CN103383395B - A kind of liquid phase chip reagent box for detection of lung cancer autoantibody - Google Patents
A kind of liquid phase chip reagent box for detection of lung cancer autoantibody Download PDFInfo
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Abstract
The invention discloses a kind of liquid phase chip reagent box for detection of lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffers, antigen protein is at least two kinds in p62, NY-ESO-1, p53 and CAGE, wherein, the microballoon of the different antigen protein of coupling has different color codings; Two resist for human immunoglobulins G.The present invention is used for the liquid phase chip reagent box of detection of lung cancer autoantibody, on the technology platform of liquid-phase chip, achieve the joint-detection to four kinds of antibody in serum, and with p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody for mark, detection accuracy is high, and the early diagnosis for lung cancer provides new thinking.
Description
Technical field
The present invention relates to field of pharmaceutical biology, particularly relate to a kind of liquid phase chip reagent box for detection of lung cancer autoantibody.
Background technology
In recent years, the incidence of China's cancer is obvious ascendant trend, accounts for more than 20% of the cause of death, occupies first of all kinds of cause of the death.The World Health Organization (WHO) predicts, to the year two thousand twenty, will have 2,000 ten thousand new cancer cases, wherein death toll reaches 1,200 ten thousand, and the overwhelming majority occurs in developing country.If do not take any effective Prevention and controls measure, expect the year two thousand twenty, the kainogenesis cancer sum that China is annual and cancer mortality sum will reach about 3,000,000, and ill sum will reach 6,600,000.
Lung cancer is the malignant tumour that in global range, M & M is the highest at present.Over nearly 20 years, owing to carrying out smoking cessation energetically, the incidence of disease of western countries' male lung cancers such as Europe and the U.S. has started to decline, but the incidence of disease of female lung cancer continues to rise.China is cigarette production and selling big country, no matter the male sex or women, the incidence of disease of lung cancer, all in lasting ascendant trend, rises faster with women's incidence of disease especially.Clinical research shows, carcinoma in situ cure rate is close to 100%, and 5 years survival rates of I phase patients with lung cancer reach 60% ~ 90%, and 5 years survival rates of IIIb and IV phase patient only 5% ~ 20%, therefore, early diagnosis early detection, early treatment reduces lung cancer mortality, extends the key of life cycle.But, owing to lacking desirable method of early diagnosis, the early diagnostic rate of lung cancer only about 14%.Therefore, how to improve lung cancer early diagnosis level and become the serious and urgent task that lung cancer preventing and controlling person faces.
The marks such as the tumour antigen in serum can induce body to produce autoantibody, tumour occur also fail by clinical examination means detect early stage, body immune system just can monitor the existence of the tumour antigen of low expression level, and cause immune response, produce a large amount of antibody, play effective bio signal amplification.
Contrast clinical conventional tumor marker proteins, the detection of autoantibody accounts for great advantage in diagnosing tumor.The first, early diagnosis can be carried out to tumour, be convenient to early treatment, improve cure rate.Multinomial research shows the existence that autoantibody can be detected before imaging examination makes a definite diagnosis the solid carcinoma several months to several years, and even before tumour is made a definite diagnosis, 2-10 just can detect in serum the antibody had for tumour antigen.The second, autoantibody is higher than corresponding tumour antigen titre, and the autoantibody for single antigen is increased in a large number by immune response, and in serum, a large amount of albumin is deposited in case of interferers, and comparatively other mark more easily detects.Therefore analyze the immune change of tumor patient, instead of oncoprotein itself becomes a very important Research approach.3rd, sample easily obtains, and testing result is relatively stable.Tumour antigen is once be secreted into blood, may be degraded soon or remove, and autoantibody is subject to protease hydrolytic effect unlike other polypeptide, can stable in serum, sustainable existence for a comparatively long period of time, and the half life period of autoantibody is very long, be approximately 7 days, fluctuation hourly is very little, stable in physicochemical property, preserves its activity almost without impact for a long time at-80C.4th, its operate reagent used and technology simple and easy to do, reproducible, just can be detected by the enzyme linked immunosorbent assay (ELISA) of routine or enzyme-linked immuno assay (EIA).
Tumour is the Carcinogenesis of a polygenes, multi-step; although tumour autoantibody is the ideal candidates index of diagnosing tumor; only diagnose by an index; usually can cause false positive and false negative; so; single tumour autoantibody detects and certainly exists some limitation, affects the clinical diagnosis of lung cancer.One, in specific tumors, the positive rate of single tumour autoantibody only has 10% usually, in optimal cancer colonies, be also only 20-30%; They are two years old, a lot of biological approaches in tumor invasion are that various diseases (comprising tumour and other autoimmune diseases) is common, generally, same tumor can produce one or more tumour autoantibodies singularly, and the histological types of different tumour or same tumor both can detect common tumour antibody, also different tumour antibodies can be detected.
Research trend is in recent years in carrying out cancer detection for associating antibody repertoire.Chapman etc. use ELISA to detect the serum antibody for 7 kinds of tumour antigens (p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1 and GBU4-5) in lung cancer (comprising non-small cell lung cancer and small-cell carcinoma of the lung) patient and normal control population's serum, result display monospecific antibody detects positive rate between 5%-36%, the specificity of diagnosis reaches 96%-100%, and the susceptibility of 7 kinds of antibody combined detections can reach 76%, specificity is 92%.This absolutely prove tumor markers individual event detect, specificity is higher, but susceptibility and accuracy lower; After joint-detection, although specificity decreases, susceptibility and accuracy all improve a lot.
Therefore the autoantibody that joint-detection kinds of tumors is relevant, form the autoantibody " finger-print " that tumour itself is exclusive, the specificity of diagnosis and prognostic and susceptibility are brought up to the single individual level that is beyond one's reach, because these autoantibodies occur usually before clinical symptoms, thus there is important diagnosis and prognosis and judge to be worth.
Summary of the invention
The invention provides a kind of liquid phase chip reagent box for detection of lung cancer autoantibody, achieve the joint-detection of Multiple Antibodies in serum, high to the detection accuracy of lung cancer.
A kind of liquid phase chip reagent box for detection of lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffers, antigen protein is p62, NY-ESO-1, p53 and CAGE, wherein, the microballoon of the different antigen protein of coupling has different color codings; Two resist for biotin labeled human immunoglobulins G.
CAGE: tumor-related gene, belong to cancer-testis (cancer-testis CT) antigen family member, that the people such as Cho in 2002 adopt recombinant clone to express the serological analysis technology of antigen, one that finds during examination testis tissue cDNA library new Cancer-testis antigen encoding gene.
P62: nucleoporin is 1 tumor associated antigen that relative molecular mass is about 62 × 103, belongs to a member of IMA-IGF2BP3-001 (IGF2) mRNA associated proteins family, also known as MP2/IGF2BP2.
NY-ESO-1: be that chen in 1997 etc. utilize SEREX technology to screen the tumour antigen obtained from cancer of the esophagus cDNA library, belongs to CTA(cancer-testis antigen) family, this family member only expresses in testis tissue and some tumor tissues.
P53:p53 gene is a kind of antioncogene, is positioned human chromosomal 17p13.1, the molecular weight of 393 amino acid composition of encoding be 53kD core in phosphorylated protein, be called as p53 albumen.
The present invention is with antigen protein p62, NY-ESO-1, the autoantibody that p53 and CAGE produces is as detected object, in liquid phase chip reagent box of the present invention, antigen protein and two anti-all can be special with antibody (the p62 antibody in corresponding Sera of Lung Cancer, NY-ESO-1 antibody, p53 antibody or CAGE antibody) combine, and SA-PE can with the combination of biotin high degree of specificity, therefore, finally can form " microballoon-antigen protein+serum antibody+two resists+SA-PE " compound for Sera of Lung Cancer antibody, detected by instrument, reaction type is determined according to microballoon color difference, phycoerythrin is excited with green laser, measure the quantity of the reporter fluorescence molecule that microballoon combines, for indirectly determining the content of the Sera of Lung Cancer antibody that microballoon combines.
Described reaction buffer can be 1%PBSB.
Described dilution buffer can be 1%PBSB.
Described antigen protein is p62, NY-ESO-1, p53 and CAGE.
Described antigen protein is by HaloTag
tMinterchangeable labelling technique is connected with microballoon.
The amino acid sequence of p62 albumen is as shown in SEQ ID NO.5; The gene order of coding p62 albumen is as shown in SEQ ID NO.1; The amino acid sequence of NY-ESO-1 albumen is as shown in SEQ ID NO.6, and the gene order of coding NY-ESO-1 albumen is as shown in SEQ ID NO.2; The amino acid sequence of p53 albumen is as shown in SEQ ID NO.7, and the gene order of coding p53 albumen is as shown in SEQ ID NO.3; The amino acid sequence of CAGE albumen is as shown in SEQ ID NO.8; , the gene order of coding CAGE albumen is as shown in SEQ ID NO.4.
Preferably, described two resist for goat anti-human immunoglobulin G, and experiment finds that mouse source antibody can produce cross reaction, and the antibody of Yang Yuan then can overcome this defect.
Described microballoon can adopt the microballoon of conventional liquid-phase chip.
When preparing the microballoon of coupled antigen albumen, the addition of antigen protein is 5 ~ 10 μ g/6.25 × 10
6individual microballoon, is preferably 6.25 μ g/6.25 × 10
6individual microballoon.
The concentration of the microballoon of often kind of coupled antigen albumen is 1.2 ~ 1.8 × 10
4individual/μ l, is preferably 1.2 × 10
4individual/μ l.
Biotin labeled two concentration resisted are 0.1 ~ 0.125 μ g/ml, are preferably 0.1 μ g/ml.
Compared with prior art, beneficial effect of the present invention is:
The present invention is used for the liquid phase chip reagent box of detection of lung cancer autoantibody, achieve the joint-detection to four kinds of antibody in serum, and with the p62 antibody in serum, NY-ESO-1 antibody, p53 antibody and CAGE antibody for mark, detection accuracy is high, and the diagnosis and prognosis for lung cancer provides new instrument and thinking.
The present invention is used for the technology platform of liquid phase chip reagent box based on liquid-phase chip of detection of lung cancer autoantibody, can high-throughoutly detect fast trace sample, liquid phase environment is more conducive to the native conformation keeping protein, is also more conducive to reaction and carries out, highly sensitive, signal to noise ratio (S/N ratio) is good.
Accompanying drawing explanation
Fig. 1 is the distribution of the unimolecule autoantibody in patients with lung cancer and normal healthy controls;
Fig. 2 is the accuracy that 3 kinds of biostatistics algorithms (CCP, DLDA, BCCP) analyze liquid phase chip reagent box of the present invention detection.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
The preparation of embodiment 1 liquid phase chip reagent box
Reagent and solution
(1) 0.1M NaH
2pO
4, pH6.2: weigh 3.5814g NaH
2pO
4in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 6.2;
(2) 0.05M MES, pH5.0: weigh 0.976g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 5.0;
(3) 0.1M MES(2-(N-morpholino) ethyl sulfonic acid), pH6.0: weigh 1.952g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 6.0;
(4) 0.1M MES, pH4.5: weigh 1.952g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 4.5;
(5) PBS:NaCl, 137mM; KCl, 2.7mM; Na
2hPO
4, 8.1mM; KH
2pO
4, 1.5mM; PH7.2-7.4,0.22um membrane filtration;
(6) 0.02%Proclin300,0.22um membrane filtration is contained in 1%PBSB:0.1%PBS;
(7) 0.1%tween-20,0.22um membrane filtration is contained in 0.1%PBST:0.1%PBS;
(8) 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC);
(9) N-hydroxy thiosuccinimide (S-NHS).
1, for detecting the liquid phase chip reagent box of autoantibody, include:
1) coupling has 55#, 47#, 27#, 33# microballoon of Halo amine (O2) ligand;
2) containing the fusion of Halo Tag: p62, NY-ESO-1, p53, CAGE;
3) SA-PE;
4) goat anti-human igg-biotin: concentration is 0.1 μ g/ml;
5) reaction buffer: 1%PBSB;
6) dilution buffer: 1%PBSB;
7) sealed membrane;
8) 96 orifice plates.
Prepare above-mentioned liquid phase chip reagent box, comprise the steps:
1, recombinant protein abduction delivering and purifying
Reagent and solution:
KRX recombinant bacterium (respectively containing, for example the gene shown in SEQ ID NO.1 ~ 4 ,-80 DEG C of preservations)
Dusty yeast (OXOID)
Peptone (OXOID)
Halo tag protein purfication resins（Promega）
Protease inhibitor（Roche）
Lysozyme（TOPBIO）
DNase I
TEV protease（Promega）
His link resin (Promega)
Purification column (BIO-RAD)
CA-630（Sigma）
LB nutrient culture media (5.0gNaCl, adjusts PH to 7.0 ~ 7.5 to be settled to 1L, 120 ° of autoclaving 20min for 10.0g peptone, 5.0g dusty yeast)
Damping fluid 1(50mM Hepes, PH7.5,150mM NaCl, 1mM DTT)
Damping fluid 2(50mM Hepes, PH7.5,150mM NaCl, 1mM DTT, 0.5mM EDTA, 0.05%CA630)
Experimental procedure:
One, a large amount of abduction delivering of recombinant bacterium
1, to spend the night amplification
Take out the KRX recombinant bacterium of preservation from-80 degree refrigerators, join 10ml and contain in the ammonia benzyl resistance LB nutrient culture media of 0.2% glucose, 37 DEG C of 275rpm spend the night amplification.
2, cultivation is expanded
The thalline spent the night is joined 1L according to 1:100 contain 37 DEG C of 275rpm in the LB nutrient culture media of ammonia benzyl resistance and be cultured to OD=0.4-0,5, add glucose and rhamnose that final concentration is 0.05% in nutrient culture media, 25 DEG C of 225rpm overnight incubation.
3, the centrifugal 15min of 5000g collects thalline, and 100ml/ manages, and carries out mark and is placed in-80 ° of preservations.
Two, KRX recombinant bacterium lysis
1, get the KRX recombinant bacterium that 1 pipe-80 ° is frozen, add that 15ml damping fluid 1 is resuspended adds 100ul Protease inhibitor, 10ul Lysozyme(100mg/ml respectively afterwards), 10ul DNase I(5mg/ml), 100ul1%CA-630; Resuspended thalline, after mixing, ice bath places 30min.
2, ultrasonication thalline (super 5s, stops 10s, 85% power, 3min), places 10min on ice.
3,12000g, 4 ° of centrifugal 30min, get supernatant, get 100ul supernatant to (wherein 50ul is labeled as S, and the TEV enzyme labeling of 50ul+10ul1:100 dilution is in addition S-TEV) in 1.5ml centrifuge tube.
4, centrifugal Halo link: the Halo link beads getting 1 pipe 2ml respectively of cleaning simultaneously, 3300rpm is centrifugal, and 5min abandons supernatant, adds the rear centrifugal 5min of 3300rpm of mixing in 2ml lavation buffer solution to beads and abandons supernatant, repeated washing 2 times.
5, joined in centrifugal bacterium liquid supernatant by the complete Halo link of cleaning, room temperature blending incubation was in conjunction with 2 hours.
6, above-mentioned mixed liquor is joined gravity in purification column and cross post, collect filtrate 100ul(FT); Then add lavation buffer solution 10ml, resuspended rear room temperature blending incubation 10min, after abandon filtrate, repeated washing 4 times.
7, close purification column, add the lavation buffer solution of 3ml, close after putting 1ml and add 5ul TEV enzyme rear enclosed purification column, 37 ° of reaction 1h.
8, cross post and collect filtrate (E1-1), add 2ml purification buffer in pillar, envelope post mixing 10min, collecting by filtration filtrate is labeled as (E1-2), after add 10ul His link resin in E1-1 and E1-2 mixed, incubated at room 1h, 1000g collected by centrifugation supernatant is labeled as E2.
2, albumen and microballoon coupling
By p62, NY-ESO-1, p53, CAGE recombinant protein of purifying respectively with the coupling of 55#, 47#, 27#, 33# microballoon.
Reagent and solution:
(1) 0.1M NaH
2pO
4, pH6.2: weigh 3.5814g NaH
2pO
4in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 6.2;
(2) 0.05M MES, pH5.0: weigh 0.976g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 5.0;
(3) 0.1M MES(2-(N-morpholino) ethyl sulfonic acid), pH6.0: weigh 1.952g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 6.0;
(4) 0.1M MES, pH4.5: weigh 1.952g MES in 90mL ddH
2in O, NaOH is settled to 100mL, 0.22um membrane filtration after regulating pH to 4.5;
(5) PBS:NaCl, 137mM; KCl, 2.7mM; Na
2hPO
4, 8.1mM; KH
2pO
4, 1.5mM; PH7.2-7.4,0.22um membrane filtration;
(6) 0.02%Proclin300,0.22um membrane filtration is contained in 1%PBSB:0.1%PBS;
(7) 0.1%tween-20,0.22um membrane filtration is contained in 0.1%PBST:0.1%PBS;
(8) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC)
(9) N-hydroxy thiosuccinimide (S-NHS).
Experimental procedure:
(1) take out microballoon stoste, vortex 20s, ultrasonic 20s, get 50ul(about 6.25 × 10
6individual microballoon) in Axygen EP pipe (mark microballoon model and title), 14000x g, 4mins;
(2) 14000g, RT(room temperature), 4min;
(3) carefully sucking supernatant, a small amount of supernatant can be remained for reducing the loss;
(4) 100ul dH is added
2o, respectively vortex and ultrasonic about 20s;
（5）14000g，RT，4min；
(6) carefully suck supernatant, a small amount of supernatant can be remained for reducing the loss, adding 80ul0.1MNaH
2pO
4, pH6.2, respectively vortex and ultrasonic about 20s;
(7) 10ul S-NHS(ddH is added rapidly
2o is dissolved to 50mg/mL), vortex mixes;
(8) 10ul EDC(ddH is added rapidly
2o is dissolved to 50mg/mL), vortex and ultrasonic about 20s respectively;
(9) RT, lucifuge rotates hatches 20min(every 10min vortex once);
（10）14000g，RT，4min；
(11) carefully suck supernatant, add 0.05M MES, pH5.0 to 125 μ L, respectively vortex and ultrasonic about 20s;
（12）14000g，RT，4min；
(13) carefully suck supernatant, repeat above-mentioned washing step once;
(14) carefully supernatant is sucked, with the resuspended microballoon of 12.5ul 0.05M MES, pH5.0, vortex and ultrasonic about 20s respectively;
(15) add albumen that 6.25ug implementation step 1 purifying obtains in above-mentioned microballoon, be 250ul 0.05M MES, pH5.0 to final volume, vortex is about 20s respectively;
(16) RT, lucifuge rotates hatches 2h;
（17）14000g，RT，4min；
(18) carefully suck supernatant, add 1mL0.01%PBST, respectively vortex and ultrasonic about 20s;
（19）14000g，RT，4min；
(20) carefully suck supernatant, repeat above-mentioned washing step once;
(21) 1mL1%PBSB is added, respectively vortex and ultrasonic about 20s;
(22) RT, lucifuge rotates hatches 30min, closes the activated carboxyl site of microsphere surface remnants;
（23）14000g，RT，4min；
(24) carefully suck supernatant, add 1mL1%PBSB, respectively vortex and ultrasonic about 20s;
(25) carefully suck supernatant, adding 1%PBSB to final volume is 37ul, respectively vortex and ultrasonic about 20s;
(26) get 2ul in 38ul1%PBSB, vortex 20s, after ultrasonic 20s, blood counting chamber counts, ultimate density (individual/mL).
(27) on Axygen centrifuge tube, microballoon concentration (1.2 × 10 is marked
4individual/μ l), coupling time, then place 4 degree keep in Dark Place.
Embodiment 2 lung cancer detection is tested
1, detection method
(1) first take out all reagent before using, place balance to room temperature;
The serum of (2)-80 DEG C of taking-ups patient (trouble lung cancer) and Healthy People, after being placed on thawed on ice respectively, vortex mixes, and get V-type 96 hole blood-coagulation-board (96 orifice plate) by serum-dilution 200 times, rifle head mixes up and down, does not produce bubble as far as possible;
(3) microballoon of 4 kinds of coupled antigen proteins in Example 1, vortex mixing 20s, ultrasonic 20s, get appropriate in 1000ul1%PBSB by 2500, every hole, vortex mixing 20s, ultrasonic 20s, and final volume is 10ul/ hole;
(5) open and wash trigger---Prime----rinse(channel2)----Prime---places 96 orifice plates---Run5:MAGX3;
(6) serum of dilution 200 times is added immediately, 30ul/ hole;
(7) 96 orifice plates are wrapped, room temperature with aluminium-foil paper, concussion incubation reaction 60min;
(8) 0.1%PBST washes three times, Run5:MAGX3;
(9) 1%PBSB dilutes goat anti-human igg-biotin (1:5000) and SAPE(1:500), 50ul/ hole;
(10) 96 orifice plates are wrapped, room temperature with aluminium-foil paper, concussion incubation reaction 60min;
(11) 1%PBST washes three times, Run5:MAGX3;
(12) the resuspended microballoon of 100ul/ hole 1%PBSB, room temperature, concussion 3-5min;
(13) on Luminex series liquid-phase chip analyser, read result, instrument can drawing standard curve automatically, and calculates the measured value of sample to be tested.
2, interpretation of result
Fig. 1 shows the distribution of the unimolecule autoantibody in 48 early stages of lung cancer and 48 normal healthy controls.
The algorithm (CCP, DLDA, BCCP) utilizing 3 kinds of biostatistics conventional is analyzed, and finds that accuracy rate can reach 72% by detecting self aborning p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody.
Claims (1)
1., for a liquid phase chip reagent box for detection of lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffers, it is characterized in that,
Antigen protein is p62, NY-ESO-1, p53 and CAGE, and wherein, the microballoon of the different antigen protein of coupling has different color codings;
Two resist for goat anti-human immunoglobulin G;
When preparing the microballoon of coupled antigen albumen, the addition of antigen protein is 5 ~ 10 μ g/6.25 × 10
6individual microballoon;
The concentration of the microballoon of often kind of coupled antigen albumen is 1.2 ~ 1.8 × 10
4individual/μ l;
Biotin labeled two concentration resisted are 0.1 ~ 0.125 μ g/ml.
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| CN109470853B (en) * | 2017-09-08 | 2022-03-29 | 广州市丹蓝生物科技有限公司 | Liquid phase protein chip for diagnosing autoimmune disease, kit and manufacturing method |
| CN109142755B (en) * | 2018-10-09 | 2021-06-22 | 福建省立医院 | Four-autoantibody combined detection kit for diagnosing early esophageal squamous cell carcinoma and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022013321A3 (en) * | 2020-07-14 | 2022-03-10 | Oncimmune Limited | Use of antigen combination for detecting autoantibodies in lung cancer |
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