcggcggcagctggccgacatggctggaa(SEQ ID NO:4)；
rP2 sequence (5 '-3'):
tgctggtgggaatggaccattggctgcggcggcggcggcagctgcgcgctgatgattgcgaacagctgcggcggcggcggcagctggccgacatggctggaa(SEQ ID NO:5)；
rP3 sequence (5 '-3'):
tgcgcgctgatgattgcgaacagctgcggcggcggcggcagctggccgacatggctggaaggcggcggcggcagctgctggtgggaatggaccattggctgc(SEQ ID NO:6)。
since the recombinant protein was small, we selected the GST-tagged vector pGEX-6p-1 for expression of the protein of interest. After analyzing the enzyme cutting sites of pGEX-6p-1, bamH I and Not I are selected as enzyme cutting sites of recombinant protein coding genes connected into a vector, so that enzyme cutting sites BamH I (5 '-GGATCC-3') and Not I (5 '-GCGGCCGC-3') and respective protection bases thereof are respectively introduced into the 5 'end and the 3' end of the gene sequence of the recombinant protein. Then, the Beijing Nosai genome research center, inc. was entrusted with the whole gene synthesis, and the synthesized gene sequence was ligated into T-Vector pMD TM 19 (Simple) vector, and sequencing analysis of the obtained recombinant vector shows that the sequence is completely correct. We will link with the coding recombinant protein nucleotide sequence of recombinant vectors named rP1-T, rP-T, rP-T. The three nucleotide sequences cloned into the T-vector are as follows, the restriction sites are underlined:
rP1 sequence with cleavage site (5 '-3'):
cgcggatcctgcgcgctgatgattgcgaacagctgcggcggcggcggcagctgctggtgggaatggaccattggctgcggcggcggcggcagctggccgacatggctggaagcggccgctaaactat(SEQ ID NO:7)；
rP2 sequence (5 '-3') with cleavage site:
cgcggatcctgctggtgggaatggaccattggctgcggcggcggcggcagctgcgcgctgatgattgcgaacagctgcggcggcggcggcagctggccgacatggctggaagcggccgctaaactat(SEQ ID NO:8)；
rP3 sequence with cleavage site (5 '-3'):
cgcggatcctgcgcgctgatgattgcgaacagctgcggcggcggcggcagctggccgacatggctggaaggcggcggcggcagctgctggtgggaatggaccattggctgcgcggccgctaaactat(SEQ ID NO:9)。
we used restriction enzymes BamH I and Not I to cut out 102bp rP1, rP2 and rP3 fragments from rP1-T, rP-T, rP-T vector. Meanwhile, pGEX-6p-1 vector was linearized with restriction enzymes BamH I (NEB, # R0136S) and Not I (NEB, # R0189S).
Enzyme digestion system:
the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3h. Using a general agarose gel recovery kit (Tiangen, DP 209), 102bp rP1, rP2, and rP3 fragments were recovered, respectively, according to the procedures described in the kit instructions. The three digested DNA fragments were then ligated into pGEX-6p-1 linear vectors using T4 DNA ligase (Takara, 2011A), respectively. The linking system is as follows:
ligation was performed overnight at 16 ℃ to give a ligation product. The ligation products were transformed into Trans 5. Alpha. Competent cells (Beijing holotype gold, CD 201-01) by heat shock method as follows: adding 10 μ L of the ligation product to the competent cells, and standing on ice for 30min; after heat shock is carried out for 45s at the temperature of 45 ℃, the centrifugal tube is quickly transferred to ice and stands for 2min; adding 500 μ L of antibiotic-free sterile LB liquid medium, shaking-culturing at 37 deg.C and 180rpm for 45min, and recovering cells; sucking 100 μ L of bacterial liquid, and spreading on 100 μ g/mL Amp + Resistant LB plate medium, 37 degrees C overnight culture. Positive single clones were selected for sequencing. Sequencing results show that the rP1, the rP2 and the rP3 fragments are respectively and successfully connected into the expression vector. Prokaryotic expression vectors for expressing the recombinant protein are named pGEX-Rp1, pGEX-Rp2 and pGEX-Rp3 respectively. The expressed protein sequence is shown as follows, wherein three epitopes are marked by underlines, the epitopes are connected by flexible peptide GGGGS, and other amino acid sequences are tag protein GST sequences.
Protein Rp1:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIPRNSCALMIANSCGGGGSCWWEWTIGCGGGGSWPTWLEVDSSGRIVTD(SEQ ID NO:10)
protein Rp2:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIPRNSCWWEWTIGCGGGGSCALMIANSCGGGGSWPTWLEVDSSGRIVTD(SEQ ID NO:11)
protein Rp3:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIPRNSCALMIANSCGGGGSWPTWLEGGGGSCWWEWTIGCVDSSGRIVTD(SEQ ID NO:12)
3. protein induced expression and purification
(1) Plasmid extraction and transformation
Recombinant plasmids pGEX-Rp1, pGEX-Rp2 and pGEX-Rp3 were extracted using a plasmid miniextraction kit (Tiangen, DP 103-02) according to the protocol of the kit instructions. The three recombinant plasmids are respectively transferred into Escherichia coli BL-21 (DE 3) cells (Beijing holotype gold, CD 601) by a heat shock method, and the recombinant bacteria are obtained by the same transformation method.
(2) Protein expression
The recombinant bacteria were inoculated into 100. Mu.g/mL Amp + Resistant LB liquid medium, shaking at 37 ℃ and 180 rpm. To be OD 600 When the concentration is approximately equal to 0.8, IPTG (isopropyl-. Beta. -D-thiogalactoside) is added to a final concentration of 1mM, and the expression is induced at 30 ℃ for 3h. Subpackaging the bacterial liquid into high-speed centrifuge bottles, centrifuging at 8000rpm for 5min, and collecting the thallus. The recombinant bacteria were lysed using JY92-II ultrasonic cell disruptor (Ningbo Xinzhi Biotech Co., ltd.) at 200W,15s × 10 times. The lysate was centrifuged at 10000rpm for 10mAnd in, taking the supernatant of the cell lysate for SDS-PAGE detection.
We used the ExPASY software (http:// web. ExPASy. Org/computer _ pi /) to predict that recombinant proteins with GST tags (Rp 1, rp2, rp 3) have a molecular weight of about 31kDa. SDS-PAGE result shows that 3 recombinant proteins are well expressed after being induced by 1mM IPTG for 3 hours, and the expression product of each induced recombinant plasmid can be clearly seen to have a heavily-stained protein band at about 31kDa, so that the size of the expression product of the induced recombinant plasmid is consistent with the size of the expected protein.
(3) Affinity chromatography purification of recombinant fusion proteins
Resuspend Sepharose 4B (Thermo Fisher), load 1mL of column, and equilibrate with 4mL of equilibration solution (pH 8.0, 50mM Tris,150mM NaCl). Filtering to remove the balance liquid, opening the bottom cover of the column, taking out the agar beads, placing the agar beads in a clean 3mL centrifuge tube, adding 1mL of the supernatant of the cell lysate of the recombinant escherichia coli, gently mixing the agar beads uniformly, placing the centrifuge tube at room temperature for 5-10 min, mixing the agar beads uniformly for several times, centrifuging the centrifuge tube at 500rpm for 10min, and removing the supernatant. Adding 2mL of Tris-HCl buffer solution for resuspension, placing at room temperature for 5-10 min, uniformly mixing for several times, centrifuging at 500rpm for 10min, and removing supernatant; repeat for 1 time. Adding 200 mu L glutathione eluent (50 mM Tris-HCl,10mM reduced glutathione, pH 8.0) into a centrifugal tube, gently mixing uniformly, placing at room temperature for 5-10 min, mixing uniformly for several times, centrifuging at 500rpm for 10min, collecting supernatant, dialyzing to remove glutathione to obtain purified recombinant proteins (Rp 1, rp2 and Rp 3), and storing at-80 ℃ for later use.
The purified recombinant protein was detected by SDS-PAGE, and the result is shown in FIG. 1, and the recombinant protein after affinity chromatography had a single band and a molecular weight of about 31kDa. Western Blot detection of the purified recombinant protein was performed using anti-GST antibody. The method comprises the following steps: gels were prepared using Red-PAGE color Rapid gel preparation kit 12% (Sevenbio, SW 145-02). mu.L of recombinant protein was added to 5. Mu.L of 5 Xprotein loading buffer (Sevenbio, SW 163-01) and boiled in boiling water for 5min. And (3) carrying out electrophoresis after sample loading, carrying out electrophoresis on the concentrated gel at a voltage of 60V, carrying out electrophoresis on the separated gel at a voltage of 80V until bromophenol blue just runs out of the gel, stopping electrophoresis, and carrying out membrane transfer. Transferring the membrane with 350mA current for 40min, taking out the membrane, and sealing with sealing solution (S) at room temperatureevenbio, SW 162-02-02) for 1h, hybridized with an anti-GST antibody (shanghai Biyun, AG 768) at 1 2 The membranes were diluted 10 XTBST (Sevenbio, SW 111-02) in O and washed 3 times for 5min each. After addition of 1. As a result, as shown in FIG. 2, a band having a molecular weight of about 31kDa was formed in the target region, and the recombinant fusion protein of interest was obtained.
The purified recombinant protein was quantitatively determined by performing an assay according to the operating instructions of the BCA-100 protein quantitation kit (Beijing Saichi Bion., 300001-B) as follows: taking the average OD value measured by the standard protein of the BCA-100 protein quantitative kit as a vertical coordinate, taking the protein concentration (mg/mL) of each hole as a horizontal coordinate to prepare a standard protein curve, obtaining a regression equation through linear regression fitting, substituting the OD values of the samples, and calculating the concentrations of the three recombinant proteins. The concentration of each of the three recombinant proteins (Rp 1, rp2, rp 3) was adjusted to 1mg/mL with PBS (pH 7.0).
Example 2 Performance testing of recombinant proteins
1. Solution preparation
pH 7.4,0.01mol/L phosphate buffer: weighing 7.9g NaCl,0.2g KCl,0.24g KH 2 PO 4 And 1.8g K 2 HPO 4 Dissolved in 800ml of distilled water, the pH of the solution is adjusted to 7.4 with HCl, and finally distilled water is added to a constant volume of 1L. Storing in refrigerator at 4 deg.C.
And (3) standard substance: rituximab (Roche, lot. USA, batch No. H0278) was diluted to final concentrations of 30ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 3000ng/mL and 10000ng/mL, respectively, with phosphate buffer at pH 7.4,0.01mol/L to give 6 concentrations of standards (S1-S6).
Washing liquid: 0.01M pH 7.4PBS buffer containing 0.05% Tween20 (v/v).
Sealing liquid: 2g bovine serum albumin was added per 100ml of phosphate buffer (pH 7.4, 0.01mol/L).
Substrate color developing solution: two-component TMB color developing solution (solibao, PR 1210).
Coating buffer (pH9.6, 0.1mol/L carbonate buffer): 3.34 g of anhydrous sodium bicarbonate and 1g of anhydrous sodium carbonate are taken, distilled water is added for full stirring, the pH value is adjusted to 9.6, and the volume is fixed to 500 ml.
2. Enzyme label plate coating
Experimental groups: the recombinant proteins Rp1, rp2, and Rp3 purified in example 1 were used to coat an enzyme plate (Corning, 42592), respectively. The coating method comprises the following steps: the purified recombinant protein (1 mg/mL) was subjected to 1:10000 dilution (namely coating concentration is 0.1 mug/mL), adding into an enzyme-labeled plate hole, reacting for 2 hours at 37 ℃ with 100 muL of each hole, shaking to dry after removing the coating liquid, adding 200 muL of confining liquid (phosphate buffer containing 2% bovine serum albumin) into each hole, reacting for 2 hours at 37 ℃, shaking to remove the confining liquid, shaking to dry, drying, and packaging and storing in vacuum by using an aluminum foil bag.
Control group: the enzyme label plate is respectively coated with artificially synthesized polypeptide 1 (CALMIANSC), polypeptide 2 (CWWEWTIGC) and polypeptide 3 (WPTWLE), the coating method is the same as the experimental group, and the coating concentration is 0.1 mu g/mL.
3. Sensitivity detection
(1) 100 mul of each standard substance S1-S6 and 100 mul of each normal human serum (K1-K10) without taking medicine are added into an enzyme label plate coated with protein or polypeptide, and the mixture is sealed by a cover plate membrane and reacted in a 37 ℃ thermostat for 60 minutes.
(2) Spin-dry the liquid in the hole, wash the plate with washing liquid for 5 times, and pat dry.
(3) 100. Mu.L of horseradish peroxidase-labeled mouse anti-human IgG (Solibao, K0001M-HRP) (diluted with blocking solution at 1.
(4) Spin-dry the liquid in the hole, wash the plate with washing liquid for 5 times, and pat dry.
(5) 50 μ L of substrate color developing solution A (carbamide peroxide) and 50 μ L of substrate color developing solution B (tetramethylbenzidine, TMB) are added into each hole, mixed evenly by gentle oscillation, and developed for 10 minutes in a constant temperature box at 37 ℃ in a dark place.
(6) 50 mu L of 2mol/L sulfuric acid stop solution is added into each hole, and the mixture is gently shaken and evenly mixed.
(7) The absorbance value (OD value) per well was measured with the microplate reader (Thermo Scientific, multiskan FC) wavelength set at 450/630 nm.
4. The result of the detection
The test data are shown in table 1, table 2 and fig. 3. It can be seen from table 1 that the detection limits of absorbance of these six antigens are 0.093, 0.085, 0.083, 0.073, 0.043 and 0.066, respectively. As can be seen from Table 2, the detection limits of the polypeptide 1, the polypeptide 2 and the polypeptide 3 on rituximab are all larger than 30ng/mL, and the detection sensitivities of the recombinant proteins Rp1, rp2 and Rp3 are all below 30 ng/mL. The protein Rp2 has the widest linear range and the best performance, and is most suitable for being used as an antigen for detecting rituximab.
Example 3 kit for Rituximab concentration detection kit Components of kit
1. Enzyme label plate coated with recombinant protein: the coating protein is the recombinant protein Rp1/Rp2/Rp3 purified in the embodiment 1, and the coating concentration can be 0.05-0.25 mu g/mL;
2. HRP-labeled mouse anti-human IgG: the working concentration of the diluent can be 1;
3. sample diluent: 50mM phosphate buffer (pH 7.4) containing 0.3M NaCl;
4. standard products S1-S6: rituximab solutions of 30ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 3000ng/mL and 10000ng/mL, respectively;
5. substrate color developing solution: the liquid A consists of a liquid A and a liquid B, and the liquid A of a substrate color development liquid is carbamide peroxide; the substrate color developing solution B is tetramethyl benzidine;
6. stopping liquid: 2mol/L sulfuric acid;
7. 20 × concentrated washing solution: the pH was 7.4, containing Proclin300 (v/v) at a final concentration of 0.05%, tween-20 at a final concentration of 2.0% (v/v), and 0.02mol/L phosphate buffer.
(II) preparation of kit
Coated enzyme label plate: the recombinant proteins Rp1, rp2 and Rp3 purified in example 1 were used to coat the elisa plates, respectively. The coating method comprises the following steps: recombinant protein (1 mg/mL) was subjected to 1:10000 dilution, adding into a hole of an enzyme label plate, reacting for 2 hours at 37 ℃ with 100 mu L of each hole; after the coating solution is thrown off, patting the solution dry, adding 200 mu L of confining solution (phosphate buffer solution containing 2% bovine serum albumin) into each hole, and reacting for 2 hours at 37 ℃; and (5) throwing off the sealing liquid, drying, and vacuum packaging and storing by using an aluminum foil bag.
Preparing enzyme-labeled antibody: the HRP-labeled mouse anti-human IgG needs to be purified by affinity chromatography, has strong species specificity, the purity of more than 95 percent and the titer of not less than 1. HRP-labeled mouse anti-human IgG (Solebao, cat # K0001M-HRP) was used diluted in commercial enzyme conjugate dilutions (Biopanda, cat # EL 0004).
Preparing sample diluent:50 mM phosphate buffer (pH 7.4) containing 0.3M NaCl, formulated as: 17.5g of NaCl,1g of KCl, and 1.2g of KH were weighed 2 PO 4 And 9g K 2 HPO 4 Dissolving in 800ml distilled water, adjusting pH value of the solution to 7.4 with HCl, and adding distilled water to a constant volume of 1L.
Preparing standard substance: a standard was prepared using a commercial rituximab drug (manufactured by roche, usa, under lot number H0278). Rituximab was diluted to final concentrations of 30ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 3000ng/mL and 10000ng/mL with phosphate buffer at pH 7.4,0.01mol/L to give standards S1-S6.
Preparation of concentrated washing liquid: phosphate buffer solution with pH 7.4 and 0.02mol/L is prepared. Then Proclin300 was added to a final concentration of 0.05% (v/v) and Tween-20 was added to a final concentration of2.0% (v/v). It is diluted 20 times with distilled water.
Kit assembly：
Kit P1: the enzyme label plate coated with the recombinant protein Rp1, the mouse anti-human IgG marked by the HRP, the sample diluent, the standard products S1-S6, the substrate developing solution, the stop solution, the concentrated washing solution and the kit use instruction are all put into the kit.
Kit P2: and (3) completely filling the enzyme label plate coated with the recombinant protein Rp2, the mouse-anti-human IgG marked by the HRP, the sample diluent, the standard substances S1-S6, the substrate developing solution, the stop solution, the concentrated washing solution and the kit use instruction into the kit.
Kit P3: the enzyme label plate coated with the recombinant protein Rp3, the mouse anti-human IgG marked by the HRP, the sample diluent, the standard products S1-S6, the substrate developing solution, the stop solution, the concentrated washing solution and the kit use instruction are all put into the kit.
Kit using method：
(1) Adding 90 mu L of sample diluent into the ELISA plate coated with the recombinant protein, adding 10 mu L of sample into each hole, adding 100 mu L of standard products S1-S6 into each reserved hole, sealing the plate by using a cover plate film, and reacting in a 37 ℃ thermostat for 60 minutes;
(2) Spin-drying the liquid in the holes, washing the plate 5 times by 1 times of washing liquid, and patting to dry;
(3) 100. Mu.L of HRP-labeled mouse anti-human IgG (diluted with an enzyme conjugate diluent according to 1;
(4) Spin-drying the liquid in the holes, washing the plate 5 times by 1 times of washing liquid, and patting to dry;
(5) Adding 50 mu L of substrate color development liquid A and 50 mu L of substrate color development liquid B into each hole, lightly shaking and uniformly mixing, and performing light-resistant color development for 10 minutes in a constant temperature box at 37 ℃;
(6) Adding 50 mu L of 2mol/L sulfuric acid stop solution into each hole, and lightly shaking and uniformly mixing;
(7) Setting the wavelength of a microplate reader at 450/630nm, and measuring the absorbance value (OD value) of each hole;
(8) And (3) taking the absorbance value and the identification concentration value of the standard as standards, performing four-parameter fitting curve, substituting the absorbance value of each detection hole into the curve, and obtaining the concentration of the rituximab in each sample.
Example 4 evaluation of the clinical test with the kit of the invention
The normal human serum samples used in the following experiments were from a healthy physical population and the patient serum samples were from clinical patients taking rituximab drugs.
1. Comparison of detection Performance between the kit of the present invention and the commercial kit
Serum samples from 45 patients taking rituximab were tested using the kits P1, P2 and P3 prepared in example 3 and a commercial kit (Theradiag, cat # LTR 002-48), respectively, to test the performance of the kit. Detection method the kit usage in example 3 was referred to. The detection results are shown in FIG. 4, the results of the kit P2 and the commercial kit are the best in accordance, and R is obtained after linear regression 2 =0.9853 better than the kits P1 and P3. The linear regression coefficients for kits P1 and P3 and the commercial kit were 0.8657 and 0.8843, respectively.
2. Specificity verification of the kit of the invention
100 normal human serum samples were tested using the kit P2 prepared in example 3 to test the specificity of the kit. The detection results are shown in table 3, among 100 normal human serum samples, the blood concentration of rituximab in 99 samples is less than 30ng/mL, and the rituximab is in an undetected state; the plasma concentration of rituximab in only 1 sample was 42ng/ml, which is a weakly positive sample.
The specificity of the kit is as high as 99%.
Sequence listing
<110> Jiangxi province people hospital
<120> artificial antigen and kit for rituximab blood concentration detection
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Claims (10)
1. The artificial antigen for detecting the blood concentration of rituximab is characterized by comprising an amino acid sequence shown in SEQ ID NO 1, SEQ ID NO.
2. The artificial antigen for blood rituximab concentration detection according to claim 1, wherein the artificial antigen further comprises a tag sequence.
3. The artificial antigen for detecting the blood concentration of rituximab of claim 2, wherein the amino acid sequence of the artificial antigen is shown as SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12.
4. A gene encoding the artificial antigen for blood concentration detection of rituximab of claim 1.
5. The gene of claim 4, wherein the gene comprises the nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO.
6. A kit comprising the artificial antigen for blood rituximab concentration detection according to any one of claims 1-3.
7. The kit of claim 6, wherein the artificial antigen is coated on the ELISA plate, and the coating concentration is 0.05-0.25 μ g/mL.
8. The kit according to claim 7, characterized by further comprising an enzyme-labeled secondary antibody, a sample diluent, a standard, a substrate developing solution, a stop solution and/or a concentrated washing solution.
9. The kit of claim 8, wherein the enzyme-labeled secondary antibody is HRP-labeled mouse anti-human IgG; the standard substance is rituximab solution with the concentration of 30ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 3000ng/mL and 10000 ng/mL; the sample diluent is 50mM phosphate buffer solution containing 0.3M NaCl; the stop solution is 2mol/L sulfuric acid; the concentrated washing solution is 0.02mol/L phosphate buffer solution with pH 7.4 and containing 0.05% (v/v) Proclin300 and 2.0% (v/v) Tween-20.
10. The method for preparing the artificial antigen for plasma rituximab concentration detection according to any one of claims 1-3, which comprises the following steps: synthesizing the coding gene of the artificial antigen, constructing an expression vector, and inducing protein expression after transforming host bacteria.
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