CN106248971B - For detecting the ELISA kit of HER2 contents, application method and purposes in human serum - Google Patents
For detecting the ELISA kit of HER2 contents, application method and purposes in human serum Download PDFInfo
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Abstract
It is used to detect the ELISA kit of HER2 contents, application method and purposes in human serum the invention discloses a kind of.The kit includes HER2 ECD standard antigens, anti-HER2 capture antibody and anti-HER2 detection antibody；The capture antibody is humanization HuA21 antibody (amino acid sequence of heavy chain is that the amino acid sequence of the light chain of sequence 1 is sequence 2), the detection antibody is the HerA antibody through biotin labeling (amino acid sequence of heavy chain is sequence 3, and the amino acid sequence of light chain is sequence 4).Provided by the present invention to detect that the enzyme linked immunological kit of HER2 contents in human serum realize the accurate quantitative analysis detection to HER2 contents in human serum based on double-antibody method, the detection method can be used as a kind of non-invasive, rapidly simplicity, diagnosis and the method for monitoring HER2 positive tumor diseases.
Description
Technical field
The invention belongs to biotechnology and medical domain, it is related to a kind of ELISA for being used to detect HER2 contents in human serum
Kit, application method and purposes.
Background technology
Tumour is to have a strong impact on one of principal disease of public health, and its effectively treatment is dependent on the early screening to tumour
With diagnosis in time.Tumor-marker analyte detection has significant role to tumor research and clinical practice, has been widely used in
Tumour early screening, diagnosis, treatment, therapeutic evaluation and Prognosis scoveillance etc..
Human epidermal growth factor receptor 2 (HER2) high expression in the kinds of tumors such as breast cancer, stomach cancer, detects tumour at present
HER2 expressions are organized to be used for the clinical diagnosis and treatment of tumour as an individual index.Traditional HER2 detections are main
By traumatic tumor tissue biopsy, using the method such as SABC (IHC) and FISH (FISH), but these
Method can not detect the HER2 levels in patient or recurrence and metastatic tumo(u)r patient's body after operative treatment.Numerous studies show,
HER2 fragments can shed into HER2 extracellular domain fragments soluble in blood, affinity antibody to SpA with swelling from tumor cell surface
There is obvious correlation in the HER2 expressions of tumor tissue.Exist in the metastatic breast cancer patients serum for having up to more than 50%
HER2 levels rise phenomenon (having research display to be more than some threshold value (such as 15ng/ml) can point out tumour to get along with), and HER2
Serum levels and its variation tendency can be used as evaluation and the prognosis evaluation index of oncotherapy effect.Therefore HER2 serum levels inspection
Survey has important application value in tumor diagnosis and therapy.
The content of the invention
It is used to detect the ELISA kit of HER2 contents, application method in human serum it is an object of the invention to provide a kind of
And purposes.
The enzyme linked immunological kit provided by the present invention for being used to detect HER2 contents in human serum is a kind of based on dual anti-
The enzyme linked immunological kit of HER2 contents in sandwich method detection human serum, including HER2-ECD standard antigens, anti-HER2 capture
The detection antibody of antibody (being used as coating antigen) and anti-HER2；The capture antibody is humanization HuA21 antibody, the detection antibody
For the HerA antibody through biotin labeling.
The amino acid sequence of the heavy chain of the humanization HuA21 antibody is specific as shown in sequence 1 in sequence table；The people source
The amino acid sequence for changing the light chain of HuA21 antibody is specific as shown in sequence 2 in sequence table.
The amino acid sequence of the heavy chain of the HerA antibody is specific as shown in sequence 3 in sequence table；The HerA antibody
The amino acid sequence of light chain is specific as shown in sequence 4 in sequence table.
The amino acid sequence of the HER2-ECD standard antigens is specific as shown in sequence 5 in sequence table.
In the present invention, the HER2-ECD standard antigens are particular by by sequence 6 in nucleotide sequence such as sequence table
Shown HER2-ECD genes carry out expressing acquisition in mammalian cell.
Further, the HER2-ECD genes are to import the mammalian cell by recombinant expression carrier；It is described
Recombinant expression carrier is specifically by SfiI the and NotI endonuclease recognized sites in pSecTag2A carriers (Invitrogen companies)
Between small fragment replace with the recombinant expression carrier obtained after the HER2-ECD genes and (be named as pSecTag2A-HER2-
ECD).Wherein, the mammalian cell concretely Chinese hamster ovary celI.
More specific, the HER2-ECD standard antigens are prepared according to the method comprised the following steps：Will
The pSecTag2A-HER2-ECD is transferred to after Chinese hamster ovary celI, is cultivated 24 hours, (is contained 0.3mg/mL with Selective agar medium
Zeocin resistance clone) is screened, resistance clone is expanded and cultivated, centrifugation (8000rpm is centrifuged 15 minutes) takes supernatant to filter (mistake
0.22 μM of filter membrane), gained filtrate is subjected to Mabselect Sure affinity purifications, the HER2-ECD standard antigens are obtained.Its
In, the filtrate is subjected to Mabselect Sure affinity purifications specific as follows：Mabselect Sure glue (GE companies) is used
After PBS balances, filtrate described in loading first elutes 5 column volumes in advance using A liquid (20mM sodium phosphates, 500mM NaCl, pH5.0),
5 column volumes are eluted using B liquid (20mM sodium acetates, 150mM NaCl, pH3.5) again, eluting peak is collected, then 30KDa is concentrated
Centrifuge tube concentration obtains the HER2-ECD standard antigens.
In the present invention, the humanization HuA21 antibody specifics are by by such as sequence table of equimolar nucleotide sequence
The encoding gene and nucleotide sequence of the heavy chain of humanization HuA21 antibody shown in middle sequence 7 are as shown in sequence 8 in sequence table
The encoding gene of the light chain of humanization HuA21 antibody carries out expressing what is obtained in mammalian cell.
Further, the light chain of the encoding gene of the heavy chain of the humanization HuA21 antibody and the humanization HuA21 antibody
Encoding gene be that the mammalian cell is imported by recombinant expression carrier 1 and recombinant expression carrier 2 respectively；It is described heavy
Group expression vector 1 is specifically by SfiI the and NotI endonuclease recognized sites in pSecTag2A carriers (Invitrogen companies)
Between small fragment replace with the recombination expression that is obtained after the encoding gene (sequence 7) of the heavy chain of the humanization HuA21 antibody and carry
Body (is named as pSecTag2A-HuA21-1)；The recombinant expression carrier 2 is specifically by pSecTag2A carriers (Invitrogen
Company) in SfiI and NotI endonuclease recognized sites between small fragment replace with the light chain of the humanization HuA21 antibody
The recombinant expression carrier (being named as pSecTag2A-HuA21-2) obtained after encoding gene (sequence 8).Wherein, the lactation is moved
Thing cell concretely Chinese hamster ovary celI.
More specific, the humanization HuA21 antibody is prepared according to the method comprised the following steps：Will etc.
Mole the pSecTag2A-HuA21-1 and the pSecTag2A-HuA21-2 be transferred to after Chinese hamster ovary celI, cultivate 24 hours, use
Selective agar medium (containing 0.3mg/mL Zeocin) screens resistance clone, and resistance clone is expanded and cultivated, and takes culture supernatant successively
Protein A affinity chromatographys and S200 sieve chromatographies are carried out, is finally concentrated with 30KDa ultra-filtration centrifuge tubes, the people source is obtained
Change HuA21 antibody.
In the present invention, the HerA antibody specifics are by by sequence 9 in equimolar nucleotide sequence such as sequence table
HerA antibody of the encoding gene and nucleotide sequence of the heavy chain of shown HerA antibody as shown in sequence 10 in sequence table it is light
The encoding gene of chain carries out expressing what is obtained in mammalian cell.
Further, the encoding gene of the light chain of the encoding gene of the heavy chain of the HerA antibody and the HerA antibody is point
The mammalian cell is not imported by recombinant expression carrier 3 and recombinant expression carrier 4；The recombinant expression carrier 3 has
Body is to replace the small fragment between SfiI the and NotI endonuclease recognized sites in pSecTag2A carriers (Invitrogen companies)
The recombinant expression carrier obtained after encoding gene (sequence 9) for the heavy chain of the HerA antibody (is named as pSecTag2A-
HerA-1)；The recombinant expression carrier 4 is specifically by the SfiI and NotI in pSecTag2A carriers (Invitrogen companies)
Small fragment between endonuclease recognized site replaces with the weight obtained after the encoding gene (sequence 10) of the light chain of the HerA antibody
Group expression vector (being named as pSecTag2A-HerA-2).Wherein, the mammalian cell concretely Chinese hamster ovary celI.
More specific, the HerA antibody is prepared according to the method comprised the following steps：Will be equimolar
The pSecTag2A-HerA-1 and the pSecTag2A-HerA-2 are transferred to after Chinese hamster ovary celI, are cultivated 24 hours, are cultivated with selection
Base (containing 0.3mg/mL Zeocin) screens resistance clone, and resistance clone is expanded and cultivated, and takes culture supernatant successively progress
Protein A affinity chromatographys and S200 sieve chromatographies, are finally concentrated with 30KDa ultra-filtration centrifuge tubes, obtain the HerA antibody.
In the present invention, the detection antibody is prepared according to the method comprised the following steps：By the HerA
Antibody and the biotin of activation are according to mol ratio 1：30 mixing, (25 DEG C) placement half an hour of room temperature, subsequent over-molecular sieve post is removed
Uncombined biotin, it is the detection antibody to obtain the HerA antibody through biotin labeling.
, can also be containing at least one of following in the enzyme linked immunological kit except above-mentioned substance：96 hole elisa Plates,
Bed board buffer solution, cleaning solution, confining liquid, Horseradish peroxidase-conjugated avidin, nitrite ion and terminate liquid.
In the present invention, the bed board buffer solution is specially the phosphate buffer that pH is 7.0.The cleaning solution is specially
The phosphate buffer that pH only containing 0.05% volumn concentration Tween 20 is 7.0.The pH delays for 7.0 phosphate
The solvent of fliud flushing is water, and solute is sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate；The sodium chloride is in the pH
Concentration in 7.0 phosphate buffer is 135mM, and the potassium chloride is dense in 7.0 phosphate buffer in the pH
Spend for 2.7mM, concentration 1.5mM of the potassium dihydrogen phosphate in the phosphate buffer that the pH is 7.0, the phosphoric acid hydrogen two
Concentration of the sodium in the phosphate buffer that the pH is 7.0 is 8mM.The confining liquid is specially only containing 10g/L BSA
The cleaning solution.The nitrite ion is specially tetramethyl benzidine.The terminate liquid is specially 1M sulfuric acid.
Application of the enzyme linked immunological kit in detection human serum in HER2 contents falls within the protection model of the present invention
Enclose.
As needed, the application may be either the application of non-diseases diagnoses and treatment, or medical diagnosis on disease treatment application.
Application of the enzyme linked immunological kit in following (A) or (B) falls within protection scope of the present invention：
(A) product for monitoring tumour generation and/or course advancement is prepared；
(B) it is used to monitor tumour generation and/or course advancement.
Wherein, the tumour is HER2 positive tumors, concretely breast cancer, stomach cancer etc..
In addition, the preparation method of the enzyme linked immunological kit falls within protection scope of the present invention.
The preparation method of the enzyme linked immunological kit, specifically may include following steps：By by nucleotide sequence such as sequence
HER2-ECD genes in list shown in sequence 6 carry out the expression acquisition HER2-ECD standards in mammalian cell and resisted
It is former；Pass through the coding base of the heavy chain of the humanization HuA21 antibody by equimolar nucleotide sequence as shown in sequence 7 in sequence table
The encoding gene of the light chain of the humanization HuA21 antibody of cause and nucleotide sequence as shown in sequence 8 in sequence table is in mammal
In cell express obtaining the humanization HuA21 antibody；By by sequence 9 in equimolar nucleotide sequence such as sequence table
HerA antibody of the encoding gene and nucleotide sequence of the heavy chain of shown HerA antibody as shown in sequence 10 in sequence table it is light
The encoding gene of chain carries out expressing obtaining the HerA antibody in mammalian cell.
It is demonstrated experimentally that the enzyme linked immunological examination provided by the present invention that HER2 contents in human serum are detected based on double-antibody method
Agent box can realize the accurate quantitative analysis detection to HER2 contents in human serum, and stability is good.The detection method can be used as one kind
Non-invasive, rapidly simplicity, diagnosis and the method for monitoring HER2 positive tumor diseases.
Brief description of the drawings
Fig. 1 is that the HER2 extracellular region standard antigens SDS-PAGE that ELISA sandwich method kits of the present invention are used (is gone back
Prototype).10% glue SDS-PAGE glue.1,2, No. 3 samples that pH3.5 is afforded.4 μ l samples/swimming lane.Molecular weight standards
(M) molecular size range is (kDa) from top to bottom：113,78,55,45,28.
Fig. 2 is the SDS-PAGE for Anti-HER 2 HuA21 and the HerA antibody that ELISA sandwich method kits of the present invention are used
Electrophoretogram.10% glue SDS-PAGE glue.Electrophoresis left figure is HuA21 non-reduced (1) and the sample for reducing (2), molecular weight standards
(M) molecular size range is (kDa) from top to bottom：120,100,80,70,60,50,30,20,15；Electrophoresis right figure is HerA non-also
Former (1) and the sample for reducing (2), molecular weight standards (M) molecular size range is (kDa) from top to bottom：116,66.2,43,31,
20.1,14.4；10 μ g samples/swimming lane.
Fig. 3 does breast cancer for Anti-HER 2 HuA21 and the HerA antibody that ELISA sandwich method kits of the present invention are used and exempted from
The result of epidemic disease histochemical method dyeing.Wherein, the result that A dyes for the immunohistochemical method of humanization HuA21 antibody；B is
The result of the immunohistochemical method dyeing of HerA antibody.
Fig. 4 is that sandwich method ELISA of the present invention is obtained using the HER2 extracellular region antigen standard concentration of gradient dilution as abscissa
The light absorption value obtained is ordinate, and carrying out five parameter nonlinear regressions by log-log coordinate calculates obtained standard curve.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Chinese hamster ovary celI：Come from Chinese Academy of Sciences's Shanghai cell bank.
The expression vector pSecTag2A-HER2-ECD of extracellular region containing HER2 (HER2-ECD) gene, its construction method is such as
Under：With DNA fragmentation shown in sequence in sequence table 6 (encoding gene of HER2-ECD standard antigens) for template, using primer ECD-N
Enter performing PCR amplification with ECD-DK-NotI, gained PCR primer is carried out after SfiI and NotI double digestions, glue reclaim with passing through same
PSecTag2A carriers (Invitrogen companies) skeleton large fragment of double digestion is connected, and obtains recombinant plasmid.It will show through sequencing
Small fragment between SfiI and NotI endonuclease recognized sites in pSecTag2A carriers (Invitrogen companies) is replaced with
" recombinant expression carrier obtained after DNA fragmentation shown in sequence 6+GGAGACGATGACGATAAG " is named as pSecTag2A-
HER2-ECD, the carrier can HER2-ECD standard antigens shown in sequence 5 in expressed sequence table, and the carrier carries
Zeocin resistant genes.Wherein, ECD-N (5 ' -3 ')：AAAGCGGCCCAGCCGGCC(lower stroke of-AGCACCCAAGTGTGCACCG
Line part be SfiI recognition sequence ,-after sequence correspond to sequence table in sequence 6)；ECD-DK-NotI(5’-3’)：
TATATGCGGCCGC(underscore part is NotI knowledge to CTTATCGTCATCGTCTCC-GGACGTCAGAGGGCTGGCTCTCTG
Other sequence ,-after sequence correspond to sequence table in sequence 6).
The expression vector pSecTag2A-HuA21-1 of the antibody-encoding genes of HuA21 containing humanization.The carrier is will
Small fragment between the restriction enzyme site SfiI and NotI of pSecTag2A carriers (Invitrogen companies) replaces with sequence in sequence table
The recombinant plasmid obtained after the encoding gene of the heavy chain of humanization HuA21 antibody shown in row 7, the plasmid can express humanization
The heavy chain (sequence 1) of HuA21 antibody, and carry Zeocin resistant genes.
The expression vector pSecTag2A-HuA21-2 of the antibody-encoding genes of HuA21 containing humanization.The carrier is will
Small fragment between the restriction enzyme site SfiI and NotI of pSecTag2A carriers (Invitrogen companies) replaces with sequence in sequence table
The recombinant plasmid obtained after the encoding gene of the light chain of humanization HuA21 antibody shown in row 8, the plasmid can express humanization
The light chain (sequence 2) of HuA21 antibody, and carry Zeocin resistant genes.
The expression vector pSecTag2A-HerA-1 of the antibody-encoding genes containing HerA.The carrier is by pSecTag2A carriers
Small fragment between the restriction enzyme site SfiI and NotI of (Invitrogen companies) replaces with the HerA in sequence table shown in sequence 9
The recombinant plasmid obtained after the encoding gene of the heavy chain of antibody, the plasmid can express the heavy chain (sequence 3) of HerA antibody, and
Carry Zeocin resistant genes.
The expression vector pSecTag2A-HerA-2 of the antibody-encoding genes containing HerA.The carrier is by pSecTag2A carriers
Small fragment between the restriction enzyme site SfiI and NotI of (Invitrogen companies) is replaced with sequence table shown in sequence 10
The recombinant plasmid obtained after the encoding gene of the light chain of HerA antibody, the plasmid can express the light chain (sequence 4) of HerA antibody,
And carry Zeocin resistant genes.
Embodiment 1, the method for preparation and use for detecting the enzyme linked immunological kit of HER2 contents in human serum
First, for detecting the preparation of the enzyme linked immunological kit of HER2 contents in human serum
The enzyme linked immunological kit provided by the present invention for being used to detect HER2 contents in human serum, including HER2-ECD marks
The capture antibody (humanization HuA21 antibody) and the anti-HER2 detection antibody (HerA through biotin labeling of quasi- antigen, anti-HER2
Antibody), and ELISA Plate, bed board buffer solution, cleaning solution, confining liquid, Horseradish peroxidase-conjugated avidin, nitrite ion and end
Only liquid etc..
(1) conventional equipment and reagent
1st, 96 hole elisa Plates (Nunc companies)；
2nd, bed board buffer solution：PH is 7.0 phosphate buffer；
3rd, cleaning solution：The only pH containing 0.05% volumn concentration Tween 20 is 7.0 phosphate buffer；
4th, confining liquid：The only BSA containing 10g/L cleaning solution.
5th, Horseradish peroxidase-conjugated avidin；
6th, chromogenic substrate：Tetramethyl benzidine；
7th, terminate liquid：1M sulfuric acid.
Wherein, the solvent for the phosphate buffer that the pH is 7.0 is water, and solute is sodium chloride, potassium chloride, biphosphate
Potassium, disodium hydrogen phosphate；Concentration of the sodium chloride in the phosphate buffer that the pH is 7.0 is 135mM, the potassium chloride
Concentration in the phosphate buffer that the pH is 7.0 is 2.7mM, and the potassium dihydrogen phosphate is in the phosphoric acid that the pH is 7.0
Concentration 1.5mM in salt buffer, concentration of the disodium hydrogen phosphate in the phosphate buffer that the pH is 7.0 is 8mM.
(2) preparation of HER2-ECD standard antigens
Chinese hamster ovary celI is inoculated with 6cm Tissue Culture Dish, extracellular region containing HER2 is transfected during cell culture to 90% density
(HER2-ECD) the expression vector pSecTag2A-HER2-ECD of gene, is cultivated 24 hours, pancreatin had digestive transfer culture to 10 after transfection
The porocyte culture plates of block 96, add and (resist containing 0.3mg/mL Zeocin on expression vector pSecTag2A skeletons containing Zeocin
Property gene) selective medium screening resistance clone.After cell clone is grown (about 2 weeks), according to HER2- in different supernatants
ECD content selects expression quantity highest clone (the specific HER2-ECD expression quantity ELISA inspections produced using R＆D companies of the U.S.
Test agent box is operated to specifications).The clone of gained height expression is expanded into culture step by step, supernatant merges about 1.8L,
8000rpm is centrifuged 15 minutes, crosses 0.22 μM of filter membrane.Take 5ml Mabselect Sure glue (GE companies) balanced with PBS after by it
Filtrate loading obtained by preceding 0.22 μM of filter membrane of mistake, applied sample amount is 2 liters of nutrient solution correspondence gained filtrates, first with A liquid (formula：Solvent is
Water, solute and concentration are：20mM sodium phosphates, 500mM NaCl, pH5.0) 5 column volumes are eluted in advance, elution flow rate is 1mg/ml,
Again with B liquid (formula：Solvent is water, and solute and concentration are：20mM sodium acetates, 150mM NaCl, pH3.5) 5 column volumes of elution,
Elution flow rate is 1mg/ml, and Ultraviolet Detector monitoring eluting peak (unique eluting peak, i.e. target product occurs) is being received in advance
100 μ l 2M Tris neutralizer (compound methods are added in collector：Tris 12.114g, plus 800ml deionized water dissolvings are taken, so
Afterwards with 1M HCl regulation pH to 8.0, deionized water is finally mended to 1000ml.) (100 μ l are often added in pipe), eluting peak is collected, altogether
Collect 3 to manage, often pipe about 5ml.SDS-PAGE electrophoresis is carried out to the sample of collection, it was demonstrated that collected sample and theoretical molecular phase
Together.Merge 3 pipe samples, 30KDa concentration centrifuge tubes are concentrated into about 4ml, ultraviolet determination concentration OD280/OD260=5.88/3.60,
Then 4 pipes are distributed into, often pipe about 1ml, concentration 0.5mg/ml, -80 DEG C of preservations.
The SDS-PAGE electrophoresis detections result (reduced form) of HER2 extracellular regions (HER2-ECD) standard antigen after purification is such as
Shown in Fig. 1.The result shows that HER2-ECD is monomer, and molecular weight 110kDa has been obtained expressing with theoretical molecular identical and produced
Thing, electrophoresis purity 95% or so.Further HER2 extracellular regions (HER2-ECD) standard antigen after purification obtained as above is entered
Row N-terminal and C-terminal sequencing, as a result show that purifying gained HER2-ECD sequences are consistent with sequence 5.
The preparation of (three) two kinds of Anti-HER 2s
Two kinds of Anti-HER 2s are respectively humanization HuA21 antibody and HerA antibody.Wherein, humanization HuA21 antibody is
A kind of novel antibodies of targeting HER2 extracellular region I, II domains, HerA antibody is protein structure and marketed drug He Sai
The recombinant antibodies of the identical another targeting HER2 extracellular region iv domains in spit of fland.Both antibody have entirely different
Variable region amino acid sequence, and recognize the entirely different epitope of HER2 extracellular regions.
Chinese hamster ovary celI is inoculated with 6cm Tissue Culture Dish, HuA21 containing humanization is transfected during cell culture to 90% density and is resisted
The expression vector (equimolar pSecTag2A-HuA21-1 and pSecTag2A-HuA21-2) of body encoding gene is anti-containing HerA
The expression vector (equimolar pSecTag2A-HerA-1 and pSecTag2A-HerA-2) of body encoding gene.24 are cultivated after transfection
Hour, pancreatin had digestive transfer culture to 10 piece of 96 porocyte culture plates adds and contains 0.3mg/mL Zeocin (expression vectors
On pSecTag2A skeletons contain Zeocin resistant genes) selective medium screening resistance clone.After cell clone is grown
(about 2 weeks), select expression quantity highest clone (specific according to the content of humanization HuA21 antibody in different supernatants, HerA antibody
To take after supernatant doubling dilution, the coated ELISA Plates of HER2-ECD are added, titre highest gram is chosen after indirect elisa method colour developing
It is grand).The clone of gained height expression is expanded into culture step by step, until roller bottle culture.The supernatant of roller bottle culture is taken, the first step is first used
Protein A affinity columns are purified.Concrete operations are：First Protein A posts (HiTrap rProtein A are balanced with PBS
FF, GE company), then culture supernatant is crossed after post, and 5-10 volume PBS washes away foreign protein, plus glycine hydrochloride (formula：Sweet ammonia
Sour 7.507g, adjusts pH to 3.0, deionized water is settled to 500ml with hydrochloric acid) elution destination protein, next step is continued after merging.The
Two steps are the purifying of S200 molecular sieve chromatographies.Concrete operations are：First S200 molecular sieve columns (SP Sepharose are balanced with PBS
FF, GE company), then add first step purification of samples to cross post, the efflux after tight 1 volume PBS elution of monitoring treats maximum suction
Receive and start to collect preceding 3 pipe (often pipe 1ml samples) after peak (280nM UV absorption devices are shown) occurs, next step is continued after merging.Most
Latter step is to be concentrated (to have run reduced form SDS-PAGE electrophoresis before concentration to demonstrate,prove with 30KDa ultra-filtration centrifuge tubes (Millipore companies)
True weight chain and light chain molecule amount are errorless, and purity is more than 95%, as a result sees Fig. 2), the humanization purified after concentration
HuA21 antibody, HerA antibody.
Left figure in Fig. 2 is HuA21 electrophoresis results：Three bands that No. 1 swimming lane is 150kDa or so, are followed successively by from top to bottom
Polymer, antibody monomer, aglycosylated antibody；No. 2 swimming lanes are the band of 50KDa, 25KDa two, respectively heavy chain and light chain, size
It is consistent completely with expection.Right figure is HerA electrophoresis results：Two bands that No. 1 swimming lane is 150kDa or so, are followed successively by from top to bottom
Antibody monomer, aglycosylated antibody；No. 2 swimming lanes are the band of 50KDa, 25KDa two, respectively heavy chain and light chain, size and expection
It is consistent completely.HuA21, HerA after purification obtained as above are further subjected to N-terminal and C-terminal sequencing, as a result shown pure
Change gained HuA21 sequences with sequence 1 and 2 to be consistent；Purifying gained HerA sequences are consistent with sequence 3 and 4.
(4) the biotinylation mark of detection antibody
Anti-HER 2 HerA prepared by above-mentioned steps (three) and biotin (the EZ-Link NHS-PEG4- of activation
Biotin kits, Pierce companies) according to mol ratio 1：30 mixing, it is (20-25 DEG C) placement half an hour of room temperature, subsequent to cross molecule
Sieve post and remove uncombined biotin (molecular sieve column that foregoing EZ-Link NHS-PEG4-Biotin kits are carried),
Obtain the HerA detection antibody of biotin labeling.
The specificity identification of (five) two kinds of Anti-HER 2s
There is good binding specificity for two kinds of Anti-HER 2s that further checking above-mentioned steps (three) are obtained, to it
The immunohistochemical method staining analysis of breast cancer tumour sample is carried out.Using 0-3+ points-scoring systems, 2+ represents breast cancer group
The medium expression of middle HER2 is knitted, 3+ represents HER2 height expression in breast cancer tissue, and cancer beside organism represents that being not affected by cancer cell invades profit
Normal galactophore tissue, this class loading HER2 levels are feminine gender.It is raw on two kinds of Anti-HER 2s mark prepared by above-mentioned steps three
Thing element (specific steps are with reference to step 4), carries out antigen-antibody binding reaction, instead with 3+ breast cancer tissues and Carcinoma side normal tissue
Dyed after the completion of answering with Horseradish peroxidase-conjugated avidin and DAB chromogenic substrates (ThermoFisher companies), it is micro-
Microscopic observation, take pictures.
As a result show, whether HuA21 or HerA antibody, can carry out specific stain to the high expression tissues of HER2,
And cancer beside organism's then dye-free, concrete outcome is shown in Fig. 3.Two kinds of antibody that the result demonstrates above-mentioned steps (three) preparation have good
Good antigen-recognition specificity.
2nd, the application method for being used to detect the enzyme linked immunological kit of HER2 contents in human serum that prepared by step one
1st, standard curve is made using HER2-ECD standard antigens
The humanization HuA21 antibody that step one is prepared to the anti-HER2 of gained is diluted with bed board buffer solution (formula is seen above)
To 2 μ g/ml, 100 μ l bed boards are added per hole, 4 DEG C overnight.Board-washing three times, adds 300 μ l confining liquids (formula is seen above), 37 DEG C per hole
Closing 1 hour.With cleaning solution (formula is seen above) board-washing three times, one HER2-ECD prepared the step of by concentration be 0.5mg/ml
Standard antigen with the serum Special sample diluted of the BSA containing 10g/L to 500pg/ml, then with 7 Ep pipes doubling dilutions into
Gradient solution (table 1), then by each two multiple holes of concentration, adds in plate per the μ l of hole 100, vibrates 1 hour.(matched somebody with somebody with cleaning solution
Side is seen above) board-washing three times, plus PBS phosphate buffers (bed board buffer solution) one warp prepared the step of be diluted to 0.25 μ g/ml
The HerA antibody of biotin labeling, per the μ l of hole 100, vibrates 1 hour.With cleaning solution (formula is seen above) board-washing three times, add per hole
1% (10g/L) BSA (solvent is PBS) dilutes 8000 times of Horseradish peroxidase-conjugated avidin
(ThermoFisher companies) 100 μ l, vibrate 1 hour.With cleaning solution (formula is seen above) board-washing three times, tetramethyl is added to join per hole
The μ l of aniline (tetramethylbenzidine, TMB) (ThermoFisher companies) 100.Lucifuge develop the color 4 minutes, plus 1M sulphur
Sour terminating reaction.With BIO-TEK ELX-800 ELIASAs read plate at 450 nm.
The light absorption value (OD450) of the HER2-ECD standard antigens of each concentration is as shown in table 1.With HER2-ECD standard antigens
The logarithm (being bottom with 10) of concentration is abscissa, using OD450 values as ordinate, draws standard curve.Obtain calibration curve equation
For Y=0.8072*X-1.786, R2=0.9953, as shown in Figure 4.Further, by the HER2-ECD standard antigens of each concentration
Light absorption value (OD450) substitutes into above-mentioned mark curvilinear equation, produces corresponding apparent concentration under each HER2-ECD standard antigens concentration,
With apparent concentration the rate of recovery is produced than upper corresponding actual concentrations.It is computed, the HER2-ECD standard antigens of each concentration are corresponding
Apparent concentration and the rate of recovery are referring to table 1.
Apparent concentration and the rate of recovery after the light absorption value of the various concentrations HER2-ECD standard antigens of table 1 and its conversion
| HER2-ECD standard antigens | Concentration (pg/ml) | Light absorption value (OD450) | Apparent concentration (pg/ml) | The rate of recovery (%) |
| S1 | 500.0 | 2.172 | 492.5 | 98 |
| S2 | 250.0 | 1.473 | 260.8 | 104 |
| S3 | 125.0 | 0.849 | 120.7 | 97 |
| S4 | 62.5 | 0.497 | 61.9 | 99 |
| S5 | 31.3 | 0.285 | 32.4 | 104 |
| S6 | 15.6 | 0.145 | 15.3 | 98 |
| S7 | 7.8 | 0.079 | 7.8 | 100 |
| S8 | 0.0 | 0.017 | 1.4 | - |
2nd, in human serum sample to be measured HER2 contents measure
HER2-ECD standard antigens in step 1 are replaced with into human serum sample to be measured, the OD450 values of gained are substituted into and walked
The calibration curve equation of rapid 1 gained, so as to obtain the content of HER2 in human serum sample to be measured.
The rate of recovery for detecting the enzyme linked immunological kit of HER2 contents in human serum prepared by embodiment 2, embodiment 1
Determine
The present inventor determines reality further using the normal human serum of addition standard antigen as testing sample
Apply the rate of recovery for being used to detect the enzyme linked immunological kit of HER2 contents in human serum of the preparation of example 1.
For normal human serum totally 18 parts (being shown in Table 2) of examination, these serum samples come from Provincial Hospital's medical center,
Volunteer is voluntary and knows.
The humanization HuA21 antibody that step one is prepared to the anti-HER2 of gained is diluted with bed board buffer solution (formula is seen above)
To 2 μ g/ml, 100 μ l bed boards are added per hole, 4 DEG C overnight.Board-washing three times, adds 300 μ l confining liquids (formula is seen above), 37 DEG C per hole
Closing 1 hour.With cleaning solution (formula is seen above) board-washing three times, concentration is prepared for the 0.5mg/ml step one of embodiment 1
HER2-ECD standard antigens are diluted after 20 times with different numbering normal human sera samples, then (same with serum Special sample dilution
On) 200 times of dilution, by each two multiple holes of sample, add in plate, vibrate 1 hour per the μ l of hole 100.With cleaning solution (on formula is shown in
Text) board-washing three times, plus PBS phosphate buffers (bed board buffer solution) the step of be diluted to 0.25 μ g/ml one prepare through biotin
The HerA antibody of mark, per the μ l of hole 100, vibrates 1 hour.With cleaning solution (formula is seen above) board-washing three times, add 1% per hole
(10g/L) BSA (solvent is PBS) dilutes 8000 times of Horseradish peroxidase-conjugated avidin (ThermoFisher
Company) 100 μ l, vibrate 1 hour.With cleaning solution (formula is seen above) board-washing three times, add tetramethyl benzidine per hole
(tetramethylbenzidine, TMB) (ThermoFisher companies) 100 μ l.Lucifuge develops the color 4 minutes, plus 1M sulfuric acid is whole
Only react.With BIO-TEK ELX-800 ELIASAs read plate at 450 nm.The OD450 values of gained are substituted into the gained of embodiment 1
Calibration curve equation, so as to obtain the actual measurement content of HER2 in each sample, HER2 background concentrations in serum are subtracted with actual measurement content
The concentration again than the HER2-ECD standard antigens of upper actual interpolation is the rate of recovery (sample namely after use plus HER2 afterwards
Measured value subtract not plus HER2 measured value, then divided by addition HER2 measure the rate of recovery).
As a result referring to table 2.Average recovery rate is up to 92%.The result shows the kit using the present invention in sandwich method
The satisfactory rate of recovery can be obtained in ELISA detections.
The kit of the present invention of table 2 does serum antigen rate of recovery test result
| Blood serum sample title | Add antigen concentration (ng/ml) | Survey antigen concentration (ng/ml) | The rate of recovery (%) |
| Serum 11 | 125.0 | 109.7 | 88 |
| Serum 29 | 125.0 | 115.4 | 92 |
| Serum 30 | 125.0 | 120.9 | 97 |
| Serum 26 | 125.0 | 110.1 | 88 |
| Serum 24 | 125.0 | 125.9 | 101 |
| Serum 14 | 125.0 | 118.5 | 95 |
| Serum 8 | 125.0 | 110.7 | 89 |
| Serum 4 | 125.0 | 110.8 | 89 |
| Serum 1 | 125.0 | 125.9 | 101 |
| Serum 33 | 125.0 | 101.0 | 81 |
| Serum 3 | 125.0 | 104.8 | 84 |
| Serum 7 | 125.0 | 124.9 | 100 |
| Serum 12 | 125.0 | 115.7 | 93 |
| Serum 19 | 125.0 | 105.5 | 84 |
| Serum 32 | 125.0 | 121.1 | 97 |
| Serum 10 | 125.0 | 124.4 | 100 |
| Serum 6 | 125.0 | 104.4 | 84 |
| Serum 2 | 125.0 | 105.5 | 84 |
| Average recovery rate | 92 |
Embodiment 3, using the kit of embodiment 1 clinical tumor patients serum is detected
For totally 42 parts of the serum sample of examination, these serum samples come from Provincial Hospital's oncology, Anhui Medical University of Science and Technology
Attached First Hospital oncology, the attached First Hospital Oncological Surgery of Bengbu Medical College are learned, donor is voluntary and knows.These are contributed
Person is patient with breast cancer, confirms that tumor tissues HER2 expression is 2+ or 3+ water with SABC through relevant hospital pathology department
Flat, according to foreign study data, this kind of patient has a certain proportion of Serum HER2 height expression, but Chinese population on earth how many compare
The high expression of example and high expression and the correlation of prognosis situation, there is no AUTHORITATIVE DATA at present.The development of this kit helps to fill out
Mend the blank of domestic this respect research.
The humanization HuA21 antibody that step one is prepared to the anti-HER2 of gained is diluted with bed board buffer solution (formula is seen above)
To 2 μ g/ml, 100 μ l bed boards are added per hole, 4 DEG C overnight.Board-washing three times, adds 300 μ l confining liquids (formula is seen above), 37 DEG C per hole
Closing 1 hour.It is with cleaning solution (formula is seen above) board-washing three times, different numberings are dilute for trying blood serum sample serum Special sample
Release liquid (ibid) and dilute 200 times, by each two multiple holes of sample, add in plate, vibrate 1 hour per the μ l of hole 100.Use cleaning solution
(formula is seen above) board-washing three times, plus PBS phosphate buffers (bed board buffer solution) one are prepared the step of be diluted to 0.25 μ g/ml
HerA antibody through biotin labeling, per the μ l of hole 100, vibrates 1 hour.With cleaning solution (formula is seen above) board-washing three times, per hole
Plus 1%, (10g/L) BSA (solvent is PBS) dilutes 8000 times of Horseradish peroxidase-conjugated avidin
(ThermoFisher companies) 100 μ l, vibrate 1 hour.With cleaning solution (formula is seen above) board-washing three times, tetramethyl is added to join per hole
The μ l of aniline (tetramethylbenzidine, TMB) (ThermoFisher companies) 100.Lucifuge develop the color 4 minutes, plus 1M sulphur
Sour terminating reaction.With BIO-TEK ELX-800 ELIASAs read plate at 450 nm.The OD450 values of gained are substituted into the institute of embodiment 1
The calibration curve equation obtained, so as to obtain the content of HER2 in each sample.
Experiment is provided with Siemens Company's clinical detection reagent box (production code member is 00915031) as the present invention simultaneously
The control of kit.Concrete operations and result determination methods are referring to kit specification.
As a result show：In 42 blood serum samples, 9 Siemens Company's clinical detection reagent boxes measure the sample of the positive originally
Invention kit there are 8 to measure Serum HER2 level is substantially higher, trend is basically identical.In addition, all Virus monitories are higher
Sample is HER2 breast cancer patients with positive, illustrates that the degree of accuracy of this kit is very high.But due to there is no Chinese population HER2 at present
Breast cancer patients with positive Serum HER2 expression reference value, so this kit can only enter with the Serum HER2 level of Healthy People
Row is relatively.42 blood serum sample comparative results are shown in Table 3.The sample (Serum 1,3,6,25,28,33,34,41) of overstriking is
The higher sample of kit detection level of the present invention, labeled as italic sample (Serum 1,3,6,15,25,28,33,34,
41) it is that (kit specification thinks that more than 15.2ng/ml is positive to Siemens Company's kit test positive, and this result is
The statistical result of west crowd)., can be with although we lack the statistical data of Chinese population seropositivity critical value
Find out that the relative height of the Serum HER2 content that detects of the present invention and Siemens Company kit are basically identical.
It is contemplated that being compared with the kit of clinic approval after confirmation has a very high reliability, do big in Chinese population
Examination is measured, a strong work is provided for the Study of China human serum HER2 relations for raising and suffering between HER2 height expression tumours
Tool.At present, the corresponding high-level Serum HER2 sample that the present invention is detected is strictly HER2 positive breast cancer patients.The present invention
Another meaning be that it belongs to liquid biopsy category, can accomplish faster, earlier, more convenient, high-throughout general sieve, to breast cancer
Early stage patient has the effect of extraordinary early diagnosis and early warning.This point will could also be demonstrate,proved by following a large amount of clinical sample researchs
It is bright.
The kit of the present invention of table 3 detects the result of clinical patient serum
Claims (7)
1. it is used to detecting the enzyme linked immunological kits of HER2 contents in human serum a kind of, including it is HER2-ECD standard antigens, anti-
HER2 capture antibody and anti-HER2 detection antibody；The capture antibody is humanization HuA21 antibody, and the detection antibody is
HerA antibody through biotin labeling；
The amino acid sequence of the heavy chain of the humanization HuA21 antibody is as shown in sequence 1 in sequence table；The humanization HuA21
The amino acid sequence of the light chain of antibody is as shown in sequence 2 in sequence table；
The amino acid sequence of the heavy chain of the HerA antibody is as shown in sequence 3 in sequence table；The ammonia of the light chain of the HerA antibody
Base acid sequence is as shown in sequence 4 in sequence table；
The amino acid sequence of the HER2-ECD standard antigens is as shown in sequence 5 in sequence table；
The humanization HuA21 antibody is by the humanization by equimolar nucleotide sequence as shown in sequence 7 in sequence table
Humanization HuA21 antibody of the encoding gene and nucleotide sequence of the heavy chain of HuA21 antibody as shown in sequence 8 in sequence table it is light
The encoding gene of chain carries out expressing what is obtained in mammalian cell.
2. enzyme linked immunological kit according to claim 1, it is characterised in that：The HER2-ECD standard antigens are to pass through
HER2-ECD gene of the nucleotide sequence as shown in sequence 6 in sequence table is carried out in mammalian cell expressing acquisition.
3. enzyme linked immunological kit according to claim 1, it is characterised in that：The HerA antibody is by by equimolar
HerA antibody of the nucleotide sequence as shown in sequence 9 in sequence table heavy chain encoding gene and nucleotide sequence such as sequence table
The encoding gene of the light chain of HerA antibody shown in middle sequence 10 carries out expressing what is obtained in mammalian cell.
4. according to any described enzyme linked immunological kit in claim 1-3, it is characterised in that：The enzyme linked immunological kit
In also containing at least one of following：ELISA Plate, bed board buffer solution, cleaning solution, confining liquid, horseradish peroxidase-labeled parent
With element, nitrite ion and terminate liquid.
5. enzyme linked immunological kit according to claim 4, it is characterised in that：The bed board buffer solution is that pH is 7.0
Phosphate buffer；
The phosphate buffer that the cleaning solution is 7.0 for the pH only containing 0.05% volumn concentration Tween 20；
The confining liquid is the only cleaning solution containing 10g/L BSA；
The nitrite ion is tetramethyl benzidine；
The terminate liquid is 1M sulfuric acid.
6. any described enzyme linked immunological kit is preparing to enter for monitoring tumour generation and/or the course of disease in claim 1-5
Application in the product of exhibition.
7. the preparation method of any described enzyme linked immunological kit, comprises the following steps in claim 1-5：
By the way that HER2-ECD gene of the nucleotide sequence as shown in sequence 6 in sequence table is expressed in mammalian cell
Obtain the HER2-ECD standard antigens；
Pass through the coding of the heavy chain of the humanization HuA21 antibody by equimolar nucleotide sequence as shown in sequence 7 in sequence table
The encoding gene of the light chain of the humanization HuA21 antibody of gene and nucleotide sequence as shown in sequence 8 in sequence table is dynamic in lactation
In thing cell express obtaining the humanization HuA21 antibody；
Pass through the encoding gene and core of the heavy chain of the HerA antibody by equimolar nucleotide sequence as shown in sequence 9 in sequence table
The encoding gene of the light chain of HerA antibody of the nucleotide sequence as shown in sequence 10 in sequence table carries out table in mammalian cell
Reach the HerA antibody.
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| CN107860925B (en) * | 2017-11-03 | 2019-07-12 | 合肥瀚科迈博生物技术有限公司 | For detecting the ELISA detection kit of HuA21 antibody in serum |
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| IL275376B2 (en) * | 2013-03-11 | 2024-01-01 | Genzyme Corp | Hyperglycosylated binding polypeptides |
| CN104225594B (en) * | 2014-09-23 | 2016-03-23 | 合肥瀚科迈博生物技术有限公司 | Anti-HER 2 humanized antibody and relevant anti-tumor compositions thereof |
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