CN105866433A - Detection method for NY-ESO-1 autoantibodies in peripheral blood - Google Patents
Detection method for NY-ESO-1 autoantibodies in peripheral blood Download PDFInfo
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Abstract
The invention discloses a detection method for NY-ESO-1 autoantibodies in peripheral blood. The method comprises the following steps: preparing NY-ESO-1 antibodies by taking an NY-ESO-1 recombinant protein as an antigen, coupling the NY-ESO-1 antibodies and latex microspheres to prepare antibody conjugates, enabling a mixed solution a of the NY-ESO-1 recombinant proteins and a peripheral blood sample to be tested to be subjected to mixing reaction for 5 to 10min, adding the antibody conjugates, detecting a light absorption value at 546nm, and obtaining the concentration of the NY-ESO-1 autoantibodies in the sample according to a standard curve. The invention also provides a chemiluminescence detection method for the NY-ESO-1 autoantibodies in the peripheral blood. According to the detection method for the NY-ESO-1 autoantibodies in the peripheral blood, a large sample can be automatically detected on a large-sized automatic instrument such as a biochemical analyzer and a chemiluminescence immunity analyzer, and the sensitivity is 1 to 10ng/mL.
Description
(1) technical field
The present invention relates to the detection method of anti-NY-ESO-1 autoantibody in a kind of peripheral blood.
(2) background technology
NY-ESO-1 is the important member in Cancer-testis antigen (cancer-testis antigen, CTA) family, swollen
Tumor antigen have more strongly immunogenic, wide expression in kinds of tumors, and express the most hardly.NY-
ESO-1 antigen initially from human esophageal carcinoma screening obtain (Chen YT, et al.Cancer J.2000,6 Suppl3:
S208-217.).Hereafter, find that NY-ESO-1 is multiple swollen in neuroblastoma, synovial sarcoma, chromoma, oophoroma etc.
Tumor tissue is all expressed higher, also have to a certain degree in colorectal cancer, liver cancer, bladder transitional cell carcinoma, Huppert's disease, lung cancer
Express (Rodolfo M, et al.Cancer Research.2003,63:6948-6955；Jungbluth AA,et
al.International J Cancer.2001,94:252-256；Barrow C,et al.Clinical Cancer
Research.2006,12:764-771；Odunsi K,et al.Cancer Research.2003,63:6076-6083；
Nakamura S,et al.J Gastroenterology and Hepatology.2006,21:1281-1285.)。NY-
ESO-1 gene family includes tri-members of NY-ESO-1, LAGE-1 and ESO3, is different from the specific table of NY-ESO-1 and LAGE-1
Reaching in tumor tissues, ESO3 wide expression, in the various normal structure of human body, does not have tumour restricted.NY-ESO-1 gene mRNA is compiled
Code section length 543bp, coded protein relative molecular weight about 18kD, it is made up of 180 amino acid, its N end has rich in glycine,
There is hydrophobic amino acid sequence in C end, has potential trans-membrane region.NY-ESO-1 has more than 20 CD4+Or CD8+T cell identification table
Position, and in kinds of tumors, can detect that spontaneous immune response (Chen JL, et al.International J
Cancer.2015,136:E590-601.).Have been found that 155-163 amino acids (QLSLLMWIT), 157-165 bit amino
Acid (SLLMWITQC) and 3 Antigenic Peptide of 157-167 amino acids (SLLMWITQCFL) can induce NY-ESO-1 positive tumor
Patient produces ctl response, can be applicable to immunotherapy of tumors (Karbach J, et al.International J
Cancer.2007,121:2042-2048.).NY-ESO-1, as the stronger CTA of immunogenicity, can activate CD4+And CD8+T drenches
Bar cell, induction body produces the cell for tumour and HI.T lymphocyte identification table in NY-ESO-1 peptide chain
Position is main based on the restricted Antigenic Peptide of HLA-A2 and HLA-DR.Do not express HLA molecule due to reproduction cell and blood-testis barrier is deposited
, the immunotherapy of tumors general normal tissue cell for CTA does not produce immunotoxicity, and current NY-ESO-1 is considered as
The preferable target antigen of immunotherapy of tumors.
NY-ESO-1, as a kind of important tumor associated antigen, at early diagnosis of tumor, develops, and result for the treatment of is commented
The aspects such as valency also have important value.Owing to NY-ESO-1 expresses in kinds of tumors tissue, and the most in the normal tissue
Expressing, it has relatively high specific in diagnosing tumor, and NY-ESO-1 judges the most significant at tumor prognosis, research
Find tumor cells expression NY-ESO-1 prompting prognosis poor (Ohue Y, et al.Oncoimmunology.2014,3:
e970032.).In medullary carcinoma of breast patient, estrogen receptor negative person's tumor tissues NY-ESO-1 positive rate is higher than estrogen
Receptor positive person, NY-ESO-1 positive patient is less than NY-ESO-1 negative patient (Ademuyiwa FO, et al.PloS life cycle
one.2012,7:e38783.).Also research is had to think that NY-ESO-1 is it after G. cephalantha patient is carried out multiplicity
The individual index (Laban S, et al.International J Cancer.2014,135:1142-1152.) of poor prognosis.
CTA participates in initial stage embryonic development and stem cell self, and it is expressed in tumor tissues and is confined to have stem cell spy
Property cell, CT gene expression cell be probably abnormal tumor stem cell clone propagation result.The tumour cell expressing CTA loses
Remove differentiation capability, and tumor recurrence and transfer (Colombo M, et al.International Reviews may have been participated in
Immunology.2015,34:188-199.).In tumor stem cell, CT gene expression has been applied to controlling of tumor recurrence and transfer
Treat.Research is had to isolate tumor stem cell, compared to noble cells, tumor stem cell from glioma cell line and tissue
Middle NY-ESO-1 expresses notable rising.Additionally, NY-ESO-1 is also one of non-small cell lung cancer prognostic marker (John T, et
al.PloS one.2013,8:e67876.)。
Owing to NY-ESO-1 is a sequestered antigen, can effectively induce again cell and the HI of body simultaneously,
The NY-ESO-1 antigen that the tumour cell expressing NY-ESO-1 discharges expression due to downright bad or secretion mode enters blood, stimulates body to exempt from
Epidemic disease cell produces the repeats itself antibody for NY-ESO-1, therefore, by specific NY-ESO-1 in detection peripheral blood certainly
Body circulating antibody can be as a tumor markers, for tumor screening, auxiliary diagnosis and Index for diagnosis, it is judged that tumour cell table
Reach NY-ESO-1 antigen status it can also be used to monitor and evaluate the tumor patient cell reactivity to immunization therapy.
(3) summary of the invention
It is an object of the present invention to provide the assay method of anti-NY-ESO-1 autoantibody in the body fluid such as a kind of human serum, be i.e. used for
Anti-NY-ESO-1 autoantibody (including IgG, IgA and IgM antibody) in the body fluid such as peripheral blood (including serum, blood plasma and whole blood)
Detection.The present invention provides a kind of new tumor markers for tumor high-risk diagnosis, is also the essence of NY-ESO-1 positive tumor
Quasi-treatment and/or the personalized immunization therapy Outcome measure such as CAR-T, TCR-T provide foundation.
The technical solution used in the present invention is:
The present invention provides the detection method of anti-NY-ESO-1 autoantibody in a kind of peripheral blood, and described method is: with NY-
ESO-1 recombinant protein is that antigen prepares anti-NY-ESO-1 antibody, and prepared by anti-NY-ESO-1 antibody and latex microsphere coupling antibody
Conjugate, then by NY-ESO-1 recombinant protein mixed liquor a and peripheral blood testing sample hybrid reaction 5-10min, adds the most again
Enter antibody coupling matter, light absorption value at detection 546nm, obtain anti-NY-ESO-1 autoantibody concentrations in sample according to calibration curve；
Described NY-ESO-1 recombinant protein mixed liquor a quality group becomes: the poly-second of NY-ESO-1 recombinant protein 100-200ng/ml, 35g/L
Glycol-6000, bovine serum albumin(BSA) (BSA) 5g/L, 0.05%Tween-20, NaN30.05%, solvent is the buffering of pH6.8
Liquid；Described calibration curve is prepared as follows: be that antigen immune rabbit prepares the anti-NY-ESO-of rabbit with NY-ESO-1 recombinant protein
1 specific IgG antibodies, more anti-for rabbit NY-ESO-1 specific IgG antibodies is pressed 1000-62.5 μ g/ml gradient concentration with containing volume
The buffer solution of concentration 0.1-1% bovine serum albumin(BSA) is diluted to dilution, with without antibody composition containing described bovine serum albumin(BSA)
Buffer solution be blank, specific antibody is prepared by anti-for rabbit NY-ESO-1 specific IgG antibodies and latex microsphere coupling even
Connection thing, then by NY-ESO-1 recombinant protein mixed liquor b and variable concentrations dilution hybrid reaction 5-10min, finally adds
Specific antibody conjugate, measures light absorption value at 546nm, respectively with light absorption value as ordinate, with the anti-NY-ESO-of rabbit in dilution
1 specific IgG antibodies concentration makes calibration curve as abscissa；Described NY-ESO-1 recombinant protein mixed liquor b forms same NY-
ESO-1 recombinant protein mixed liquor a (NY-ESO-1 recombinant protein mixed liquor a, NY-ESO-1 recombinant protein mixed liquor of the present invention
B is NY-ESO-1 recombinant protein mixed liquor, names for ease of distinguishing different step consumption difference, and letter does not contain itself
Justice).
Further, described latex microsphere is with a diameter of 40-230nm, and microsphere surface activated group is-COOH or-NH2Matter
The form of amount concentration 10% latex solution adds, preferably purchased from Bangs company of the U.S..
Further, described antibody coupling matter preparation method is: (1) latex microsphere activates: by latex microsphere and pH5.0-
7.5,0.01-0.5mol/L MES buffer solution (preferably pH6.5,0.05mol/L), water, Tween-20 mixing, vortex oscillation mixes,
Prepare latex mixed liquor；In described latex mixed liquor, add the 50mg/mL NHS aqueous solution and 10mg/mL carbodiimide is water-soluble
Liquid, vortex oscillation 15-45 minute, more ultrasonic 5-15 time, each ultrasonic 5-10 second, it is spaced 15-30 second, ultrasonic energy 6000-
9000kJ (the most ultrasonic 5 times, the most ultrasonic 10 seconds, be spaced 15 seconds, ultrasonic energy 9000kJ)；Mixed liquor mistake after ultrasonic end
Sephadex G-25 sephadex column, washes with water to 2 times that final volume is initially use latex volume, it is thus achieved that the glue of activation
Emulsion；Described latex microsphere is with diameter 40-230nm, and microsphere surface activated group is-COOH or-NH210wt% latex solution shape
Formula adds；Described latex microsphere and described buffer solution volume ratio are 0.4:1, and described water with described buffer solution volume ratio is
1.5:1, described Tween-20 and described buffer solution volume ratio are 1-2:100 (preferably 2:100)；The described NHS aqueous solution is with described
Buffer solution volume ratio be 1:10-20 (preferably 1:16), the described carbodiimide aqueous solution and described buffer solution volume ratio are 1:
10-20 (preferably 1:16)；(2) coupling: anti-NY-ESO-1 antibody is dissolved in pH7.4,100-200mM PBS (preferably
Make antibody-solutions in 100mM), after latex solution mixing antibody-solutions and step (1) activated, react 1-in vertical mixed instrument
4 hours (preferably 4h)；Add pH6.8-8.0,0.5-4.0mol/L glycine buffer (preferably pH7.4,1mol/L) in
Vertical mixed instrument mixing 10-60 minute (preferably 60min), is centrifuged 30-120 minute (preferably by reactant liquor 15000-30000rpm
20000rpm is centrifuged 30 minutes), abandon supernatant, retain precipitation, by the resuspended precipitation of cleaning solution, 15000-30000rpm is centrifuged 15-120
Minute (preferably 20000rpm is centrifuged 30 minutes), repeats resuspended and centrifugal 3-5 time, takes last centrifugal precipitation and be antibody coupling
Thing；Described cleaning solution refers to that pH7.4,50-500mM glycine buffer containing volumetric concentration 0.1% bovine serum albumin(BSA) is (excellent
Select pH7.4,100mM)；The latex solution volumetric usage of described activation is calculated as 0.1-1.0ml/mg with anti-NY-ESO-1 antibody mass.
Further, described calibration curve is prepared as follows: be antigen immune rabbit system with NY-ESO-1 recombinant protein
Standby rabbit anti-NY-ESO-1 specific IgG antibodies, then by anti-for rabbit NY-ESO-1 specific IgG antibodies with containing volumetric concentration 0.25% N
Sero-abluminous buffer solution is diluted to 1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, the dilution of 62.5 μ g/ml
Liquid, with the buffer solution containing described bovine serum albumin(BSA) without antibody composition as blank, by anti-for rabbit NY-ESO-1 specific IgG
Antibody and latex microsphere coupling prepare specific antibody conjugate, then by dense from different for NY-ESO-1 recombinant protein mixed liquor b
Degree dilution hybrid reaction 5-10min, finally adds specific antibody conjugate, measures light absorption value at 546nm respectively, to inhale
Light value is ordinate, makes calibration curve using rabbit anti-NY-ESO-1 specific IgG antibodies concentration as abscissa；Described restructuring egg
White mixed liquor b quality group becomes: NY-ESO-1 recombinant protein 100-200ng/ml, 35g/L PEG-4000, ox blood is pure
Albumen 5g/L, 0.05%Tween-20, NaN30.05%, solvent is the Tris-HCl buffer solution of pH6.8,50mM.
Further, described anti-NY-ESO-1 autoantibody is one of following: anti-NY-ESO-1 autoantibody IgG, anti-NY-
ESO-1 autoantibody IgA or anti-NY-ESO-1 autoantibody IgM, the most anti-NY-ESO-1 autoantibody IgG.
Additionally, the present invention also provides for detection method (the i.e. chemiluminescence of anti-NY-ESO-1 autoantibody in a kind of peripheral blood
Method detects), described method: using NY-ESO-1 recombinant protein is envelope antigen, adds testing sample, add anti-human igg and resist
Body is directly and 2', 6'-dimethyl-carbonyl phenyl 10-methyl-9-acridine formic acid esters 4'-N-succinimide ester trifluoromethane sulfonic acid
Labelled antibody prepared by salt (being called for short acridinium ester DMAE-NHS) (CAS:115853-74-2) coupling, adds substrate solution A and substrate solution
B carries out luminescence-producing reaction, detects reactant liquor luminous intensity, according to people's anti-NY-ESO-1IgG antibody calibration curve, it is thus achieved that testing sample
In anti-NY-ESO-1 autoantibody concentrations；Described substrate solution A consists of: mass concentration 0.1-0.25% carbamide peroxide, volume
Concentration 0.01%Triton X-100, solvent is 100mM, pH8.0Tris buffer solution, and substrate solution B consists of: 100mM NaOH,
The Triton X-100 of volumetric concentration 2%, solvent is water；The amount of described anti-human IgG antibodies and acridinium ester DMAE-NHS material it
Ratio is 1:3-5；Described anti-human IgG antibodies is goat anti-human igg antibody or rabbit anti-human igg's antibody；Described calibration curve is by such as lower section
Prepared by method: the people anti-NY-ESO-1IgG antibody 100mM containing volumetric concentration 0.25%BSA, the Tris-HCl buffer solution of pH7.4
It is diluted to 1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, the dilution of 62.5 μ g/ml, examines as this chemiluminescence
The calibration object of survey method or standard items, with the buffer solution containing described bovine serum albumin(BSA) without antibody composition as blank, use
NY-ESO-1 recombinant protein is envelope antigen, is separately added into described variable concentrations calibration object or standard items, adds anti-human igg and resists
The labelled antibody that body is directly prepared with acridinium ester DMAE-NHS coupling, adds substrate solution A and substrate solution B and carries out luminescence-producing reaction, inspection
Measured reaction liquid luminous intensity, with people's anti-NY-ESO-1IgG AC as abscissa, luminous intensity is ordinate, draws standard
Curve；Described substrate solution A and substrate solution B composition is with testing sample detection substrate solution A and substrate solution B used；The anti-NY-of described people
ESO-1IgG preparation method for antibody is: collect the human serum of anti-NY-ESO-1 Autologous IgG antibody positive, recombinates egg with NY-ESO-1
-Sepharose 4B affinity column purifies the specific antibody of anti-NY-ESO-1 recombinant protein in vain, dilute after quantification of protein
Release.
Further, preferred carbamide peroxide mass concentration 0.1%.
Latex immunoenhancement reaction principle is: first with NY-ESO-1 recombinant protein coupling latex microsphere, then with being somebody's turn to do
The latex microsphere of coupling recombinant protein combines with the antibody in sample, occurs that because of agglutinating reaction turbidity changes, and can pass through
Detect turbidity intensity of variation and speculate the autoantibody in sample.
Latex Immune-enhancing effect suppression method reaction principle is: be first that the antibody in sample is anti-with the NY-ESO-1 that excess is added
Primary life is specific binding, then detects the NY-ESO-of residual ionization with the latex microsphere of anti-NY-ESO-1IgG antibody coupling
1 antigen.NY-ESO-1 amount of antigen owing to adding is known, therefore, can come according to the NY-ESO-1 amount of antigen of residual ionization
Speculate the autoantibody in sample.This reaction pattern is A competitive inhibition method, the highest.
Anti-NY-ESO-1 autoantibody in ELISA and chemoluminescence method detection sample uses indirect method.
NY-ESO-1 recombinant protein of the present invention be prepared as approach well known, preferably by PCR method by NY-
ESO-1 DNA sequence dna is connected to protokaryon/eukaryotic expression vector, with E.coli DE3 and/or HEK293 or Chinese hamster ovary celI table
Reach, produce and purify NY-ESO-1 recombinant protein.
The detection method of anti-NY-ESO-1 autoantibody of the present invention, it is also possible to using NY-ESO-1 recombinant protein as bag
By antigen, with sheep or rabbit anti-human igg's antibody and horseradish peroxidase or acridinium ester conjugate as tracer antibody, set up indirect method
Anti-NY-ESO-1 autoantibody detection method in the body fluid such as heterogeneous serum.
Compared with prior art, the present invention has the advantages that: anti-NY-ESO-1 in peripheral blood of the present invention
The detection method of autoantibody can be carried out greatly on the large automatic instrument such as Biochemical Analyzer and chemical illumination immunity analysis instrument
Sample Aulomatizeted Detect.The inventive method detection sensitivity 1-10ng/mL.
(4) accompanying drawing explanation
Fig. 1 is reaction principle schematic diagram, and A is that latex immunoenhancement detects the signal of anti-NY-ESO-1 autoantibody principle
Figure, B is that latex Immune-enhancing effect suppression method detects anti-NY-ESO-1 autoantibody principle schematic, and C is that ELISA method detects anti-NY-
ESO-1 autoantibody principle schematic, D is that chemoluminescence method detects anti-NY-ESO-1 autoantibody principle schematic；
Fig. 2 is reaction normal curve, and A is that latex immunoenhancement detects anti-NY-ESO-1 autoantibody calibration curve, and B is
Latex Immune-enhancing effect suppression method detects anti-NY-ESO-1 autoantibody calibration curve, and C is that ELISA method detects anti-NY-ESO-1 self
Antibody calibration curve, D is that chemoluminescence method detects anti-NY-ESO-1 autoantibody calibration curve.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
Embodiment 1
Prepared by 1.NY-ESO-1 recombinant protein
1.1 NY-ESO-1 DNA sequence dnas obtain
1.1.1 from the tumour cell that NY-ESO-1 expresses, obtain NY-ESO-1 DNA sequence dna
From tumour cell, NY-ESO-1 positive cell strain is screened by antigen in rinsed fluid method, 2 × 106Express NY-ESO-1
Tumour cell (such as A375 MC and MCF7 breast cancer cell line, the present embodiment selects A375 MC)
With RNAeasy kit (purchased from Qiagen) isolated and purified total serum IgE, through Oligo dT15Primer (purchased from Takara) is inverse by total serum IgE
It is transcribed into cDNA, is therefrom expanded acquisition NY-ESO-1 DNA sequence dna (nucleotide sequence by PCR (PCR) method
Shown in SEQ ID NO.1, amino acid sequence is shown in SEQ ID NO.2), DNA sequencing is verified.
1.1.2 chemical synthesis NY-ESO-1 DNA sequence dna
The NY-ESO-1 DNA sequence dna (protein ID:P78358) provided according to Genbank, entrusts commercial company
(nucleotides sequence is classified as shown in SEQ ID NO.1, and amino acid sequence to learn by SEQ ID NO.2 synthesis NY-ESO-1 DNA sequence dna
Show), and it is cloned into suitable Clonal carrier (such as pUC19, pUC57 etc., the present embodiment selects pUC57).
1.2 pass through procaryotic cell expression NY-ESO-1 recombinant protein
With step 1.1 obtain NY-ESO-1 DNA as template, with PCR (PCR) by NY-ESO-1 DNA
Open reading nucleotide sequence (ORF) is cloned into the NdeI/BamHI restriction endonuclease of procaryotic cell expression carrier pET15b or pET14b
Site, after DNA sequencing checking, converts Rosetta DE3 Escherichia coli, selects the bacterial clone of high expressed NY-ESO-1, expand
When cultivating to OD value to 0.4-0.8, inducing 4 hours at 37 DEG C with 1.0mM IPTG, 10000rpm is centrifuged 15 minutes and collects bacterium
Precipitation.
NY-ESO-1 renaturation and purifying: illustrate as a example by processing 150g bacterial precipitation.150g bacterial precipitation is added
1.5L inclusion body lysate I (pH8.0,50mM Tris-HCl buffer solution contains 1mM EDTA and 100mM NaCl) laggard horizontal high voltage
Ultrasonication (pressure 10MPa, supersonic frequency 20000Hz, ultrasonically treated 5min), centrifugal, precipitation 700ml inclusion body lysate
II (pH8.0,50mM Tris-HCl buffer solution contains 1mM EDTA, 100mM NaCl and 0.5%Triton X-100) washes
Washing, wash 2 times, 10000rpm is centrifuged 10min, and the precipitation of generation 0.5M urea (uses pH8.0,50mM Tris-HCl buffer solution
Prepare containing 1mM EDTA, 100mM NaCl) wash 1 time, 10000rpm is centrifuged 10min.Precipitation 200ml, 8M urea (is used
PH8.0,50mM Tris-HCl buffer solution is prepared containing 1mM EDTA, 100mM NaCl) resuspended, after magnetic agitation 30min,
10000rpm is centrifuged 10min, supernatant (200ml) pH 10.7,50mM K2HPO4+ 1mM EDTA+50mM NaCl solution dilutes
10 times, standing 30min, then adjust pH to 8.0 with 2M HCl after adjusting pH10.7 with 1M NaOH, 10000rpm is centrifuged 10min, by upper
Clearly (2L) loading bag filter carries out renaturation dialysis.Dislysate be pH8.0,50mM Tris-HCl buffer solution containing 1mM EDTA,
100mM NaCl, 10mM reduced glutathione, 2mM oxidized form of glutathione and 1mM dithiothreitol (DTT) (DTT).4 DEG C of renaturation are saturating
Analysis 48h, then dialyses with pH8.0,50mM Tris-HCl buffer solution NaCl Han 100mM, and mistake nickel post after 3 times of dialysing is carried out
Purifying, eluent is pH8.0,50mM Na2HPO4Buffer solution includes 150mM NaCl and 300mM imidazoles.Pure with AKTA further
Changing recombinant protein peak, Lorry ' s phenol reagent carries out quantification of protein.
1.3 eukaryotics produce NY-ESO-1 recombinant protein
The NY-ESO-1 DNA obtained with step 1.1, as template, is cloned into the eukaryotic expression vector pcDNA3.1 (U.S.
Invitrogen company), transfect the engineering cell Chinese hamster ovary celI (U.S. by the method (particle gun or liposome) of physical transfection
ATCC company), 5%CO2In bioreactor, serum-free medium (Gibco company of the U.S.) is cultivated 7-10 days, collects supernatant,
Concentrate, with AKTA purification of recombinant proteins peak.Lorry ' s phenol reagent carries out quantification of protein.
The most anti-NY-ESO-1 recombinant protein rabbit source property preparation method of polyclonal antibody
2.1 animal immune
With the NY-ESO-1 recombinant protein of 1.2 or 1.3 preparations, with 1mg/ time/NY-ESO-1 recombinant protein and complete good fortune
Family name's adjuvant equal-volume mixes, the immunity of rabbit subcutaneous multi-point injection.Every 2 weeks 1 time, continuous 5 times, separation neck after being spaced 1 week, is used to move
Arteries and veins, collect blood about 80-120 milliliter, room temperature place 2 hours, 4 DEG C, 10000rpm be centrifuged 30min, separate and collect serum.
2.2 antibody purification
First prepare NY-ESO-1 recombinant protein-Sepharose 4B affinity column: take step 1.2 or the nothing of 1.3 preparations
The Sepharose 4B 2g that high-purity (90%) the NY-ESO-1 recombinant protein 25mg that free amine group pollutes activates with hydrogen bromide
(purchased from Pharmacia) cross-links, and cross-links by operational manual, it is thus achieved that NY-ESO-1 recombinant protein-Sepharose
4B affinity column.The most above-mentioned serum (step 2.1 is collected) NY-ESO-1 recombinant protein-Sepharose 4B affinity chromatography
Post purifies the specific IgG type antibody of anti-NY-ESO-1 recombinant protein, finally detects purity with 8%SDS-PAGE, need to reach
90%, it is thus achieved that rabbit anti-NY-ESO-1 specific IgG antibodies.IgG PBS is diluted to that < 1mg/ml enters by ultraviolet spectrophotometry
Row is quantitatively.IgG measures (mg/ml)=(1.42 × OD280nm-0.74×OD403nm) × extension rate.
Specific binding reaction is there is in anti-NY-ESO-1 Anti-TNF-α physical efficiency prepared by the present embodiment with NY-ESO-1 albumen,
And not with non-NY-ESO-1 albumen generation nonspecific reaction.
2.3 rabbit anti-NY-ESO-1 specific IgG antibodies is as calibration object or standard items
Above-mentioned steps 2.2 obtain rabbit anti-NY-ESO-1 specific IgG antibodies after quantification of protein with containing volumetric concentration
The Tris-HCl buffer solution of the 100mM of 0.25%BSA, pH7.4 be diluted to containing rabbit anti-NY-ESO-1IgG antibody 1000,500,
250,125,62.5 μ g/ml dilution not etc., as calibration object or the standard items of this latex detection method.
Above-mentioned steps 2.2 obtain rabbit anti-NY-ESO-1 specific IgG antibodies after quantification of protein with containing 10% human blood
The Tris-HCl buffer solution of clear 100mM, pH7.4 is diluted to containing rabbit anti-NY-ESO-1 specific IgG antibodies 700,400,100 μ
The dilution that g/ml does not waits, as the quality-control product of this latex detection method.
2.4 people anti-NY-ESO-1 autoantibody is as calibration object or standard items
Collect the serum 100mL of anti-NY-ESO-1 Autologous IgG antibody positive, with NY-ESO-1 recombinant protein-Sepharose
4B affinity column purifies the specific antibody of anti-NY-ESO-1 recombinant protein, finally detects purity with 8%SDS-PAGE, need to reach
To 90%, it is thus achieved that people's anti-NY-ESO-1 autoantibody.Lorry ' s phenol reagent carries out quantification of protein.Use after quantification of protein
The Tris-HCl buffer solution of the 100mM containing volumetric concentration 0.25%BSA, pH7.4 is diluted to containing anti-NY-ESO-1 autoantibody
1000,500,250,125,62.5 μ g/ml dilution not etc., as calibration object or the standard items of this latex detection method.
3. anti-NY-ESO-1 repeats itself antibody during latex immunoenhancement detects the body fluid such as human serum
3.1 latex Immune-enhancing effect suppression methods
Latex microsphere crosslinking rabbit source property polyclone IgG antibody, says as a example by 1% latex-antibody of activation crosslinking 4mL
Bright.
3.1.1 latex activation
3.1.1.1 0.05mol/L MES buffer solution (pH6.5) 1ml, diameter 40-230nm, microsphere surface activated group
For 10% latex (Bangs company of the U.S.) 0.4ml, water 2.1mL, the 20 μ l Tween-20 of-COOH, vortex oscillation mixes.
3.1.1.2 50mg/mL NHS aqueous solution 0.25ml, 10mg/mL carbodiimide (EDAC) aqueous solution 0.25mL are added,
Vortex oscillation 30 minutes.
3.1.1.3 ultrasonic 5 times of mixed liquor, the most ultrasonic 10 seconds, are spaced 15 seconds, ultrasonic energy 9000kJ.Cross Sephadex
G-25 sephadex column (purchased from Pharmacia), washes with water to final volume 10-20ml, it is thus achieved that the latex solution of activation.
The optimization that the most anti-NY-ESO-1 specific IgG antibodies cross-links with latex
3.1.2.1 the latex solution 1.0ml of above-mentioned steps 3.1.1 activation, respectively with 0.1,0.2,0.4,0.6,0.8,1.0,
1.2,1.4,1.6,1.8,2.0mg rabbit anti-NY-ESO-1 specific IgG antibodies (is dissolved in 1.0ml pH7.4,100mM PB slow respectively
Rush in liquid) mixing after, react 4 hours in vertical mixed instrument (Ningbo medical instrument companies of Toshiba).
3.1.2.2 it is separately added into 1.0mol/L glycine buffer (pH7.4) 1ml and mixes 60 points in vertical mixed instrument
Clock, to close the active group of residual, it is thus achieved that antibody-latex coupling liquid.
3.1.3 the separation of cross-linking agent
3.1.3.1 the antibody that prepared by above-mentioned steps 3.1.2.2-latex coupling liquid 20000rpm is centrifuged 30 minutes, abandons supernatant,
Retain precipitation.
3.1.3.2 with the resuspended precipitation of cleaning solution 40ml (cleaning solution: pH7.4,100mM glycine buffer includes 0.1%
BSA), 20000rpm is centrifuged 30 minutes.
3.1.3.3 repeat 3.3.2 step 3-5 time.
The most anti-NY-ESO-1 specific IgG antibodies and the preservation of latex cross-linking agent
The latex precipitation that above-mentioned steps 3.1.3.3 obtains adds 4ml containing 0.25%BSA, 0.3%PVP, 0.05%NaN3
PH7.4,500mM glycine buffer, ultrasonic (9000J) 15 seconds mixing, 4 DEG C of preservations.
3.2 latex immunoenhancements
Latex microsphere crosslinking NY-ESO-1 recombinant protein, is carried out as a example by the 1% latex-recombinant protein of activation crosslinking 4mL
Explanation.
3.2.1 latex activation
10% latex (the U.S. of 3.2.1.1 0.05mol/L MES buffer solution (pH6.5) 1ml, diameter 80-400nm
Bangs company) 0.4ml, water 2.1mL, Tween-20 20 μ l, vortex oscillation mixes.
3.2.1.2 50mg/mL NHS aqueous solution 0.25ml, 10mg/mL carbodiimide (EDAC) aqueous solution 0.25mL are added,
Vortex oscillation 30 minutes.
3.2.1.3 ultrasonic 5 times of mixed liquor, the most ultrasonic 10 seconds, are spaced 15 seconds, ultrasonic energy 9000kJ.Cross Sephadex
G-25 sephadex column (purchased from Pharmacia), washes with water to final volume 10-20ml, it is thus achieved that the latex solution of activation.
3.2.2 the optimization that NY-ESO-1 recombinant protein cross-links with latex
3.2.2.1 the latex solution 1.0ml of above-mentioned steps 3.2.1 activation, respectively with 0.1,0.2,0.4,0.6,0.8,1.0,
1.2,1.4,1.6,1.8,2.0mg NY-ESO-1 recombinant proteins (step 3.1.4) (are dissolved in 1.0ml pH7.4,100mM respectively
In PB buffer solution) mixing after, in vertical mixed instrument react 4 hours.
3.2.2.2 it is separately added into 5mol/L glycine buffer (pH7.4) 1ml to mix 60 minutes in vertical mixed instrument,
To close the active group of residual, it is thus achieved that NY-ESO-1 recombinant protein-latex mixed liquor.
3.2.3 the separation of cross-linking agent
3.2.3.1 NY-ESO-1 recombinant protein-latex mixed liquor 20000rpm that above-mentioned steps 3.2.2.2 obtains is centrifuged 30
Minute, abandon supernatant, retain precipitation.
3.2.3.2 with the resuspended precipitation of cleaning solution 40ml (cleaning solution: pH7.4,500mM glycine buffer includes 0.1%
BSA), 20000rpm is centrifuged 30 minutes.
3.2.3.3 repeat 3.3.2 step 3-5 time.
3.2.4 NY-ESO-1 recombinant protein and the preservation of latex cross-linking agent
The latex precipitation that above-mentioned steps 3.2.3.3 obtains adds 4ml containing 0.25%BSA, 0.3%PVP, 0.05%NaN3
PH7.4,100mM glycine buffer, ultrasonic 15 seconds mixing, 4 DEG C of preservations.
3.3 anti-NY-ESO-1 specific IgG antibodies/NY-ESO-1 recombinant proteins cross-link the determination of optium concentration with latex
3.3.1 main agents constituent
The suppression method detection of latex Immune-enhancing effect
Trishydroxymethylaminomethane (Tris-HCl) buffer solution of reagent R1:50mM, pH6.8 contains:
PEG(MW 6000)35g/L；BSA 5g/L；NY-ESO-1 recombinant protein 200 ng/mL；
Tween-20 0.05%；NaN30.05%.
The latex microsphere cross-linking anti-NY-ESO-1 specific IgG antibodies that reagent R2: step 3.1 obtains.
Latex immunoenhancement detects
Trishydroxymethylaminomethane (Tris-HCl) buffer solution of reagent R1:50mM, pH6.8 contains:
PEG(MW 6000)35g/L；BSA5g/L；Tween-20 0.05%；NaN30.05%.
The latex microsphere of the crosslinking NY-ESO-1 recombinant protein that reagent R2: step 3.2 obtains.
3.3.2 detecting step
Reaction type: performance rate method temperature: 37 DEG C of cuvette optical path: 0.6cm
Master/slave wavelength: 546nm/800nm unit: U/ml the Direction of Reaction: rise
3.3.3 result determines:
Calculate the difference (A calibration-A is blank) of calibration object absorbance, set up suitable mathematical modulo (non-linear) such as Logit-
Log etc., fit to the calibration curve of multiple spot calibration.Reactions change amplitude according to each latex calibration curve, the range of linearity, relevant
Coefficient and reaction repeatability, determine that optimal antibody with latex ratio is: 1mg antibody: 0.1-1.0ml latex (10%)；Live
The latex solution volumetric usage changed is calculated as 0.1-1.0ml/mg with anti-NY-ESO-1 antibody mass.
3.4 peripheral blood anti-NY-ESO-1 autoantibody detects
3.4.1 main agents constituent
Latex Immune-enhancing effect suppression method
Trishydroxymethylaminomethane (Tris-HCl) buffer solution of reagent R1:50mM, pH6.8 contains:
PEG(MW 6000)35g/L；BSA 5g/L；NY-ESO-1 recombinant protein 200 ng/mL；
Tween-20 0.05%；NaN30.05%.
The latex microsphere cross-linking anti-NY-ESO-1 specific IgG antibodies that reagent R2: step 3.1 obtains.
Latex immunoenhancement
Trishydroxymethylaminomethane (Tris-HCl) buffer solution of reagent R1:50mM, pH6.8 contains:
PEG(MW 6000)35g/L；BSA 5g/L；Tween-20 0.05%；NaN30.05%.
The latex microsphere of the crosslinking NY-ESO-1 recombinant protein that reagent R2: step 3.2 obtains.
3.4.2 detecting step
Reaction type: 2 method temperature of set time: 37 DEG C of cuvette optical path: 1.0cm
Master/slave wavelength: 546nm/800nm unit: U/ml the Direction of Reaction: rise
3.4.3 result calculates:
Calculate the difference (A calibration-A is blank) of calibration object absorbance, set up suitable mathematical modulo (non-linear) such as Logit-
Log etc., fit to the calibration curve of multiple spot calibration.According to the absorbance difference of sample (A sample-A is blank), on working curve
Trying to achieve the content of anti-NY-ESO-1 antibody in sample, calibration curve is as shown in Figure 2.
Latex immunoturbidimetry prepared by the present embodiment is for detecting peripheral blood or serum anti-NY-ESO-1 autoantibody water
Flat, when in sample containing NY-ESO-1, the latex microsphere of crosslinking specific antibody or antigen can be due to antigen and antibody specific
In conjunction with there is aggegation, causing turbidity to increase, changing by measuring sample absorbance, reference standard curve, in quantitative analysis sample
Anti-NY-ESO-1 autoantibody content, detection usefulness and enzyme linked immunosorbent assay (ELISA), chemiluminescence immunoassay detection method are close
Or equivalence.The results are shown in Table shown in 1.
4. anti-NY-ESO-1 autoantibody during enzyme linked immunosorbent assay (ELISA) detects the body fluid such as human serum
4.1 biotin labeling NY-ESO-1 recombinant protein
10mg NY-ESO-1 recombinant protein (step 1.1 or 1.2 preparations) is dissolved in 1ml 0.01M carbonate buffer solution,
Add 5mg NHS activated biotin, after room temperature lucifuge is gently mixed 1-2 hour, loads in bag filter, use 0.15M pH7.4 for 4 DEG C
PBS, after changing liquid 4-6 time, 10000-20000rpm is centrifuged 15-30min and goes precipitation.Add final concentration 30% glycerine-20 DEG C.
Biotinylation NY-ESO-1 recombinant protein is most preferably coated concentration and determines.Dilute with the carbonate buffer solution (CBS) of 50mM
Release Streptavidin to after 10 μ g/mL, be coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, with containing 0.05%
Phosphate buffer (PBS) or the Tris-HCl buffer solution (PBS) of Tween-20 wash 3 times, by 10% calf serum room temperature envelope
Close 30min, add through with the phosphate buffer (PBS) containing 0.05%Tween-20 and 5% calf serum or Tris-HCl
The biotinylation NY-ESO-1 recombinant protein 100 μ L/ hole of buffer solution (TBS) 1:1000 dilution, room temperature reaction 30-60min, with containing
Phosphate buffer (PBS) or the Tris-HCl buffer solution (TBS) of 0.05%Tween-20 wash 3 times, add containing 0.05%
The rabbit of the phosphate buffer (PBS) of Tween-20 and 5% calf serum or Tris-HCl buffer solution (TBS) 1:3000 dilution resists
NY-ESO-1 recombinant protein IgG antibody 100 μ L/ hole, room temperature reaction 30-60min, delays with the phosphate containing 0.05%Tween-20
Rushing liquid (PBS) or Tris-HCl buffer solution (PBS) washs 3 times, the HRP adding 100 μ L/ holes marks the goat anti-rabbit igg (U.S.
Jackson company) room temperature reaction 15-30min, with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl
Buffer solution (PBS) washs 3 times, is eventually adding the colour developing of tetramethyl biphenyl ammonia (TMB) substrate and observes.So that obvious blue reacting hole to occur
Highest dilution hole be optimal diluted concentration.Present invention discover that biotinylation NY-ESO-1 recombinant protein be most preferably coated concentration with
1 μ g/mL is preferred.
4.2 are coated and close
After the carbonate buffer solution dilution NY-ESO-1 recombinant protein of 50mM to 5 μ g/mL, it is coated reaction with 100 μ L/ holes
Micropore, 4 DEG C of overnight or 37 DEG C of 120min, buffer with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl
Liquid (TBS) washs 3 times, closes 30min by 10% calf serum room temperature, dries, and can directly carry out next step operation, it is possible to through filling
Dividing after dehydrating, sealing is put 4 DEG C and can be preserved for a long time.
When using biotin-avidin system (step 4.1), dilute Streptavidin extremely with the carbonate buffer solution of 50mM
After 10 μ g/mL, being coated reaction micropore with 100 μ L/ holes, 4 DEG C of overnight or 37 DEG C of 120min, with the phosphoric acid containing 0.05%Tween-20
Salt buffer (PBS) or Tris-HCl buffer solution (TBS) wash 3 times, close 30min by 10% calf serum room temperature, dry, warp
The most dried, sealing is put 4 DEG C and can be preserved for a long time.
4.3 sample-adding
Add 100 μ L/ hole serum samples to be detected, as time quantitative, to set up 5 concentration be 1000 the most simultaneously, 500,250,
125,62.5 μ g/ml people anti-NY-ESO-1 standard items or calibration object (step 2.4), room temperature or 37 DEG C of reaction 60min or 4 DEG C of mistakes
Night.
During as used Streptavidin to be coated reaction micropore, it is simultaneously introduced biotin labeled NY-ESO-1 recombinant protein
(step 4.1), mixing.
4.4 add enzyme labelled antibody
Above-mentioned reaction the micropore phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer solution
(TBS) washing 3 times, finally add goat-anti people's enzyme mark IgG antibody (purchased from Jackson company of the U.S.), room temperature reaction 30-
120min。
4.5 colour developing
Above-mentioned reaction the micropore phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer solution
(PBS) washing 3 times, add 100 μ l/ holes tetramethyl biphenyl ammonia (TMB) or o-dihydroxy ammon (OPD) substrate lucifuge colour developing 5-15min
Observe.As time quantitative, then add the hydrochloric acid of the 1.8M in 50 μ l/ holes or sulfuric acid to terminate reaction.Each hole is surveyed at 450nm with ELIASA
Or the absorbance OD value at 495nm.
4.6 results judge
Qualitative: all obvious blue or yellow reaction person occur for the positive, is otherwise negative.
Quantitative: according to the absorbance of anti-NY-ESO-1 gauge orifice, draw calibration curve, calibration curve as shown in Figure 2, calculates
The content of various kinds sample wells.The results are shown in Table shown in 1.
5. anti-NY-ESO-1 repeats itself antibody during Chemiluminescence immunoassay detects the body fluid such as human serum
5.1 acridinium ester label specific antibodies
Use direct labelling method.10mg sheep or rabbit anti-human igg's antibody, be preferential with goat anti-human igg antibody, be dissolved in respectively
With 160 μ l acridinium ester solution (1mg 2', 6'-dimethyl-carbonyl phenyl in the carbonate buffer solution of 10ml 100mM, pH 9.0
10-methyl-9-acridine formic acid esters 4'-N-succinimide ester trifluoromethyl sulfonic acid (being called for short acridinium ester DMAE-NHS) (CAS:
115853-74-2) being dissolved in 1.0ml dimethylformamide, acridinium ester DMAE-NHS is purchased from lark prestige Science and Technology Ltd.), sheep
Or rabbit anti-human igg's antibody is 1:4 with acridinium ester DMAE-NHS mol ratio.Being gently mixed mixing, it is little that room temperature lucifuge is gently mixed 3
Time.Cross Sephadex G-50 gel column (purchased from Pharmacia), elute with pH 9.0,100mM phosphate buffer, receive
JiIgGFeng, it is thus achieved that sheep or rabbit anti-human igg's antibody-acridinium ester, i.e. labelled antibody.IgG measures (mg/ml)=(OD280nm-OD403nm×
0.3)×0.62。
The optimal diluted concentration of acridinium ester labeling antibody determines.Human IgG antigen is diluted with the carbonate buffer solution of pH 9.6,50mM
(purchased from Jackson company of the U.S.), to after 1 μ g/mL, is coated reaction micropore, 4 DEG C overnight or 37 DEG C placements with 100 μ L/ holes
120min, washs 3 times, with 10% with the pH 7.4 containing volumetric concentration 0.05%Tween-20,50mM phosphate buffer (PBS)
Calf serum room temperature closes 30min, then is separately added into 100 μ l through the pH 7.4 containing 5% calf serum, 50mM phosphate buffer
(PBS) with sheep or the rabbit anti-human igg's antibody-acridinium ester room temperature of 1:1000,1:2500,1:5000,1:10000,1:20000 dilution
Reaction 60min, washs 3 times with the PBS (pH7.4,50mM) containing 0.05%Tween-20, adds containing mass concentration 0.1%H2O2's
0.1M NaOH solution 100 μ l/ hole, surveys each hole with Roche E601 chemical illumination immunity analysis instrument (Roche company of the U.S.) luminous strong
Degree.It is optimal diluted concentration with the highest dilution hole of the higher reacting hole of luminous intensity.Present invention discover that and be preferred with 1:5000.
5.2 are coated and close
After the carbonate buffer solution dilution NY-ESO-1 recombinant protein of pH 9.6,50mM to 1 μ g/mL, with 100 μ L/ holes
It is coated reaction micropore, places 120min, with the phosphate buffer containing volumetric concentration 0.05%Tween-20 for 4 DEG C overnight or 37 DEG C
(PBS, pH 7.4,50mM) or pH 7.4Tris-HCl buffer solution (TBS) wash 3 times, close by 10% calf serum room temperature
30min, dries, and through the most dried, sealing is put 4 DEG C and can be preserved for a long time.
When using biotin-avidin system (step 4.1), dilute strepto-with the carbonate buffer solution of pH 9.6,50mM
Avidin, to after 10 μ g/mL, is coated reaction micropore with 100 μ L/ holes, places 120min, with containing volumetric concentration for 4 DEG C overnight or 37 DEG C
Phosphate buffer (pH 7.4PBS) or the Tris-HCl buffer solution (pH 7.4TBS) of 0.05%Tween-20 wash 3 times, use
10% calf serum room temperature closing 30min, dries, and through the most dried, sealing is put 4 DEG C and can be preserved for a long time.
5.3 sample-adding
Adding 100 μ L/ hole serum samples to be detected, as time quantitative, 5 concentration the most simultaneously setting up step 2.4 to prepare are
1000,500,250,125,62.5 μ g/ml people anti-NY-ESO-1 autoantibody standard items or calibration object, room temperature or 37 DEG C of reactions
60min or 4 DEG C are overnight.
During as used Streptavidin to be coated reaction micropore (step 4.1), it is simultaneously introduced 100 μ L/ holes biotin labeled
NY-ESO-1 recombinant protein 1 μ g/ml, mixing.
5.4 add acridinium ester label antibody
Above-mentioned reaction micropore with the phosphate buffer (pH 7.4PBS) containing volumetric concentration 0.05%Tween-20 or
Tris-HCl buffer solution (pH7.4TBS) washs 3 times, finally adds the sheep through acridinium ester label or rabbit anti-human igg's antibody 100 μ
L/ hole, room temperature reaction 60min.
5.5 strengthen luminescence
Above-mentioned each hole adds 50 μ L substrate solution A, and (carbamide peroxide including mass concentration 0.1-0.25% (with 0.1% is
Good, carbamide peroxide is Sigma Co., USA's product) and the Triton X-100 of volumetric concentration 0.01%, solvent is 100mM,
The Tris buffer solution of pH8.0) and 50 μ L substrate solution B (include the Triton X-100 of 10mM NaOH and volumetric concentration 2%, solvent
For water), mixing, survey each hole luminous intensity with Roche E601 chemical illumination immunity analysis instrument (Roche company of the U.S.) at once.
5.6 results judge
Quantitative: according to the luminous intensity of anti-NY-ESO-1 gauge orifice, with people's anti-NY-ESO-1 antibody of step 2.4 preparation
Correction product concentration is abscissa, and luminous intensity is ordinate, draws calibration curve, and calibration curve as shown in Figure 2, calculates each sample
The content in hole.The results are shown in Table shown in 1.The method sensitivity 1-10ng/mL, with ELISA correlation r2=0.97.
Table 1 clinical serum sample is with 4 kinds of distinct methods testing results (unit: μ g/mL)
Claims (10)
1. the detection method of anti-NY-ESO-1 autoantibody in a peripheral blood, it is characterised in that described method is: with NY-ESO-
1 recombinant protein is that antigen prepares anti-NY-ESO-1 antibody, and prepared by anti-NY-ESO-1 antibody and latex microsphere coupling antibody coupling
Thing, then mixes NY-ESO-1 recombinant protein mixed liquor a with peripheral blood testing sample, room temperature reaction 5-10min, adds
Enter antibody coupling matter, light absorption value at detection 546nm, obtain anti-NY-ESO-1 self in peripheral blood testing sample according to calibration curve
AC；Described NY-ESO-1 recombinant protein mixed liquor a quality group becomes: NY-ESO-1 recombinant protein 100-200ng/ml,
35g/L PEG-4000, bovine serum albumin(BSA) 5g/L, 0.05%Tween-20, NaN30.05%, solvent is pH6.8's
Buffer solution；Described calibration curve is prepared as follows: be that antigen immune rabbit prepares the anti-NY-of rabbit with NY-ESO-1 recombinant protein
ESO-1 specific IgG antibodies, more anti-for rabbit NY-ESO-1 specific IgG antibodies is pressed respectively 1000-62.5 μ g/ml gradient concentration
It is diluted to dilution, to buffer containing described bovine serum albumin(BSA) with the buffer solution containing volumetric concentration 0.1-1% bovine serum albumin(BSA)
Liquid is blank, and prepared by anti-for rabbit NY-ESO-1 specific IgG antibodies and latex microsphere coupling specific antibody conjugate, so
After the dilution of NY-ESO-1 recombinant protein mixed liquor b and different gradients is mixed, react 5-10min, finally add special
Property antibody coupling matter, respectively measure 546nm locate light absorption value, with light absorption value as ordinate, with the anti-NY-ESO-1 of rabbit in dilution spy
Heterogenetic antibody IgG concentration makes calibration curve as abscissa；Described NY-ESO-1 recombinant protein mixed liquor b forms same NY-
ESO-1 recombinant protein mixed liquor a.
2. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described latex
Microballoon is with a diameter of 40-230nm, and microsphere surface activated group is-COOH or-NH2The form of mass concentration 10% latex solution
Add.
3. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described antibody
Conjugate preparation method is: (1) latex microsphere activates: buffered with pH5.0-7.5,0.01-0.5mol/L MES by latex microsphere
Liquid, water, Tween-20 mix, and vortex oscillation mixes, and prepare latex mixed liquor；50mg/mL is added in described latex mixed liquor
The NHS aqueous solution and the 10mg/mL carbodiimide aqueous solution, vortex oscillation 15-45 minute, more ultrasonic 5-15 time, each ultrasonic 5-10
Second, it is spaced 15-30 second, ultrasonic energy 6000-9000kJ；After ultrasonic end, Sephadex G-25 sephadex crossed by mixed liquor
Post, washes with water to 2 times that final volume is initially use latex volume, it is thus achieved that the latex solution of activation；Described latex microsphere is with diameter
For 40-230nm, microsphere surface activated group is-COOH or-NH2Mass concentration 10% latex solution form add；Described latex
Microballoon and buffer solution volume ratio are 0.4:1, and described water and buffer solution volume ratio are 1.5:1, described Tween-20 and described buffer solution
Volume ratio is 1-2:100；The described NHS aqueous solution and described buffer solution volume ratio are 1:10-20, the described carbodiimide aqueous solution with
Described buffer solution volume ratio is 1:10-20；(2) coupling: anti-NY-ESO-1 antibody is dissolved in pH7.4,100-200mM PBS buffering
Liquid is made antibody-solutions, after the latex solution mixing that antibody-solutions and step (1) are activated, little in vertical mixed instrument reaction 1-4
Time；Add pH6.8-8.0,0.5-4.0mol/L glycine buffer in vertical mixed instrument mixing 10-60 minute, general's reaction
Liquid 15000-30000rpm is centrifuged 30-120 minute, abandons supernatant, retains precipitation, with the resuspended precipitation of cleaning solution, 15000-
30000rpm is centrifuged 15-120 minute, repeats resuspended and centrifugal 3-5 time, takes last centrifugal precipitation and is antibody coupling matter；Institute
State cleaning solution and refer to pH7.4,50-500mM glycine buffer containing volumetric concentration 0.1% bovine serum albumin(BSA)；Described work
The latex solution volumetric usage changed is calculated as 0.1-1.0ml/mg with anti-NY-ESO-1 antibody mass.
4. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described standard
Curve is prepared as follows: be that antigen immune rabbit prepares rabbit anti-NY-ESO-1 specific IgG with NY-ESO-1 recombinant protein
Antibody, more anti-for the rabbit NY-ESO-1 specific IgG antibodies buffer solution containing volumetric concentration 0.25% bovine serum albumin(BSA) is diluted
Become 1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, the dilution of 62.5 μ g/ml, with without antibody composition containing described
Bovine serum albumin(BSA) buffer solution be blank, anti-for rabbit NY-ESO-1 specific IgG antibodies is prepared with latex microsphere coupling
Specific antibody conjugate, then by NY-ESO-1 recombinant protein mixed liquor b and variable concentrations dilution hybrid reaction 5-
10min, finally adds specific antibody conjugate, measures light absorption value at 546nm respectively, with light absorption value as ordinate, with dilute
Release the anti-NY-ESO-1 specific IgG antibodies concentration of rabbit in liquid and make calibration curve as abscissa；Described recombinant protein mixed liquor b
Quality group becomes: NY-ESO-1 recombinant protein 100-200ng/ml, 35g/L PEG-4000, bovine serum albumin(BSA) 5g/L,
0.05%Tween-20, NaN30.05%, solvent is the Tris-HCl buffer solution of pH6.8,50mM.
5. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described anti-NY-
ESO-1 autoantibody is one of following: anti-NY-ESO-1 autoantibody IgG, anti-NY-ESO-1 autoantibody IgA or anti-NY-
ESO-1 autoantibody IgM.
6. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described antibody
Conjugate and NY-ESO-1 recombinant protein mixed liquor a volume ratio are 1:3, described peripheral blood testing sample and antibody coupling matter volume
Ratio is 3:50.
7. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described preparation
The Tris-HCl buffer solution that solvent is pH6.8,50mM of NY-ESO-1 recombinant protein mixed liquor a.
8. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 1, it is characterised in that described dilution
The Tris-HCl buffer solution that buffer solution is pH6.8,50mM of rabbit anti-NY-ESO-1 specific IgG antibodies.
9. the detection method of anti-NY-ESO-1 autoantibody in a peripheral blood, it is characterised in that described method: use NY-ESO-
1 recombinant protein is envelope antigen, adds testing sample, adds anti-human IgG antibodies directly and acridinium ester DMAE-NHS coupling system
Standby labelled antibody, adds substrate solution A and substrate solution B and carries out luminescence-producing reaction, detect reactant liquor luminous intensity, according to the anti-NY-of people
ESO-1IgG antibody calibration curve, it is thus achieved that anti-NY-ESO-1 autoantibody concentrations in testing sample；Described anti-human IgG antibodies and a word used for translation
The ratio of the amount of pyridine ester DMAE-NHS material is 1:3-5；Described anti-human IgG antibodies is that goat anti-human igg antibody or rabbit anti-human igg resist
Body；Described substrate solution A consists of: mass concentration 0.1-0.25% carbamide peroxide, volumetric concentration 0.01%Triton X-100,
Solvent is 100mM, pH8.0Tris buffer solution, and substrate solution B consists of: 10mM NaOH, the Triton X-of volumetric concentration 2%
100, solvent is water；Described people anti-NY-ESO-1IgG antibody calibration curve preparation method is: people's anti-NY-ESO-1IgG antibody is used
Tris-HCl buffer solution containing the 100mM of volumetric concentration 0.25% bovine serum albumin(BSA), pH7.4 is diluted to 1000 μ g/ml, 500 μ
G/ml, 250 μ g/ml, 125 μ g/ml, the dilution of 62.5 μ g/ml, with the delaying containing described bovine serum albumin(BSA) without antibody composition
Rushing liquid is blank, is envelope antigen with NY-ESO-1 recombinant protein, is separately added into described dilution, adds anti-human igg
The labelled antibody that antibody is directly prepared with acridinium ester DMAE-NHS coupling, adds substrate solution A and substrate solution B and carries out luminescence-producing reaction,
Detection reactant liquor luminous intensity, the AC of people anti-NY-ESO-1IgG in dilution is as abscissa, and luminous intensity is sat for vertical
Mark, draws calibration curve；Described substrate solution A and substrate solution B composition is with testing sample detection substrate solution A and substrate solution B used.
10. the detection method of anti-NY-ESO-1 autoantibody in peripheral blood as claimed in claim 9, it is characterised in that described peroxide
Changing urea quality concentration is 0.1%.
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| CN201610319075.5A CN105866433A (en) | 2016-05-13 | 2016-05-13 | Detection method for NY-ESO-1 autoantibodies in peripheral blood |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077304A (en) * | 2019-12-31 | 2020-04-28 | 深圳市瀚德标检生物工程有限公司 | Coupling method of carboxyl latex microspheres and antibody |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102346191A (en) * | 2011-06-17 | 2012-02-08 | 福建省农业科学院畜牧兽医研究所 | Muscovy duck gosling plague latex particle agglutination reagent and preparation method thereof |
| CN103383395A (en) * | 2013-07-10 | 2013-11-06 | 杭州市第一人民医院 | Liquid chip kit for detecting lung cancer autoantibody |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN105352958A (en) * | 2015-11-28 | 2016-02-24 | 宁波美康生物科技股份有限公司 | Detection reagent kit for overall 25-hydroxy-vitamin-D |
| CN105353117A (en) * | 2015-11-13 | 2016-02-24 | 浙江大学 | A preparing method of a latex microsphere crosslinked specific monoclonal or polyclonal antibody for detecting soluble abnormally glycosylated CD58 molecules |
-
2016
- 2016-05-13 CN CN201610319075.5A patent/CN105866433A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102346191A (en) * | 2011-06-17 | 2012-02-08 | 福建省农业科学院畜牧兽医研究所 | Muscovy duck gosling plague latex particle agglutination reagent and preparation method thereof |
| CN103383395A (en) * | 2013-07-10 | 2013-11-06 | 杭州市第一人民医院 | Liquid chip kit for detecting lung cancer autoantibody |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN105353117A (en) * | 2015-11-13 | 2016-02-24 | 浙江大学 | A preparing method of a latex microsphere crosslinked specific monoclonal or polyclonal antibody for detecting soluble abnormally glycosylated CD58 molecules |
| CN105352958A (en) * | 2015-11-28 | 2016-02-24 | 宁波美康生物科技股份有限公司 | Detection reagent kit for overall 25-hydroxy-vitamin-D |
Non-Patent Citations (2)
| Title |
|---|
| 张文敏等: "人NY-ESO-1基因的原核表达、纯化和初步应用", 《中国肿瘤临床》 * |
| 虞淦军等: "NY-ESO-1抗原的基础与临床研究进展", 《中国肿瘤生物治疗杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077304A (en) * | 2019-12-31 | 2020-04-28 | 深圳市瀚德标检生物工程有限公司 | Coupling method of carboxyl latex microspheres and antibody |
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