CN109709337B - Immunohistochemical detection kit for human CD26 and clinical application thereof - Google Patents

Abstract

The invention relates to an immunohistochemical detection kit for human CD26 and clinical application thereof, belonging to the field of in vitro diagnosis. The immunohistochemical detection kit disclosed by the invention adopts a brand-new anti-human CD26 antibody, and the heavy chain variable region of the antibody contains the following complementarity determining regions: the amino acid sequences are respectively shown as SEQ ID NO: 1.2, 3, HCDR1, HCDR2, HCDR 3; and a light chain variable region sequence thereof comprising the following complementarity determining regions: respectively shown in SEQ ID NO: 4. LCDR1, LCDR2 and LCDR3 shown in FIGS. 5 and 6. The anti-human CD26 antibody specificity and other related detection performances adopted by the invention can meet the performance requirements of an immunohistochemical kit, and can be a murine antibody or a recombinant expression chimeric human antibody, which is convenient for mass production, wherein the recombinant expression chimeric human antibody reduces the human anti-mouse antibody reaction, reduces the non-specific adsorption, and can also meet the requirements of large-scale clinical application. When the CD26 target medicine is applied, the immunohistochemical kit can be used along with the application of the immunohistochemical kit to achieve the effect of accurate treatment.

Description

Immunohistochemical detection kit for human CD26 and clinical application thereof

Technical Field

The invention belongs to the field of in vitro diagnosis, and particularly relates to an anti-human CD26 antibody and application thereof in an immunohistochemical detection kit.

Background

CD26 is a ubiquitous multifunctional type II transmembrane protein, has multiple biological functions, and can also exist in plasma in a dissolved form. CD26 often exists as a homodimer, the monomer of which contains 766 amino acids and has a relative molecular mass of about 110 kDa. The CD26 amino acid residues are divided into 5 parts from the inside to the outside: an intracellular domain (aa 1-6), a transmembrane domain (aa 7-28), a hyperglycosylation domain (aa29-323), a cysteine-rich domain (aa 324-551) and a C-terminal catalytic domain (552-766). The C-terminal catalytic domain of CD26 plays a role in dipeptidyl peptidase IV (DPPIV) activity, can hydrolyze various substrates in vivo to play a biological role, and the cysteine-rich region can interact with various molecules in vivo so as to participate in immune functions in vivo. The role of CD26 in immune regulation has been extensively studied, and CD26 is a molecular marker of T cell activation, also acts as a costimulatory molecule in T cell signaling, and is involved in a variety of T cell functions, including T cell maturation and migration, cytokine secretion, T cell-dependent antibody production, B cell immunoglobulin transformation, and the like. CD26 plays an important role in the occurrence and development of autoimmune diseases, has become a molecular marker of clinical diseases, and is considered as a therapeutic or diagnostic target of some immunological diseases.

The research shows that part of cancer patients have no CD26 expression in the tumor tissues, and the patients have no curative effect when using CD26 targeted anticancer drugs. Therefore, before the CD26 target medicine is used, the CD26 protein expression condition of the tumor tissues of cancer patients is detected, and the effect of accurate treatment can be achieved. The CD26 antigen immunohistochemical detection kit can achieve the effect.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an antibody capable of effectively and specifically binding to human CD26, and apply the antibody to an immunohistochemical detection kit. More specifically:

the first object of the present invention is to provide an anti-human CD26 antibody,

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO: 2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;

and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

Preferably, the amino acid sequence of the heavy chain variable region of the anti-human CD26 antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

The second purpose of the invention is to provide a chimeric antibody, wherein the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 9, the nucleotide sequence of the coding antibody heavy chain is shown as SEQ ID NO. 10, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 11, and the nucleotide sequence of the coding antibody light chain is shown as SEQ ID NO. 12.

The third purpose of the invention is to provide an expression vector containing the chimeric antibody nucleotide sequence.

The fourth object of the present invention is to provide a recombinant cell line containing the chimeric antibody expression vector.

It is a fifth object of the present invention to provide a method for producing the above chimeric antibody, comprising:

(1) constructing a mammalian host cell strain expressing an anti-human CD26 antibody as described above;

(2) inoculating the cell strain into a culture medium for suspension culture;

(3) purification preparation of anti-human CD26 chimeric antibody.

The vector is preferably pCHO1.0. The mammalian cell CHO is preferably a CHO-S cell.

The sixth purpose of the present invention is to provide the use of the above anti-human CD26 antibody in detecting CD26 protein in human tissues.

The seventh purpose of the invention is to provide an immunohistochemical kit for detecting the expression of CD26 protein in human tumor tissue, comprising an anti-human CD26 antibody, an isotype control negative antibody, an antibody diluent and a quality control chip for monitoring the staining quality, wherein the heavy chain variable region of the anti-human CD26 antibody comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO: 2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;

and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

Preferably, the amino acid sequence of the heavy chain variable region of the anti-human CD26 antibody is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

Preferably, the anti-human CD26 antibody is a chimeric antibody, and the amino acid sequence of the chimeric antibody is formed by fusing a heavy chain variable region shown in SEQ ID NO. 7 with a human monoclonal antibody IgG1 heavy chain constant region and fusing a light chain variable region shown in SEQ ID NO. 8 with a human monoclonal antibody IgG1 light chain constant region.

Preferably, the antibody dilution is preferably 0.05M TBS containing 1% BSA and 10% goat serum.

Preferably, the working concentration of the antibody is 2.5-10 ug/mL.

Preferably, the tissue fixative used with the kit is 4% paraformaldehyde or 10% neutral buffered formalin.

Preferably, the antigen retrieval solution used with the kit is a citrate buffer solution with pH 6.0.

When the kit is applied, the antibody and a target antigen (CD26) are combined and then present a target antigen site in a tissue slice through a color reaction, the distribution and the content of the target antigen can be determined through an optical microscope, and the result can provide clinical application guidance for screening clinically applicable population for CD26 target anti-cancer drugs so as to realize the effect of accurate treatment.

The antibody specificity and other related detection performances provided by the invention can meet the performance requirements of the immunohistochemical kit, and the recombinant expression antibody is convenient for mass production, reduces the human anti-mouse antibody reaction, reduces non-specific adsorption, and can meet the requirements of large-scale clinical application.

Drawings

FIG. 1 shows the electrophoresis of the variable region gene of heavy and light chains of mouse anti-human CD 26. Lane 1 is 200DNA Ladder, Lane 1 is heavy chain variable region PCR amplification gene, and Lane 3 is light chain variable region PCR amplification gene.

FIG. 2 is an agarose gel electrophoresis of PCR products of heavy and light chains of the chimeric antibody against human CD 26. Lane 1 is protein Marker, lane 2 is heavy chain gene PCR product; lane 3 is the light chain gene PCR product.

FIG. 3 shows SDS-PAGE electrophoresis of anti-human CD26 chimeric antibody by two-step chromatography. Wherein, Lane 1 is 10-100KD non-pre-staining protein Marker, Lane 2 is anti-human CD26 chimeric antibody.

FIG. 4 is a graph showing the effect of antibody-specific detection (Western Blot). Lane 1 is standard protein (Marker); lane 2 is recombinant CD26 protein

FIG. 5 shows the results of detecting cells with high expression and no (or low) expression of CD26 by the kit of the present invention (under microscope, 400X observation).

FIG. 5.1 is a staining pattern of detection antibody in CD 26-highly expressed cells; 5.2 staining of negative control antibody in CD 26-highly expressing cells; FIG. 5.3 staining pattern of detection antibody in CD26 non- (or low-) expressing cells; 5.4 staining of negative control antibody in CD26 non- (or low-) expressing cells.

FIG. 6 is a graph of the staining of antibodies in normal human tissue (200X under a microscope).

FIG. 6.1 is a graph of the staining of the test antibody in normal human brain tissue; 6.2 staining of negative control antibodies in normal human brain tissue; FIG. 6.3 detection of staining of antibodies in normal human thyroid tissue; FIG. 6.4 staining of negative control antibody in normal human thyroid tissue; FIG. 6.5 is a staining pattern of a detection antibody in normal human adrenal tissue; 6.6 staining of negative control antibodies in normal human adrenal tissue; FIG. 6.7 detection of antibody staining in normal human small intestine tissue; FIG. 6.8 staining of negative control antibodies in normal human small intestine tissue; FIG. 6.9 detection of antibody staining in normal human liver tissue;

FIG. 6.10 staining of negative control antibody in normal human liver tissue.

FIG. 7 shows the results of the detection of tissues of tumor patients by the kit of the present invention (under microscope 400X observation).

FIG. 7.1 staining pattern of detection antibody in human kidney cancer tissue; 7.2 staining of negative control antibody in human renal cancer tissue; FIG. 7.3 detection of antibody staining in human kidney cancer adjacent tissues; figure 7.4 staining of negative control antibody in human kidney cancer-adjacent tissues.

Detailed Description

Definition of

"antibody", also known as immunoglobulin, is a large Y-shaped protein secreted by B lymphocytes, and is an immunoglobulin molecule capable of specifically binding to a target antigen, such as a protein, a sugar, a polynucleotide, a lipid, a polypeptide, a small molecule compound, etc., through complementary sites (antigen-binding sites) at the two bifurcated tips of the Y.

Complementary-determining regions (CDRs), also called hypervariable regions. Patterned at the amino acid end of the antibody monomer is the most critical region for binding of the target antigen to the antibody, and in immune network theory, the complementarity determining regions of each antibody are also called idiotypes or genotypes.

A "chimeric antibody" is formed by fusing the variable region of a murine antibody with the constant region of a human antibody.

The present invention will be described in further detail below by way of examples, but the present invention is not limited to these examples.

Example 1 preparation of anti-human CD26 hybridoma cell line

1. Animal immunization

BALB/c female mice (purchased from Kyowa Kavens laboratory animals Co., Ltd.) were immunized with recombinant human CD26 protein (prepared by this company) following the general immunization protocol. For specific immunization, see "guidelines for antibody preparation and use". And tracking the serum titer of the immune mice by adopting an indirect ELISA method, selecting the immune mice with the highest serum titer, and performing fusion experiments on the spleen cells and myeloma cells of the mice.

2. Cell fusion

(1) Preparation of spleen cells immunized mice were harvested from the eyeball, sacrificed by cervical spondylolysis, soaked in 75% (v/v) alcohol for 10 minutes, their spleens were removed from the sterile console, placed in a cell screen, the cells were ground thoroughly, screened, washed several times by centrifugation with sterile 1640 medium (purchased from Gibco), resuspended to prepare single cell suspensions, and counted for use.

(2) Preparing feeder cells, namely taking one female BALB/c mouse with the age of 8-10 weeks, picking an eyeball to obtain negative serum, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies at the cervical vertebra; the abdominal skin was aseptically peeled, the peritoneum was exposed, and about 10mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of the mouse with a syringe, and the abdomen was gently massaged and air-blown several times. Sucking the culture medium containing the macrophages and injecting the culture medium into 20% 1640HAT culture medium for later use;

taking one female BALB/c mouse with the age of 2-3 weeks, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies after cervical vertebra breakage; aseptically placing thymus into a cell screen, grinding, sieving to obtain thymocytes, and placing the thymocytes into the 20% 1640HAT culture medium containing macrophages for later use.

(3) Cell fusion

Mouse myeloma cell line SP2/0 was selected at the logarithmic growth phase and collected and counted. Get about 10⁸The above spleen cells were combined with 2X 10⁷Each of the above SP2/0 cell lines was mixed in a fusion tube, centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded (discarded as clean as possible), and the fusion tube was gently rubbed back and forth on the palm of the hand to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added slowly and quickly over 60 seconds, 30mL of 1640HT medium was added and stopped, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the precipitate was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.

Mixing the HAT culture medium, subpackaging at 200 μ L/well into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in a cell culture box. After one week, 20% 1640HAT medium was replaced with 10% 1640HT medium, and after 3 days, the supernatant was examined.

3. Screening of anti-human CD26 protein specific hybridoma strain

(1) Preparation of the test plate: diluting the recombinant human CD26 protein (prepared by the company) to 1 mu g/mL by using CB coating solution, coating a 96-hole ELISA plate with 100 mu L/hole, coating overnight at 2-8 ℃, washing and patting dry once; PBST buffer containing 2% bovine serum albumin was blocked (200 ul/well) for 2 hours at 37 ℃; patting dry for later use.

(2) Screening of positive clones: adding 100 μ L/well of cell culture supernatant to be detected into the detection plate, performing action at 37 deg.C for 30 min, washing, drying, adding 100 μ L/well HRP-labeled goat anti-mouse IgG, performing action at 37 deg.C for 30 min, washing, drying, adding 100 μ L/well TMB color development solution, performing light-shielding development at 37 deg.C for 15min, adding 50 μ L of 2M H per well₂SO₄The reaction was stopped and the value read at OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1. Selecting positive clone strains to carry out cell cloning screening. After three to four rounds of cloning screening, the positive rate of the monoclonal cell strain is determined to be a stable cell strain with 100 percent of positive rate, and the cell strain is determined. Hybridoma cell line C29 has high titer, and can be used for subsequent treatmentAnd (3) carrying out antibody variable region sequence sequencing analysis on the hybridoma cell strain in one step.

Example 2 determination of variable region sequences of anti-human CD26 hybridoma cell line antibodies

1. Extraction of anti-human CD26 hybridoma cell total RNA

The anti-human CD26 hybridoma cell line was subcultured in a 75T flask until the cells were confluent to about 90%, digested, centrifuged to collect the cells, and subjected to Total RNA extraction using a High Pure RNA Isolation Kit (Roche) against a human CD26 monoclonal hybridoma cell line. Then using Total RNA of anti-human CD26 as a template, reverse transcribing and amplifying the First Strand of cDNA by using a RevertAID First Strand cDNA Synthesis Kit (Thermo), storing the reaction product at-20 ℃ for a long time, and storing the reaction product at-70 ℃.

PCR amplification of heavy and light chain variable region genes

Using the first strand cDNA of anti-human CD26 hybridoma as a template, 1. mu.L cDNA, 5. mu.L 10 XPCR buffer, 1. mu.L each of the upstream and downstream primers (25pmo1), dNTP 1. mu.L, 25mmol/L MgCl were added to a 50. mu.L reaction system₂ 1μL，H₂O39 mu L, pre-denaturation at 95 ℃ for 10min, adding 1 mu L of Taq enzyme, entering temperature cycle, and carrying out PCR amplification. The reaction conditions were 94 ℃. Denaturation for 1min, 58 ℃. Annealing for 1min, extending for 1.5min at 72 deg.C for 30 cycles, and keeping the temperature at 72 deg.C for 10 min. mu.L of the PCR product was subjected to 1.2% agarose gel electrophoresis, as shown in FIG. 1.

3. Cloning and sequencing of heavy and light chain variable region genes

According to the pGM-T Fast ligation kit (Beijing Tian and Biochemical technology Co., Ltd., VT207-02), the heavy and light chain variable region genes are respectively ligated with pGM-T vectors, transformed into escherichia coli Top10 competent cells, screened with blue and white spots, and cultured at 37 ℃ for 12-16 h.

The resulting white colonies were inoculated into 1-5mL LB medium containing ampicillin at a final concentration of 100. mu.L, shaken on a shaker at 37 ℃ for 3-4 hours, and then PCR-screened for clones with the correct sequence inserted. Meanwhile, the PCR screened positive clones are sent to Nanjing Kingsler Biotech Co., Ltd for sequencing, and the heavy chain variable region gene (SEQ ID No:7) and the light chain variable region gene (SEQ ID No:8) of the antibody are obtained through the comparative analysis of the computer network gene bank Kabat, Chothia and IMGT. The amino acid sequences of the complementarity determining regions of the heavy chain variable region are respectively: as shown in sequence SEQ ID NO:1, as shown in sequence SEQ ID NO: 2 and a CDR2 as set forth in sequence SEQ ID NO: 3, CDR 3; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 4, CDR1 as set forth in sequence SEQ ID NO: 5 and a CDR2 as set forth in sequence SEQ ID NO: 6, CDR3 shown.

Example 4 anti-human CD26 chimeric antibody expression

1. Molecular design and codon optimization of anti-human CD26 chimeric antibody

The anti-human CD26 hybridoma heavy chain variable region sequence in step 3 of example 3 was fused directly to the human monoclonal antibody IgG1 heavy chain constant region to obtain an anti-human CD26 chimeric antibody heavy chain amino acid sequence (SEQ ID No:9), the anti-human CD26 hybridoma light chain sequence was fused directly to the human monoclonal antibody IgG1 light chain constant region to obtain an anti-human CD26 chimeric antibody light chain amino acid sequence (SEQ ID No:11), and the anti-human CD26 hybridoma heavy and light chain genes were each fused separately with Optimum Gene^TMThe anti-human CD26 hybridoma heavy chain gene (SEQ ID No:10) and light chain gene (SEQ ID No:12) are obtained after codon optimization is carried out by codon optimization software.

An AvrII enzyme digestion site sequence is introduced into the 5 'end of the optimized antihuman CD26 hybridoma heavy chain gene, a BstZ7I enzyme digestion site sequence is introduced into the 3' end of the optimized antihuman CD26 hybridoma heavy chain gene, whole gene synthesis is carried out, and the synthesized gene fragment is constructed into a pUC57 plasmid (provided by Nanjing Kingsley science and technology Co., Ltd.) to obtain a long-term storage plasmid which is marked as pUC57-CD26-HC plasmid.

Introducing an EcoRV enzyme cutting site sequence into the 5 'end of the optimized antihuman CD26 hybridoma light chain gene, introducing a PacI enzyme cutting site sequence into the 3' end, carrying out whole-gene synthesis, and constructing the synthesized gene fragment into a pUC57 plasmid to obtain a long-term storage plasmid, which is marked as pUC57-CD26-LC plasmid.

2. Construction of CD26 chimeric antibody expression vector

PCR amplification was performed using pUC57-CD26-HC and pUC57-CD26-LC plasmids as templates, respectively, and the primer sequences used were as follows:

upstream primer M13F: CGC CAG GGT TTT CCC AGT CAC GAC

Downstream primer M13R: AGC GGA TAA CAA TTT CAC ACA GGA

The total reaction volume was 50. mu.L, wherein 2.5. mu.L of each primer was added at a concentration of 10. mu. mol/L, 1. mu.L of dNTP at a concentration of 10mmol/L, and Q5, 2U/. mu.L, was used as DNA polymerase, and 0.5. mu.L was added. The reaction conditions were 98 ℃ for 5 seconds, 55 ℃ for 45 seconds, and 72 ℃ for 30 seconds, and after 25 cycles, the product was analyzed by 1.0% agarose gel electrophoresis, and the results showed that the size of the product was consistent with the expected size (the results are shown in FIG. 2).

After double digestion with AvrII (# R0174S, from New England BioLabs) and BstZ17I (# R0594S, from New England BioLabs), 1% agarose was electrophoresed, and the resulting gene product was purified using a DNA gel recovery kit (DP214, from Beijing Tiangen Biotech Co., Ltd.). The cells were ligated to pCHO1.0 plasmid (available from Life) using T4 ligase (# M0202S, available from New England BioLabs), transformed into Top10 competent cells (CB104, available from Kyoto Biotech Co., Ltd.), and cultured overnight at 37 ℃ in LB solid medium containing kanamycin (0408, available from Amresco). And selecting positive clone bacteria for PCR identification on the next day, and sequencing and comparing positive results to obtain a heavy chain expression plasmid which is marked as pCHO1.0-HC and is completely consistent with an expected sequence.

After double cleavage with EcoRV (# R3195S, from New England BioLabs) and PacI (# R0547S, from New England BioLabs), the resulting gene products were subjected to electrophoresis in 1% agarose, and the resulting gene products were purified using a DNA gel recovery kit. The plasmid was ligated into pCHO1.0-HC using T4 ligase, transformed into Top10 competent cells, and cultured overnight at 37 ℃ in LB solid medium containing kanamycin. And selecting positive clone bacteria for PCR identification on the next day, and sequencing and comparing positive results to obtain an expression plasmid of the chimeric monoclonal antibody, wherein the expression plasmid is marked as pCHO1.0-D29 and is completely consistent with an expected sequence.

3. Screening of cell line stably expressing CD26 chimeric antibody

The correct pCHO1.0-D29 plasmid will be sequencedThe plasmid is linearized by NruI restriction endonuclease, and after electrotransfection into CHO-S host cell, certain concentration Puromycin and MTX are added into carbon dioxide incubator at 37 deg.C and 8% CO₂And (5) performing pressurized screening. Calculating cell viability after 10 days, transferring into shaking table at 37 deg.C and 8% CO when cell viability is more than 30%₂And suspension culture at 130rpm to obtain a cell line stably expressing the anti-human CD26 chimeric antibody.

4. Preparation of chimeric CD26 antibody

The cell line stably expressing the chimeric antibody against human CD26 was inoculated into Dynamis medium at 37 ℃ with 8% CO₂The cells were cultured on a shaker at 130 rpm. When the cell density reaches 5X 10⁶After cells/mL, glucose and feed medium were added to the shake flask. Samples were taken daily for cell density calculation and glucose and feed medium were added to the flask every other day until cell viability was less than 85% and supernatant culture was collected. Centrifuging the above supernatant culture solution at 12000rpm for 15min, collecting supernatant, filtering with 0.22 μm filter membrane to obtain supernatant of the treated culture solution, and purifying by chromatography.

The column used for affinity chromatography was HiTrapDiabselect Sure (11-0034-94, available from GE healthcare), all Fc domain-bearing antibodies were captured from the expression supernatant using a Mabselect Sure affinity column, and equilibration buffer (20mM NaH) was used₂PO₄150mM NaCl, pH7.2), and then passed through an affinity column and eluted with an elution buffer (100mM citric acid, pH 3.0). Purification and exchange of the target antibody was achieved by cation exchange chromatography using HiTrappCont S (17-5441-22, available from GE healthcare), using equilibration buffer A (50mM NaH)₂PO₄pH 5.0) after equilibration of the column, the sample was diluted to a conductivity between 3.0 and 3.5ms with double distilled water, bound to a HiTrappCatt S column, and eluted with elution buffer B (50mM NaH)₂PO₄1M NaCl, pH 6.0). The purified chimeric antibody was analyzed by SDS-PAGE gel electrophoresis and was more than 95% pure, as shown in FIG. 3.

Example 4 evaluation of antibody Performance

1. Western blot identification of antibodies

a. Polyacrylamide gel electrophoresis: preparing 12% separation gel and 5% concentrated gel, loading standard protein and human recombinant CD26 protein (prepared by the company), and performing electrophoresis at constant pressure for 1 hour;

b. film transfer: the membrane was rotated for 1 hour under constant current (35 mA/membrane) to transfer the proteins on the polyacrylamide gel to a nitrocellulose membrane. Staining SDS-PAGE gel subjected to membrane transfer by Coomassie brilliant blue G250, and observing the residual condition of protein;

c. and (3) sealing: TBST buffer containing 5% skimmed milk was blocked (blocking solution) overnight at 4 ℃; washing with a washing solution (TBST, for details, TBST buffer of TaKaRa) once for 10 minutes after blocking;

d. antigen-antibody reaction: diluting a blocking solution (according to a volume ratio of 1: 1000) with horseradish peroxidase labeled antibody (1mg/mL, labeled by a classical sodium periodate method in the company, and the same is carried out below), adding into the nitrocellulose membrane, and reacting for 1 hour at room temperature; TBST washes 5 times for 10 minutes each;

e. and (3) color development and photographing: sucking up residual liquid on the nitrocellulose membrane, adding a mixed solution (purchased from Thermo company) of 2mL of a stable peroxidase solution (1mL) and a luminol/enhancer solution (1mL) into the nitrocellulose membrane, uniformly wetting the surface of the nitrocellulose membrane, carrying out a reaction at room temperature in a dark place for one minute, and then photographing in a gel imaging system (purchased from GE company) (figure 4) to obtain a result.

As seen in the detection result, the antibody of the invention has better specificity and can specifically detect the human CD26 protein.

Example 5 preparation of immunohistochemical kit

The immunohistochemical detection kit comprises an HRP marked detection antibody (the CD26 antibody) specifically combined with target protein, an isotype control negative antibody (IgG 1 type other antibodies consistent with the subtype of the detection antibody are used as negative control antibodies), an antibody diluent and a negative and positive cytoplasm control sheet capable of monitoring the dyeing quality,

1. quality control tablet and preparation of tissue slice

The following examples are given by way of illustration and not by way of limitation to the methods by which negative and positive cytoplasmic control plates of the invention may be made using conventional methods, and those skilled in the art will recognize that other well known methods may be used.

(1) Fixing

Firstly, fixing cells in the quality control tablet

Collecting in vitro cultured cells (antigen high-expression cells or low-expression cells), centrifuging (250g for 5min), removing cell supernatant, adding a proper amount of 0.01M PBS to wash the cells, centrifuging (250g for 5min), removing washing liquid, washing for 2 times as described above, uniformly blowing cell masses at the bottom of a centrifuge tube by using a pipette, adding a fixing solution (4% paraformaldehyde or 10% neutral buffer formalin) with the volume about 25 times of the volume of the cell masses, and shaking at 100rpm to fix the cell masses for 24 hours; centrifuging (250g for 5min), removing the stationary liquid, adding purified water with the same volume as the stationary liquid, shaking and cleaning at 100rpm for 2h, centrifuging (250g for 5min), removing the cleaning liquid, blowing and beating the bottom cell mass uniformly by using a pipetting gun, sucking and placing in filter paper, wrapping, and placing in an embedding box for dewatering.

② fixation of tissue sample to be examined

The method comprises the following steps of (1) trimming a freshly collected tissue into small blocks with the thickness of 3-5 mm, placing the small blocks into a fixing solution with the volume 25 times that of the tissue, shaking at 100rpm for fixing for 24 hours, removing the tissue blocks after the fixing is finished, placing the tissue blocks into an embedding box, flushing for 2 hours by using running water, taking out, draining and dewatering.

(2) Dewatering

The samples to be dehydrated (cells or tissues in the embedding cassette) were dehydrated and wax-soaked together using a fully automatic dehydration apparatus according to the dehydration procedure of table 1.

TABLE 1

Container numbering	Content agent	Time of dehydration
			1	70% ethanol	45 to 80 minutes
2	80% ethanol	45 to 80 minutes
			3	95% ethanol	45 to 80 minutes
4	95% ethanol	45 to 80 minutes
			5	100% ethanol	45 to 80 minutes
6	100% ethanol	45 to 80 minutes
			7	Xylene	45 to 80 minutes
8	Xylene	45 to 80 minutes
			11	Paraffin (60 ℃ C.)	60 to 105 minutes
12	Paraffin (60 ℃ C.)	60 to 105 minutes

(3) Embedding

The dehydrated and cleared and waxed cells or tissues were subjected to paraffin block preparation on a paraffin embedding machine (Leica Histocore Arcadia embedding System).

(4) Slicing

Paraffin cell or tissue blocks were prepared as thin sections (3-5 microns) using a tissue microtome (Leica R2235 rotary microtome) for immunohistochemical staining.

2. Detection of

Immunohistochemical detection was carried out using the paraffin sections prepared above according to the following immunohistochemical staining procedure (again, the following examples are given only for illustration and not for limitation of the method of use and reagents used in the kit of the present invention, and those skilled in the art know that other reagents of the same type are available).

Baking wax: slicing and baking wax at 60 ℃;

dewaxing: the slices are dewaxed and hydrated in a series of dewaxing (e.g., xylene, gradient alcohol and purified water) reagents;

antigen retrieval: heating the antigen repairing liquid to the boiling point by using a water bath kettle, putting the slices into the water bath kettle, heating for 15min, taking out the slices, and naturally cooling. The inventor investigates the influence of different antigen repairing solutions on the detection result, and the influence is shown in a table 3;

endogenous peroxidase removal: endogenous peroxidase remover (e.g., 3% H) is used₂O₂0.05M TBS) to remove endogenous peroxidase from the tissue sections, washing solutions (e.g.: 0.05M TBS containing 0.025% Triton X-100);

and (3) sealing: tissue sections are blocked using blocking solution (e.g., 0.05M TBS containing 1% BSA and 10% goat serum);

detecting an antibody: the blocking solution was discarded, the CD26 detection antibody and the negative control antibody were diluted with 0.05M TBS containing 1% BSA and 10% goat serum, and the mixture was overlaid on the tissue sections dropwise and incubated at 2-8 ℃ for 16h-18h, and then washed with a washing solution (e.g., 0.05M TBS containing 0.025% Triton X-100), and the inventors examined the effect of different antibody concentrations on the detection results, as shown in Table 3 below;

color development: DAB color reagent (DAB kit, New Biotechnology Inc. of Fuzhou, for example) is dripped on the tissue section for color development, and the tissue section is observed by naked eyes, and the tissue section is washed by washing liquid (0.05M TBS containing 0.025% Triton X-100) after the color development is generally carried out for 3 to 10 minutes;

counterdyeing: washing the slices with running water, placing in hematoxylin staining solution (such as Mayer's hematoxylin, Kyoto Biotech Co., Ltd.) for 3-5 min, taking out, washing under running water to turn blue, and air drying;

sealing: placing the slices in xylene, and sealing with sealing glue (such as national medicine group, neutral gum);

reading the film: the positive staining is evaluated by three parts of staining intensity, staining density and staining pattern (part). The staining pattern to be tested should be either cytoplasmic or cytoplasmic staining, with the scoring criteria shown in Table 2.

TABLE 2

3. The specificity and sensitivity of the assay under different conditions were examined and the detailed evaluation was found in example 6, with the results shown in Table 3 below.

TABLE 3 comparison results of kits prepared under different conditions

From the comparison results, it can be seen that: in the selection of the fixing solution, 4% of paraformaldehyde and 10% of neutral buffered formalin can be used as sample fixing solution; in the selection of the antigen retrieval liquid, the citrate buffer solution with the pH value of 6.0 is used as the antigen retrieval liquid, the positive signal is stronger, and the negative signal is weakest; from the antibody concentration, it can be seen that at the working concentration of 10ug/mL, the positive control signal is strongest and the negative control does not produce any signal.

Example 6 immunohistochemical assay kit Performance test

1. Specificity of

Staining was performed in negative and positive quality control cells using IgG1 chimeric antibody as a negative control antibody, which is identical to the detection antibody subtype (see fig. 5); as can be seen from the pictures, the detection antibody in the kit of the present invention has strong positive coloration (composite score + +) in CD26 high expression cells (786-0), and does not have any brown-yellow coloration (composite score-) in CD26 non- (or low) expression cells (A375); the negative control antibody in the kit does not have any brown-yellow coloration (comprehensive score-) in CD26 high-expression cells or non- (or low) -expression cells, and the result shows that the kit has good specificity.

2. Immunogenicity

Staining was performed using a primary antibody in normal human tissue (3 per tissue, purchased from sienna biotechnology limited) to confirm cross-reactivity of the primary antibody in normal human tissue. A total of 19 normal tissues were stained for primary antibody (see fig. 6 and table 4):

TABLE 4 detection of antibody Cross-reactivity in Normal tissues

The staining results, which show cross-reactivity of the primary antibody in adrenal gland, ovary, pituitary, tonsil, heart, stomach, small intestine, liver, tongue, prostate, uterus, striated muscle and eye tissue, provide a reference for the cross-reactivity that would be present in normal tissue by the kit user using a tumor specimen with normal tissue under staining with a detection antibody.

3. Within-batch to batch repeatability

3 sections from 5 human tumor tissue samples and negative and positive quality control samples were used for staining with CD26 antibody and negative control antibody (see fig. 7 and table 5), and 3 consecutive batches of assays were performed; the results show that 3 sections of each sample in one batch have no significant difference in staining depth, staining density and staining pattern under the staining of the detection antibody and the negative antibody, and no difference exists between batches, and the results show that the kit has good repeatability.

TABLE 5 Intra-batch repeatability

The results show that the detection kit has good detection performance and can be used for detecting the CD26 antigen in tissues or cells.

Sequence listing

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Gly Tyr Ile Tyr Pro Gly Asn Gly Tyr Thr Val Tyr Asn Glu Lys Phe

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Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

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Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

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Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

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Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

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Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

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Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

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Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

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Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

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Claims (8)

1. An immunohistochemical kit for detecting CD26 protein expression in human tumor tissues, comprising an anti-human CD26 antibody, an isotype control negative antibody, an antibody diluent and a quality control chip for monitoring the staining quality, wherein the heavy chain variable region of the anti-human CD26 antibody comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO: 2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;

and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

2. The immunohistochemistry kit of claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the anti-human CD26 antibody is represented by SEQ ID NO. 7, and the amino acid sequence of the variable region of the light chain is represented by SEQ ID NO. 8.

3. The immunohistochemistry kit of claim 2, wherein the anti-human CD26 antibody is a chimeric antibody, and the amino acid sequence thereof is composed of a heavy chain variable region shown by SEQ ID NO. 7 fused with a human monoclonal antibody IgG1 heavy chain constant region, and a light chain variable region shown by SEQ ID NO. 8 fused with a human monoclonal antibody IgG1 light chain constant region.

4. The immunohistochemistry kit of claim 3, wherein said anti-human CD26 antibody is recombinantly expressed from a mammalian host cell.

5. The immunohistochemistry kit of any one of claims 1 to 4, wherein the antibody diluent in said kit comprises 0.05M TBS with 1% BSA and 10% goat serum.

6. The immunohistochemistry kit of any one of claims 1 to 4, wherein the working concentration of antibody in the kit is 2.5-10 ug/mL.

7. The immunohistochemistry kit of any one of claims 1 to 4, wherein the tissue fixative used with the kit is 4% paraformaldehyde or 10% neutral buffered formalin.

8. The immunohistochemistry kit of any one of claims 1 to 4, wherein the antigen retrieval solution used with the kit is citrate buffer solution at pH 6.0.

Images