CN109709337A

CN109709337A - The immunologic combined detection reagent kit of people CD26 and its clinical application

Info

Publication number: CN109709337A
Application number: CN201811623085.3A
Authority: CN
Inventors: 马永; 徐银妹; 赵利利
Original assignee: ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: ZONHON BIOPHARMA INSTITUTE Inc
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2019-05-03
Anticipated expiration: 2038-12-28
Also published as: CN109709337B

Abstract

The present invention relates to the immunologic combined detection reagent kit of people CD26 a kind of and its clinical applications, belong to in-vitro diagnosis field.For immunologic combined detection reagent kit disclosed by the invention using a kind of completely new anti-human CD26 antibody, heavy chain variable region contains complementary determining region below: amino acid sequence HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO:1,2,3 respectively；And its light-chain variable sequence contains complementary determining region below: LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO:4,5,6 respectively.The coherent detections performances such as the anti-human CD26 antibody specificity that the present invention uses can meet the performance requirement of immunohistochemical kit, it can be source of mouse antibody and be also possible to the chimeric human antibody of recombinant expression convenient for mass production, the chimeric human antibody wherein recombinantly expressed reduces human anti-mouse antibody reaction, non-specific adsorption is reduced, can also meet the needs of larger scale clinical application.In application CD26 target drug, with immunohistochemical kit of the invention is used, the effect precisely treated can reach.

Description

The immunologic combined detection reagent kit of people CD26 and its clinical application

Technical field

The invention belongs to in-vitro diagnosis field, especially a kind of anti-human CD26 antibody and its Immunohistochemical detection try Application in agent box.

Background technique

CD26 is a kind of generally existing Multifunctional type II transmembrane protein, and there are many biological functions, can also be with dissolution Form is present in blood plasma.CD26 often exists in the form of homodimer, and monomer contains 766 amino acid, relative molecular mass About 110kDa.CD26 amino acid residue is divided into 5 parts: intracellular region (aa1~6), transmembrane region (aa7~28), height from inside to outside Degree glycosylation area (aa29-323) is rich in cysteine area (aa324~551) and C-terminal catalyst structure domain (552~766).CD26 C-terminal catalyst structure domain play DPP IV (Dipeptidylpeptidase4, DPPIV) activity, can hydrolyze in vivo A variety of substrates play biological action, can interact with internal different kinds of molecules rich in cysteine area, to participate in internal Immune function.Effect of the CD26 in immunological regulation has been widely studied, and CD26 is the molecular marker of T cell activation, also thin in T Costimulatory molecules are used as during born of the same parents' signal transduction, further relate to a variety of T cell functions, including mature and migration occurs for T cell, carefully Intracellular cytokine secretion, the antibody that T cell relies on generate, b cell immunoglobulin transition etc..Hair of the CD26 in autoimmune disease It plays a significant role during hair tonic exhibition, it has also become the molecular marker of clinical disease, and be considered as certain immunity diseases Treatment or diagnosis target spot.

Studies have shown that part cancer patient's tumor tissues are expressed without CD26, such patient is being used using CD26 as target spot Anticancer drug inefficacy.Therefore, before using CD26 target drug, CD26 protein expression feelings are carried out to cancer patient tumor tissues Condition detection, can reach the effect precisely treated.CD26 antigen Immunohistochemical detection kit according to the present invention then may be used Reach said effect.

Summary of the invention

The technical problem to be solved in the present invention is to provide an energy effectively, the antibody of specific binding people CD26, and will It is applied in Immunohistochemical detection kit.More specifically:

The first object of the present invention is to provide a kind of anti-human CD26 antibody,

Its heavy chain variable region contains complementary determining region below: amino acid sequence is as shown in sequence SEQ ID NO:1 HCDR1, the HCDR2 as shown in sequence SEQ ID NO:2 and the HCDR3 as shown in sequence SEQ ID NO:3；

And its light-chain variable sequence contains complementary determining region below: amino acid sequence such as sequence SEQ ID NO:4 Shown in LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and the LCDR3 as shown in sequence SEQ ID NO:6.

The amino acid sequence of preferably above-mentioned anti-human CD26 antibody heavy chain variable region is as shown in SEQ ID NO:7, light chain The amino acid sequence of variable region is as shown in SEQ ID NO:8.

Second purpose of the invention is to provide a kind of chimeric antibody, the heavy chain of antibody amino acid sequence such as SEQ ID NO: Shown in 9, the nucleotide sequence of encoding antibody heavy is as shown in SEQ ID NO:10, the antibody light chain amino acid sequence such as SEQ Shown in ID NO:11, the nucleotide sequence of encoding antibody light is as shown in SEQ ID NO:12.

Third purpose of the present invention is to provide a kind of expression vector containing above-mentioned chimeric antibody nucleotide sequence.

4th purpose of the invention is to provide a kind of recombinant cell strain containing above-mentioned chimeric antibody expression vector.

5th purpose of the invention is to provide a kind of method for producing above-mentioned chimeric antibody, comprising:

(1) the mammalian host cell strain of anti-human CD26 antibody is expressed in building as described above；

(2) above-mentioned cell strain is inoculated in suspend in culture medium and is cultivated；

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

Carrier described above is preferably pCHO1.0.Mammalian cell CHO described above is preferably CHO-S cell.

Of the invention the 6th is designed to provide above-mentioned anti-human CD26 antibody CD26 albumen in detection tissue Using.

7th purpose of the invention is to provide a kind of for detecting the immune group of the CD26 protein expression in human tumour tissue Change kit, including anti-human CD26 antibody, Isotype control feminine gender antibody, antibody diluent and monitor dyeing quality Quality Control piece, The anti-human CD26 antibody, heavy chain variable region contain complementary determining region below: amino acid sequence such as sequence SEQ ID NO:1 Shown in HCDR1, the HCDR2 as shown in sequence SEQ ID NO:2 and the HCDR3 as shown in sequence SEQ ID NO:3；

Preferably, for the amino acid sequence of above-mentioned anti-human CD26 antibody heavy chain variable region as shown in SEQ ID NO:7, light chain can Become the amino acid sequence in area as shown in SEQ ID NO:8.

Preferably, the anti-human CD26 antibody is chimeric antibody, amino acid sequence is as shown in SEQ ID NO:7 Heavy chain variable region and 1 heavy chain constant domain of people's monoclonal antibody IgG, the light chain variable region and people as described in shown in SEQ ID NO:8 are single The fusion of 1 constant region of light chain of anti-igg is constituted.

Preferably, the antibody diluent preferably comprises the 0.05M TBS of 1%BSA and 10% lowlenthal serum.

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

Preferably, being 4% paraformaldehyde or 10% neutral buffered Fu Er with the matching used tissue fixative solution of kit Malin.

Preferably, the citrate buffer for being pH6.0 with the matching used antigen retrieval buffers of kit.

In application kit of the invention, tissue is presented by chromogenic reaction after antibody and target antigen (CD26) combination Target antigen site in slice, can determine the distribution and content of target antigen by optical microscopy, and result can be CD26 target-point anti-cancer drug screening clinic target user simultaneously provides clinical application guidance, to realize the effect precisely treated.

The coherent detections such as antibody specificity provided by the invention performance can meet the performance requirement of immunohistochemical kit, and And recombinant expression antibody is convenient for mass production, reduces human anti-mouse antibody reaction, reduces non-specific adsorption, can meet and face on a large scale The demand of bed application.

Detailed description of the invention

The anti-heavy chain of the anti-human CD26 mouse of Fig. 1 and light-chain variable region gene electrophoretogram.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.

The agarose gel electrophoresis figure of Fig. 2 anti-human CD26 chimeric antibody heavy and light chain PCR product.Swimming lane 1 is albumen Marker, swimming lane 2 are heavy chain gene PCR product；Swimming lane 3 is light chain gene PCR product.

The anti-human CD26 chimeric antibody of Fig. 3 chromatographs SDS-PAGE electrophoresis by two steps.Wherein, swimming lane 1 is that 10-100KD is non- Pre-dyed albumen Marker, swimming lane 2 are anti-human CD26 chimeric antibody.

Fig. 4 antibody specificity detection effect figure (Western Blot).Swimming lane 1 is standard protein (Marker)；Swimming lane 2 To recombinate CD26 albumen

Fig. 5 kit detection CD26 high expression of the present invention and not (or low) expression cell result (400X sight under microscope It examines).

Fig. 5 .1 detects colored graph of the antibody in CD26 high expressing cell；5.2 negative control antibodies are thin in CD26 high expression Dyeing in born of the same parents；Fig. 5 .3 detects colored graph of the antibody in CD26 not (or low) expression cell；5.4 negative control antibodies exist The CD26 not dyeing in (or low) expression cell.

Fig. 6 detects colored graph of the antibody in normal human tissue (200X is observed under microscope).

Fig. 6 .1 detects colored graph of the antibody in normal human's cerebral tissue；6.2 negative control antibodies are big in normal human Dyeing in brain tissue；Fig. 6 .3 detects dyeing of the antibody in normal human's parathyroid tissue；Fig. 6 .4 negative control antibody exists Dyeing in normal human's parathyroid tissue；Fig. 6 .5 detects colored graph of the antibody in normal human adrenal tissue；6.6 negative Dyeing of the property control antibodies in normal human adrenal tissue；Fig. 6 .7 detects dye of the antibody in normal human small intestine Color；Dyeing of Fig. 6 .8 negative control antibody in normal human small intestine；Fig. 6 .9 detects antibody in normal human's liver group Dyeing in knitting；

Dyeing of Fig. 6 .10 negative control antibody in normal human's liver organization.

Fig. 7 kit detection tumour patient tissue result of the present invention (400X is observed under microscope).

Fig. 7 .1 detects colored graph of the antibody in people renal carcinoma tissue；Dye of 7.2 negative control antibodies in people renal carcinoma tissue Color；Fig. 7 .3 detects dyeing of the antibody in Ren Shen cancer beside organism；Dye of Fig. 7 .4 negative control antibody in Ren Shen cancer beside organism Color.

Specific embodiment

Definition

" antibody " is also known as immunoglobulin, is a kind of large-scale Y shape protein secreted by bone-marrow-derived lymphocyte, can pass through Y shape Two of them bifurcated top complementary site (antigen junction coincidence) specific binding target antigen immunoglobulin molecules, institute State target antigen such as protein, sugar, polynucleotides, rouge, polypeptide, small molecule compound etc..

Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Molding It is most critical zone of the target antigen in conjunction with antibody in the end of antibody monomer amino acid, in Artificial Immune Network Theory, Mei Gekang The complementary determining region of body is otherwise known as idiotype or genotype.

" chimeric antibody " is formed by the variable region of source of mouse antibody and the constant domain of human antibody.

The present invention is described in further detail below by embodiment, but the present invention is not limited to these embodiments.

The preparation of the anti-human CD26 hybridoma cell strain of embodiment 1.

1. animal immune

BALB/c female mice is immunized (purchased from normal according to general immune programme with recombined human CD26 albumen (our company's preparation) This experimental animal Co., Ltd of state Cavan).Specific Immunity is referring to " Antibody preparation with use experiment guide ".Using indirect ELISA method tracks immune serum titre, chooses the highest immune mouse of serum titer, carries out mouse boosting cell and Mouse Bone Myeloma cells carry out fusion experiment.

2. cell fusion

(1) immune mouse is plucked eyeball and takes blood by the preparation of spleen cell, is placed on 75% (v/v's) through disconnected cervical vertebra execution It is impregnated 10 minutes in alcohol, its spleen is taken out in aseptic operating platform, is placed in cell screen clothes, be fully ground cell, cross sieve, With sterile 1640 culture medium (be purchased from Gibco company) centrifuge washing for several times after, cell is resuspended so that single cell suspension is made, and count Number, it is spare.

(2) preparation of feeder cells takes female BAl BIc/c mouse one of 8~10 week old, plucks eyeball and obtains negative serum, It puts to death in postposition 75% (v/v) alcohol and impregnates 10 minutes through disconnected cervical vertebra；Sterile to open skin of abdomen, exposure peritonaeum uses syringe By about 10mL 1640HT culture medium (be purchased from SIGMA company) injection mouse peritoneal, gently abdomen massage and blow and beat for several times.It draws It is spare in culture medium injection 20%1640HAT culture medium containing macrophage；

Female BAl BIc/c the mouse one for taking 2~3 week old is placed in 75% (v/v) alcohol through disconnected cervical vertebra execution and impregnates 10 minutes；Sterile to take thymus gland in cell screen clothes, sieve is crossed in grinding, obtain thymocyte be placed in it is above-mentioned containing macrophage It is spare in 20%1640HAT culture medium.

(3) cell fusion

Selection is in the murine myeloma cell strain SP2/0 of logarithmic growth phase, collects and counts.Take about 10⁸A above-mentioned spleen Cell and 2 × 10⁷A above-mentioned SP2/0 cell strain, which is added in fusion pipe, to be mixed, and 1000rpm centrifugation abandons supernatant (as far as possible after ten minutes Abandon net), fusion pipe is set and is gently rubbed back and forth on palm so that precipitating is loose.1mL preheating is added after elder generation is slow in 60 seconds fastly PEG1450 (polyethylene glycol 1450 is purchased from SIGMA company), is added 1640HT culture medium 30mL and terminates, 1000rpm is centrifuged 10 points Clock removes supernatant, and gently friction keeps precipitating loose, is added in the 1640HAT culture medium of step 2 obtained 20%.

It after above-mentioned HAT culture medium is mixed well, is dispensed with 200 holes μ L/ into 96 porocyte culture plates, sets 37 DEG C, 5% CO₂Cell incubator in cultivate.20%1640HAT culture medium is replaced with 10%1640HT culture medium after a week, is taken after 3 days It is detected clearly.

3. anti-human CD26 protein-specific hybridoma cell strains screening

(1) preparation of detection plate: with CB coating buffer dilution recombined human CD26 albumen (our company's preparation) to 1 μ g/mL, packet By 96 hole ELISA ELISA Plates, 100 holes μ L/, 2~8 DEG C of coatings overnight, washed once and pat dry；Containing 2% bovine serum albumin(BSA) PBST buffer blind (hole 200ul/), 37 DEG C are closed 2 hours；It pats dry, it is spare.

(2) screening of positive colony: 100 hole μ L/ of cells and supernatant to be checked is added in above-mentioned detection plate, in 37 DEG C Effect was washed and is patted dry after 30 minutes, and the sheep anti-mouse igg of the HRP label in 100 holes μ L/ is added, washes after acting on 30 minutes in 37 DEG C It washs and pats dry, the TMB developing solution in 100 holes μ L/ is added, colour developing 15 minutes is protected from light in 37 DEG C, the 2M H of 50 μ L is added in every hole₂SO₄Eventually It only reacts, and the reading numerical values at OD450.Positive hole determines principle: OD450 value/negative control value >=2.1.Choose positive gram Grand strain carries out Cell-cloned screening.After three to four-wheel cloning screening, monoclonal cell strain positive rate 100% is i.e. true It is set to stable cell line, singling is carried out to cell strain.Hybridoma cell strain C29 all has higher potency, then subsequent further Antibody variable sequences sequencing analysis is carried out to above-mentioned hybridoma cell strain.

The measurement of 2. anti-human CD26 hybridoma cell strain antibody variable sequences of embodiment

1. the extraction of anti-human CD26 hybridoma total serum IgE

Anti-human CD26 hybridoma cell strain is passaged in 75T culture bottle and is cultivated, until cell confluency to 90% or so digestion, Cell is collected by centrifugation, using High Pure RNA Isolation Kit (Roche) to anti-human CD26 monoclonal hybridoma Strain carries out Total RNA extraction.Then using the Total RNA of anti-human CD26 as template, RevertAid First is used Its first chain of cDNA of Strand cDNA Synthesis Kit (Thermo) reverse transcription amplification, reaction product deposit in -20 DEG C, Long term storage needs to be stored in -70 DEG C.

2.PCR amplification weight, light-chain variable region gene

Using the first chain of anti-human CD26 hybridoma cDNA as template, in 50 μ L reaction systems, it is separately added into 1 μ of cDNA 5 μ L of L, 10 × PCR buffer, upstream and each 1 μ L (25pmo1) of downstream primer, dNTP1 μ L, 25mmol/L MgCl₂1 μ L, H₂O 39 μ L, 95 DEG C of initial denaturation 10min, add 1 μ L of Taq enzyme, into temperature cycles, carry out PCR amplification.Reaction condition is 94 DEG C.Denaturation 1min, 58 DEG C.Anneal 1min, 72 DEG C of extension 1.5min, totally 30 circulations, then 72 DEG C of heat preservation 10min.Take 5 μ L PCR products 1.2% agarose gel electrophoresis is carried out, sees Fig. 1.

3. the clone and sequencing of heavy chain and light-chain variable region gene

It, will according to pGM-T Fast connection kit specification (Beijing day is with biochemical technology Co., Ltd, VT207-02) Weight, light-chain variable region gene are connect with pGM-T carrier respectively, are converted in Escherichia coli Top10 competent cell, blue hickie sieve Choosing, 37 DEG C of culture 12-16h.

The inoculation of obtained white colony is contained in the ampicillin 1-5mL LB culture medium of final concentration of 100 μ L, 37 After DEG C shaking table oscillation 3-4h, correct sequence clone is inserted into PCR screening.PCR screening positive clone is sent to Nanjing Jin Sirui simultaneously Biotechnology Co., Ltd is sequenced, and measurement result is compared by computer network gene pool Kabat, Chothia and IMGT Analysis obtains the heavy chain variable region gene (SEQ ID No:7) and light-chain variable region gene (SEQ ID No:8) of antibody.Through data Above-mentioned sequence is analyzed in library, and the amino acid sequence of each complementary determining region of heavy chain variable region is respectively: such as sequence SEQ ID NO:1 Shown in CDR1, the CDR2 as shown in sequence SEQ ID NO:2 and the CDR3 as shown in sequence SEQ ID NO:3；Its light chain can The amino acid sequence for each complementary determining region for becoming area is: CDR1, such as sequence SEQ ID NO as shown in sequence SEQ ID NO:4: CDR2 shown in the 5 and CDR3 as shown in sequence SEQ ID NO:6.

The expression of the anti-human CD26 chimeric antibody of embodiment 4.

1, anti-human CD26 chimeric antibody molecules design and codon optimization

By CD26 hybridoma weight chain variabl area sequence anti-human in 3 step 3 of embodiment directly with 1 heavy chain of people's monoclonal antibody IgG Constant domain obtains anti-human CD26 chimeric antibody heavy amino acid sequence (SEQ ID No:9), and anti-human CD26 hybridoma is thin Born of the same parents' sequence of light chain is directly merged with 1 constant region of light chain of people's monoclonal antibody IgG, obtains anti-human CD26 chimeric antibody light amino acid sequence (SEQ ID No:11), and anti-human CD26 hybridoma heavy chain and light chain gene are used into Optimum Gene respectively respectively^TMIt is close Numeral optimization software carry out codon optimization after obtain anti-human CD26 hybridoma heavy chain gene of the invention (SEQ ID No: And light chain gene (SEQ ID No:12) 10).

Anti-human CD26 hybridoma heavy chain gene after optimization is introduced into AvrII restriction enzyme site sequence at 5 ' ends, 3 ' End introduces BstZ7I restriction enzyme site sequence, and carries out full genome synthesis, by the genetic fragment of synthesis, be building up to pUC57 plasmid (by Nanjing Jin Sirui Science and Technology Ltd. provides) in, a kind of long-term preservation plasmid is obtained, pUC57-CD26-HC plasmid is denoted as.

Anti-human CD26 hybridoma light chain gene after optimization is introduced into EcoRV restriction enzyme site sequence at 5 ' ends, 3 ' End introduces PacI restriction enzyme site sequence, and carries out full genome synthesis, by the genetic fragment of synthesis, is building up in pUC57 plasmid, obtains To a kind of long-term preservation plasmid, it is denoted as pUC57-CD26-LC plasmid.

2, CD26 chimeric antibody expression vector constructs

Respectively using pUC57-CD26-HC and pUC57-CD26-LC plasmid as template, PCR amplification, the primer sequence are carried out It is as follows:

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

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

50 μ L of total volume is reacted, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, archaeal dna polymerase used are Q5, and 2U/ μ L adds 0.5 μ L.Reaction condition be 98 DEG C 5 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 25 After circulation, product is analyzed through 1.0% agarose gel electrophoresis, as the result is shown primer size and expected (result such as Fig. 2 in the same size It is shown).

With AvrII (#R0174S is purchased from New England BioLabs company) and BstZ17I, (#R0594S is purchased from New England BioLabs company) after double digestion, 1% agarose electrophoresis, obtained gene product DNA gel QIAquick Gel Extraction Kit (DP214 is purchased from Beijing Tiangeng biochemical technology Co., Ltd) purifying.With T4 ligase, (#M0202S is purchased from New England BioLabs it) is connected in pCHO1.0 plasmid (purchased from Life company), is transformed into Top10 competent cell (CB104, purchased from north Capital Tiangeng biochemical technology Co., Ltd) in, the LB solid medium in (0408, be purchased from Amresco company) containing kanamycin In 37 DEG C of overnight incubations.Second day picking positive colony bacterium carries out PCR identification, and positive findings are carried out sequencing comparison, and pre- Phase sequence is completely the same to get heavy chain expression plasmid is arrived, and is denoted as pCHO1.0-HC.

With EcoRV (#R3195S is purchased from New England BioLabs company) and PacI, (#R0547S is purchased from New England BioLabs company) after double digestion, 1% agarose electrophoresis, obtained gene product DNA gel QIAquick Gel Extraction Kit Purifying.It is connected in pCHO1.0-HC plasmid, is transformed into Top10 competent cell, containing kanamycin with T4 ligase LB solid medium in 37 DEG C of overnight incubations.Picking positive colony bacterium carries out PCR identification within second day, and positive findings are carried out Sequencing compares, completely the same to get the expression plasmid for arriving chimeric monoclonal antibody with expected sequence, is denoted as pCHO1.0-D29.

3, stablize expression CD26 chimeric antibody cell strain screening

Correct pCHO1.0-D29 plasmid NruI limitation restriction endonuclease linearisation will be sequenced, electrotransfection is thin to CHO-S host After in born of the same parents, it is separately added into certain density Puromycin and MTX and is placed in carbon dioxide incubator 37 DEG C, 8%CO₂Added Pressure screening.Cell viability is calculated after 10 days, is transferred in shaking table when Cell viability is greater than 30% or more, 37 DEG C, 8%CO₂, 130rpm, which suspends, to be cultivated, and is obtained to stablize and is expressed anti-human CD26 chimeric antibody cell strain.

4, prepared by CD26 chimeric antibody

The anti-human CD26 chimeric antibody cell strain of expression will be stablized to be inoculated in Dynamis culture medium, 37 DEG C, 8%CO₂, It is cultivated in 130rpm shaking table.When cell density reaches 5 × 10⁶After cells/mL, glucose and supplemented medium is added to shaking flask In.Sampling calculates cell density daily, and glucose and supplemented medium is every other day added into shaking flask, until cell viability Supernatant culture solution is collected lower than 85%.By above-mentioned supernatant culture solution, 12000rpm, 15min low-temperature centrifugation collects supernatant, and 0.22 μm Membrane filtration can carry out chromatographic purifying up to culture solution supernatant after processing.

The chromatographic column that affinity chromatography is selected is that (11-0034-94 is purchased from GE to HiTrapMabSelectSuRe Healthcare), the antibody of all band Fc structural domains is captured from expression supernatant using MabselectSuRe affinity column, With equilibration buffer (20mM NaH₂PO₄, 150mM NaCl, pH7.2) and after balance chromatographic column, affinity column is crossed, it is slow with elution Fliud flushing (100mM citric acid, pH3.0) elution.Pass through HiTrapCapto S (17-5441-22 is purchased from GE healthcare) sun Ion-exchange chromatography realizes that target antibody is consummate and changes liquid, with equilibration buffer A (50mM NaH₂PO₄, pH 5.0) and balance chromatography After column, sample distilled water dilutes conductance to after between 3.0-3.5ms, crossing the combination of HiTrapCapto S pillar, uses elution buffer Liquid B (50mMNaH₂PO₄, 1M NaCl, pH 6.0) and linear elution.Chimeric antibody after purification carries out PAGE gel electrophoresis point Analysis, purity are shown in Fig. 3 95% or more.

The performance evaluation of 4. antibody of embodiment

1. the Western blot of antibody is identified

A. polyacrylamide gel electrophoresis: 12% separation gel of configuration, 5% concentration glue, respectively loading standard protein and people Recombinant C D26 albumen (our company's preparation), electrophoresis 1 hour under constant pressure；

B. transferring film: transferring film 1 hour under the conditions of constant current (35mA/ film), extremely by the Protein transfer on polyacrylamide gel On nitrocellulose filter.Coomassie brilliant blue G250 dyes the SDS-PAGE glue for completing transferring film, observes the residual feelings of albumen Condition；

C. close: the TBST buffer blind (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning solution after closing (TBST, It is detailed in TaKaRa company's T BST buffer) it washed once, 10 minutes；

D. antigen-antibody reaction: confining liquid dilution (pressing 1:1000 volume ratio) Horseradish Peroxidase Conjugates (1mg/ ML, our company is using classical Over-voltage protection label, similarly hereinafter), it is added in above-mentioned nitrocellulose filter, reacts at room temperature 1 hour； TBST is washed 5 times, every time 10 minutes；

E. it develops the color and takes pictures: blotting residual liquid on nitrocellulose filter, 2mL stable type peroxide is added in nitrocellulose filter Compound enzyme solutions (1mL) and luminol/enhancing agent solution (1mL) mixed liquor (purchase is in Thermo company), uniform wet nitre The surface of acid cellulose film, room temperature be protected from light one minute after in gel imaging system (purchase in GE company) take pictures (Fig. 4), stay Take result.

Testing result, can specific detection people's CD26 albumen as it can be seen that antibody of the invention has preferable specificity.

The preparation of 5 immunohistochemical kit of embodiment

Immunologic combined detection reagent kit of the invention includes the detection antibody for specifically binding the HRP label of destination protein (above-mentioned CD26 antibody), Isotype control feminine gender antibody are (with consistent other antibody of IgG1 type of detection antibody subtype as negative right According to antibody), antibody diluent and the yin and yang attribute cytoplasm control wafer that dyeing quality can be monitored,

1, the preparation of Quality Control piece and histotomy

It is prepared using conventional method, citing, which is merely to illustrate the yin and yang attribute cytoplasm control wafer that is not intended to limit the present invention, below to adopt Method, other known methods are available as known to those skilled in the art.

(1) fixed

1. the fixation of cell in Quality Control piece

It collects cultured cell in vitro (antigen high expressing cell or low expression cell), is discarded after centrifugation (250g is centrifuged 5min) Cell supernatant is added suitable 0.01M PBS washing cell, discards cleaning solution, institute as above after centrifugation (250g is centrifuged 5min) Washing 2 times is stated, about 25 times of bodies of cell mass volume are added after blowing and beating the cell mass for being centrifuged bottom of the tube uniformly using liquid-transfering gun Long-pending fixer (4% paraformaldehyde or 10% neutral buffered formalin), 100rpm, which rocks, to be fixed for 24 hours；It is centrifuged (250g Centrifugation 5min) after discard fixer, be added and the purified water of fixer equal volume, 100rpm rock cleaning 2h, be centrifuged (250g Centrifugation 5min) after discard cleaning solution, using liquid-transfering gun by bottom cell mass blow and beat uniformly after, absorption be placed in filter paper, wrap Wrap be put into it is to be drained off in embedded box.

2. the fixation of tissue samples to be checked

The tissue of fresh acquisition is repaired and takes into 3-5 millimeters of thickness of fritter, is placed in the fixer of 25 times of volumes of tissue volume In, 100rpm, which rocks, to be fixed for 24 hours, and tissue block removal is put into embedded box after fixed, is pulled out after rinsing 2h using flowing water It drains to be drained off.

(2) it is dehydrated

It is de- according to table 1 that above-mentioned sample (cell or tissue in embedded box) to be drained off is used together fully-automatic dewatering instrument Water program carries out being dehydrated transparent waxdip.

Table 1

Container number	Content reagent	Dewatering time
			1	70% ethyl alcohol	45 minutes to 80 minutes
2	80% ethyl alcohol	45 minutes to 80 minutes
			3	95% ethyl alcohol	45 minutes to 80 minutes
4	95% ethyl alcohol	45 minutes to 80 minutes
			5	100% ethyl alcohol	45 minutes to 80 minutes
6	100% ethyl alcohol	45 minutes to 80 minutes
			7	Dimethylbenzene	45 minutes to 80 minutes
8	Dimethylbenzene	45 minutes to 80 minutes
			11	Paraffin (60 DEG C)	60 minutes to 105 minutes
12	Paraffin (60 DEG C)	60 minutes to 105 minutes

(3) it embeds

Cell or tissue that transparent waxdip finishes will be dehydrated in paraffin wax embedding (Leica HistoCore Arcadia packet Bury system) on carry out paraffin mass preparation.

(4) it is sliced

Paraffin cell block or tissue block be prepared into using histotome (Leica R2235 cycle type slicer) thinly-sliced Piece (3-5 microns) is used for immunohistochemical staining.

2, it detects

Immunohistochemistry detection is carried out (equally according to following immunohistochemical staining processes using the paraffin section of above-mentioned preparation Ground, citing is merely to illustrate the application method and agents useful for same for the kit that is not intended to limit the present invention, those skilled in the art below Know that other similar reagents are available).

Dry wax: 60 DEG C of baking waxes of slice；

Dewaxing: slice carries out dewaxing aquation in a series of dewaxing (such as: dimethylbenzene, graded ethanol and pure water) reagents；

Antigen retrieval: using water-bath coctoantigen repair liquid to face after boiling point slice is put into heating 15min after take out Natural cooling.Inventor has investigated influence of the different antigen retrieval buffers to testing result, is shown in Table 3；

Intrinsic oversxidase removal: using intrinsic oversxidase remover (such as: contain 3% H₂O₂0.05M TBS) remove histotomy in intrinsic oversxidase after using cleaning solution (such as: contain 0.025%Triton X-100's 0.05M TBS) it is washed；

Closing: using confining liquid (such as: the 0.05M TBS containing 1%BSA and 10% lowlenthal serum) to histotomy into Row closing；

Detection antibody: getting rid of confining liquid, is examined using the 0.05M TBS dilution CD26 containing 1%BSA and 10% lowlenthal serum Antibody and negative control antibody are surveyed, dropwise addition is covered on histotomy and uses cleaning solution (example after 2-8 DEG C of incubation 16h-18h of progress It such as: the 0.05M TBS containing 0.025%Triton X-100) is washed, inventor has investigated different antibodies concentration to detection As a result influence, see the table below 3；

Colour developing: DAB color developing agent (such as: Foochow steps neoformation Technology Co., Ltd., DAB kit) dropwise addition is covered on group Knit and develop the color on slice, visually observe, general colour developing used after 3-10 minute cleaning solution (such as: contain 0.025%Triton The 0.05M TBS of X-100) it is washed；

Redye: will slice under flowing water rinse be placed on haematoxylin dyeing liquid (such as: the limited public affairs of Zhuhai shellfish rope biotechnology Department, Mayer ' s haematoxylin) in 3-5 minute, take out be placed on flowing water undershoot be washed till return basket after natural air drying；

Mounting: slice is placed in dimethylbenzene, carries out mounting using mounting glue (such as: Chinese medicines group, neutral gum)；

Diagosis: it is just setting optical microscopy and is carrying out diagosis, blue is nucleus, and brown color is positive coloring, and positive coloring is logical Tinctorial strength, colorant density and color mode (position) three parts are crossed to be assessed.This detection color mode should be after birth or born of the same parents Slurry coloring, standards of grading are shown in Table 2.

Table 2

3, the detection specificity and sensibility, specific evaluation method investigated under different condition are shown in embodiment 6, as a result see the table below 3。

3 different condition reagent preparation box comparing result of table

As can be seen from the comparison result: in the selection of fixer, 4% paraformaldehyde and 10% neutral buffered formalin It can be used as sample fixer；In the selection of antigen retrieval buffers, pH6.0 citrate buffer is as antigen retrieval buffers, sun Property signal is stronger, and negative signal is most weak；From antibody concentration it can be seen that under 10ug/mL working concentration, positive control signal is most By force, and negative control does not generate any signal.

6 immunologic combined detection reagent kit performance test of embodiment

1, specific

The consistent IgG1 chimeric antibody of antibody subtype is used and detected as negative control antibody, in yin and yang attribute Quality Control cell In dyed (see Fig. 5)；Picture can be seen that the detection antibody in kit of the present invention in CD26 high expressing cell (786- 0) there are stronger positive coloring (comprehensive score +++) in, there is no any in CD26 not (or low) expression cell (A375) Brownish discoloration (comprehensive score -)；Negative control antibody in kit is thin in CD26 high expressing cell or not (or low) expression Any brownish discoloration (comprehensive score -) is not present in born of the same parents, it is good special that the above results show that kit of the present invention has Property.

2, immunogenicity

Using primary antibody, in normal human tissue, (every kind of group is woven with 3, buys in the limited public affairs of Xi'an Ai Lina biotechnology Department) it is dyed, confirm cross reactivity of the primary antibody in normal human tissue.19 kinds of normal tissues are used for the dye of primary antibody altogether Color (see Fig. 6 and table 4):

Table 4 detects the cross reaction of antibody in the normal tissue

Coloration result shows primary antibody in adrenal gland, ovary, pituitary, tonsillotome, heart, stomach, small intestine, liver, tongue, forefront There are cross reaction in gland, uterus, striated muscle and eye tissue, above-mentioned coloration result can be used for kit user with just Cross reaction existing for meeting provides reference to the tumor specimen often organized in normal tissue under the dyeing of detection antibody.

3, repeatability between being criticized in criticizing

It has used and has carried out CD26 antibody from 3 slices of 5 body tumor tissue's samples and yin and yang attribute Quality Control sample With the dyeing (see Fig. 7 and table 5) of negative control antibody, the detection of continuous 3 batches has been carried out；As the result is shown in a batch 3 of each sample slices under the dyeing of detection antibody and negative antibody, color depth, colorant density and color mode without significant Difference, difference is also not present between batch, and the above results show that kit of the present invention has good repeatability.

Repeatability between being criticized in 5 batches, table

The above results show that detection kit of the present invention has good detection performance, can be used in tissue or cell The detection of CD26 antigen.

Sequence table

<110>Jiangsu Zhonghong Biopharma Institute Inc.

<120>immunologic combined detection reagent kit of people CD26 and its clinical application

<130>immunologic combined detection reagent kit of people CD26 and its clinical application

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Ile Tyr Pro Gly Asn Gly Tyr Thr

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

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Leu Gln Ile Arg Ser Leu Thr Ser Asp Asp Ser Ser Ile Phe Phe Cys

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

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

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

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Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

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Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 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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gaggtgcagc tgcagcagtc tggagctgag ctgggcagac ctggatccag cgtgaacctg 60

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cctggacagg gactggagtg gatcggctac atctatccag gcaacggcta caccgtgtat 180

aatgagaagt tccagggcaa ggccaccctg acatctgaca cctcttccag cacagtgttt 240

ctgcagatca ggtctctgac atccgacgat tcttccatct tcttttgcgc tcgggaccac 300

ggcgattact ggggacaggg aaccacactg accgtgagct ctgcctctac aaagggcccc 360

tccgtgtttc cactggctcc atccagcaag tccaccagcg gaggaacagc cgctctgggc 420

tgtctggtga aggattattt ccccgagcct gtgaccgtga gctggaattc tggcgccctg 480

acctccggcg tgcatacatt tccagctgtg ctgcagtctt ccggcctgta cagcctgagc 540

tctgtggtga cagtgccctc cagctctctg ggcacccaga catatatctg taacgttaat 600

cacaagccat ctaataccaa ggtggacaag aaggtggagc ccaagtcctg tgataagaca 660

catacctgcc caccttgtcc tgctccagag ctgctgggcg gaccatccgt gttcctgttt 720

ccacccaagc ctaaggacac cctgatgatc tccagaacac cagaggtgac ctgcgtggtg 780

gtggacgtga gccacgagga tcccgaggtg aagttcaact ggtacgtgga tggcgtggag 840

gtgcataatg ctaagaccaa gccaagggag gagcagtaca atagcacata tcgggtggtg 900

tctgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtataa gtgcaaggtg 960

tctaataagg ccctgcccgc tcctatcgag aagacaatct ccaaggccaa gggccagcct 1020

agagagccac aggtgtacac cctgcctcca tcccgcgacg agctgacaaa gaaccaggtg 1080

agcctgacct gtctggtgaa gggcttctat ccctctgaca tcgctgtgga gtgggagtcc 1140

aatggccagc ctgagaacaa ttacaagacc acaccccctg tgctggacag cgatggctct 1200

ttctttctgt attccaagct gacagtggat aagagccgct ggcagcaggg caacgtgttt 1260

tcctgtagcg tgatgcatga ggctctgcac aatcattaca cccagaagtc tctgtccctg 1320

agccccggca agtga 1335

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Pro Lys Leu Leu Ile Tyr Lys Val Phe Lys Arg Leu Ser Gly Val Pro

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Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly

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Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Thr Leu Glu Thr Lys

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gacgtgctga tgacccagac accactgtcc ctgcccgtga gcctgggcga tcaggccagc 60

atctcttgct ccagctctag gaacatcgtg cactctgacg gcaatacata cctggattgg 120

tatctgcaga agcctggcca gtccccaaag ctgctgatct acaaggtgtt caagaggctg 180

agcggagtgc cagaccggtt ctccggaagc ggatctggaa ccgacttcac cctgaagatc 240

accagagtgg aggccgagga tctgggcgtg tacttctgct ttcagggctc ccatgtgccc 300

tatacattcg gcggaggaac cacactggag accaagcgca cagtggccgc tcctagcgtg 360

ttcatctttc ccccttctga cgagcagctg aagtctggca ccgcttccgt ggtgtgcctg 420

ctgaacaact tctacccaag ggaggccaag gtgcagtgga aggtggataa cgctctgcag 480

tccggcaata gccaggagtc tgtgaccgag caggactcca aggatagcac atattctctg 540

tccagcaccc tgacactgtc taaggccgac tacgagaagc acaaggtgta tgcttgcgag 600

gtgacccatc agggcctgtc ttcccccgtg acaaagtcct ttaacagagg cgagtgttga 660

Claims

1. it is a kind of for detecting the immunohistochemical kit of the CD26 protein expression in human tumour tissue, including anti-human CD26 anti- Body, Isotype control feminine gender antibody, antibody diluent and the Quality Control piece for monitoring dyeing quality, which is characterized in that the anti-human CD26 Antibody, heavy chain variable region contain complementary determining region below: amino acid sequence HCDR1 as shown in sequence SEQ ID NO:1, The HCDR2 as shown in the sequence SEQ ID NO:2 and HCDR3 as shown in sequence SEQ ID NO:3；

And its light-chain variable sequence contains complementary determining region below: amino acid sequence is as shown in sequence SEQ ID NO:4 LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and the LCDR3 as shown in sequence SEQ ID NO:6.

2. immunohistochemical kit as described in claim 1, which is characterized in that the amino of anti-human CD26 antibody heavy chain variable region Acid sequence is as shown in SEQ ID NO:7, and the amino acid sequence of light chain variable region is as shown in SEQ ID NO:8.

3. immunohistochemical kit as claimed in claim 2, which is characterized in that the anti-human CD26 antibody is chimeric antibody, Its amino acid sequence is as the heavy chain variable region as shown in SEQ ID NO:7 and 1 heavy chain constant domain of people's monoclonal antibody IgG, such as SEQ The light chain variable region shown in ID NO:8 merges composition with 1 constant region of light chain of people's monoclonal antibody IgG.

4. immunohistochemical kit as claimed in claim 3, which is characterized in that the anti-human CD26 antibody is by mammal place Chief cell recombinantly expresses to obtain.

5. immunohistochemical kit according to any one of claims 1 to 4, which is characterized in that anti-in the kit Body dilution preferably comprises the 0.05M TBS of 1%BSA and 10% lowlenthal serum.

6. immunohistochemical kit according to any one of claims 1 to 4, which is characterized in that anti-in the kit Body running concentration is 2.5-10ug/mL.

7. immunohistochemical kit according to any one of claims 1 to 4, which is characterized in that mating with the kit The tissue fixative solution used is 4% paraformaldehyde or 10% neutral buffered formalin.

8. immunohistochemical kit according to any one of claims 1 to 4, which is characterized in that mating with the kit The antigen retrieval buffers used are the citrate buffer of pH6.0.