CN101063683A - Liquid phase chip reagent kit detecting various anti EB viral antigen antibody - Google Patents

Liquid phase chip reagent kit detecting various anti EB viral antigen antibody Download PDF

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CN101063683A
CN101063683A CN 200710027841 CN200710027841A CN101063683A CN 101063683 A CN101063683 A CN 101063683A CN 200710027841 CN200710027841 CN 200710027841 CN 200710027841 A CN200710027841 A CN 200710027841A CN 101063683 A CN101063683 A CN 101063683A
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CN101063683B (en
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曾益新
谷爱娣
谢彦博
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TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
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TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
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Abstract

This invention discloses one liquid phase chip test agent case and its process method for multiple kinds of anti-EB virus antibody, which comprises the following parts: coupling affinity element multi-color microball covering on polypeptide; at least one kind of coupling hydroxyl group multi-color microball covering on natural or gene antigen, wherein the antigen is EB virus crack object or shell antigen VCA; gene antigen is of one amino acid in list of No. 33-No. 37; the said integration polypeptide is one from No. 1-No. 32.

Description

Detect liquid phase chip reagent box of multiple anti EB virus antigen-antibody and preparation method thereof
Technical field
The invention belongs to the medical biotechnology field, relate to the liquid phase chip reagent box and the preparation method that detect multiple anti EB virus antigen-antibody specifically.
Background technology
Nasopharyngeal carcinoma is to be popular in the southern a kind of malignant tumour of China, and wherein common with Guangdong again, annual morbidity reaches 30-50/10 ten thousand.5 years survival rates can reach 80-90% behind the early stage nasopharyngeal carcinoma radiotherapy; And advanced nasopharyngeal carcinoma (NPC) only is 20%.Therefore strengthen the secondary prevention of nasopharyngeal carcinoma, i.e. discovery early, diagnosis morning, early treatment are the keys that improves the nasopharyngeal carcinoma result of treatment.
The generation and the ebv infection of nasopharyngeal carcinoma are closely related.Infect though set up the lifelong latence of Epstein-Barr virus among the general population more than 90%, nasopharyngeal carcinoma patient's multiple Epstein-Barr virus antibody horizontal will be apparently higher than non-patient.Studies show that, when nasopharyngeal cancer patient the first three years (on average) occurs in clinical symptoms, just can be observed that multiple anti EB virus antigen-antibody significantly raises in its serum, thus immunoserology to detect ebv infection be the most frequently used means of present screening for nasopharyngeal cancer.The detection index of generally using is VCA/IgA, EA/IgA clinically.VCA/IgA, its detection has very high sensitivity, but lacks specificity, is normally used for examination to get rid of the diagnosis of nasopharyngeal carcinoma; And EA/IgA detects and to have very high specificity, but lacks sensitivity and be used to nasopharyngeal carcinoma qualitative detection really, and both can replenish mutually.In the last few years, multiple Epstein-Barr virus genetic engineering recombinant antigen or chemically synthesized polypeptide be used to serology and had detected, and the researcher has found some kinds of Epstein-Barr virus antigens with potential diagnostic value such as EBNA1, p18, p23, Rta, archaeal dna polymerase.Because the antibody repertoire of different nasopharyngeal cancer patients is incomplete same, therefore the joint-detection of multiple antigen-antibody can further improve its diagnosis performance.When EBNA1/IgA, EBNA1/IgG, Zta/IgG detected separately, its susceptibility and specificity are all at 80-88%, and be lower than traditional VCA/IgA detection sensitivity (93%), but higher specificity is arranged; And lower than the specificity (97%) of EA/IgA, but susceptibility is higher.When three's use in conjunction, its susceptibility and specificity not only improve greatly, be respectively 92%, 93-99%, and can be used for the risk assessment of nasopharyngeal carcinoma district occurred frequently healthy population examination, during two or three negative antibody, its risk factor is 0.009-0.3, and the risk factor of two kinds of positives is 4-10, and the three when all positive risk factor reached 138.This shows that multiple antibody combined detection is than detecting better effects if separately.
After the relation of Epstein-Barr virus and nasopharyngeal carcinoma was revealed, indirect immunofluorescence (IFA) detected serum VCA/IgA, EA/IgA and is widely adopted.The advantage of IFA is that the comparison sensitivity is special, but its result's judgement has subjectivity, is difficult for standardization, and wastes time and energy.Along with the continuous development innovation of inspection technology, the researcher is making great efforts to replace IFA with other technology such as immunodotting, Western blotting, ELISA etc., and wherein the ELISA method is paid attention to most.ELISA is simple, quick, responsive, and is easy to robotization, is suitable for extensive Screening Samples.Reported that the EBV antigen that is used for ELISA comprises extract, the EBV albumen of reorganization and the synthetic polypeptide that infects the EBV cell.ELISA generally can only analyze a kind of antigen/antibody at every turn, and single index is not enough to all nasopharyngeal carcinoma are carried out efficient diagnosis.Obtained ideal effect though there is research to detect two kinds of antigens simultaneously, wrapped simultaneously by same elisa plate more than three kinds and yet there are no report with the ELISA method.If multiple antigen-antibody is analyzed simultaneously, workload will increase considerably, and the difficulty of Quality Control also increases.
How efficient apace the antibody of multiple Epstein-Barr virus is detected, be a focus in the research at present.Multi-functional liquid phase suspending chip (Multi-Analyte Suspension Array) is the high-throughout molecular diagnosis platform of development recent years, its organic combination flow cytometry, elisa technique and chip technology, once can analyze 100 kinds of different factors simultaneously.Advantages such as compare with other detection method, liquid-phase chip technology has highly sensitive, and dirigibility is good, and is easy and simple to handle, quick, and standard curve range is wide.At present external existing various clinical detection kit emerges, as the fast detecting of tumor markers detection, anaphylactogen screening, pathogen etc.Because ebv infection causes that nasopharyngeal carcinoma has very strong region characteristic, main occurred frequently in south China, so the technology of its early diagnosis does not attract great attention in American-European countries.Because ebv infection causes that nasopharyngeal carcinoma has very strong region characteristic, main occurred frequently in south China, so the technology of its early diagnosis does not attract great attention in American-European countries.Also be not used in the liquid phase chip reagent box commodity of early diagnosis nasopharyngeal carcinoma at present both at home and abroad.
Summary of the invention
One of technical issues that need to address of the present invention are the deficiencies that exists on the existing serology detection method, and the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of a kind of accurate fast detecting is provided.
The technical scheme that realizes above-mentioned technical matters is as follows:
A kind of liquid phase chip reagent box that detects multiple anti EB virus antigen-antibody mainly includes: at least a above coupling hydroxyl microballoon natural or gene engineering antigen that is coated with; And/or be coated with the microballoon that the coupling of synthesizing polypeptide has Avidin more than at least a, wherein, described native antigen is total antigen lysate of Epstein-Barr virus or shell antigen VCA; Gene engineering antigen is the amino acid series of any among NO.33～NO.37 in the series of tables; Described synthetic polypeptide is to have in the series of tables any amino acid series among NO.1～NO.32.
Preferably, mainly include: the coupling that is coated with the synthetic polypeptide of NO.9 in the series of tables, NO.10, NO.12, NO.15, NO.18, NO.21, NO.23, NO.26 or NO.30 respectively has the polychrome microballoon of Avidin; Be coated with the polychrome microballoon of the coupling hydroxyl of the amino acid series of NO.33～NO.37 in the total antigen lysate of Epstein-Barr virus, shell antigen VCA or the series of tables respectively.
Further purpose of the present invention provides the preparation method of the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of above-mentioned detection.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of method for preparing the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of above-mentioned detection mainly may further comprise the steps:
(1) bag is by natural or gene engineering antigen: 0.5ml (is contained 1.1 * 10 altogether 6Individual-1.3 * 10 6Individual microballoon) the polychrome microballoon of coupling hydroxyl is put into the Ep pipe, and room temperature is centrifugal; Abandon supernatant, be resuspended among the activation buffer; Add 10 μ l 50mg/mlS-NHS, 10 μ l 50mg/ml EDC, mixing, the room temperature lucifuge is hatched 30 ± 15min; Centrifugal; Abandon supernatant, with coupling buffer washing, each 100-300 μ l is centrifugal; Add 250 μ l in the described natural or gene engineering antigen of arbitrary final concentration 50-200 μ g/ml, mixing, room temperature lucifuge reaction 1-3hr; Centrifugal, abandon supernatant; Wash 1-3 time with wash buffer, 12000rpm, 4min is centrifugal; Add blocking buffer 200 μ l, polychrome microballoon final concentration is 2000-2500/μ l, room temperature, and shaking table shakes 30 ± 15min; Centrifugal, abandon supernatant; Obtain natural or the gene engineering antigen bag by in different coupling hydroxyl microballoons; And/or (2) bag is synthesized polypeptide: the polychrome microballoon that takes out 0.1-0.4ml coupling Avidin is put into the Ep pipe; The final concentration that adds arbitrary isopyknic storage buffer dilution is the above-mentioned synthetic polypeptide of 8-12 μ g/ml), 4 ℃ of lucifuges are spent the night; Centrifugal, abandon supernatant; Wash 1-5 time with elution buffer, centrifugal; Add storage buffer 200-500 μ l, polychrome microballoon final concentration is 1200-1500/μ l; The coupling that obtains polypeptide bag quilt has the microballoon of Avidin.
The liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of detection of the present invention relates to 39 kinds of antigens, wherein, comprises 2 kinds of natural components, i.e. total antigen lysate of Epstein-Barr virus and shell antigen VCA; 5 kinds of gene engineering antigens, EA-D (being NO.33 in the series of tables), EBNA-1 total length (being NO.37 in the series of tables), EBNA-1 fragment (being NO.34 in the series of tables), p18 (being NO.35 in the series of tables), p23 (being NO.36 in the series of tables); 32 kinds of synthetic polypeptide comprise 2 of alkaline DNA enzymes, 2 of thymus gland kinases, 2 of archaeal dna polymerases, 2 of Zebra (BZLF1), Gp350/220 (BLLF1), P54/47 (BMRF1), EBNA1 (BKRF1), gp110/gp125 (BALF4), P18 (BFRF3), gN (BLRF1), EBNA2 (BYRF1), EBNA-3a (BLRF3), EBNA-3c (BERF3), EBNA-LP, RNA (ribonucleic acid) reductase (BORF2), p138 (BALF2), Rta (BRLF1), p150/160 (BcLF1), p143 (BNRF1), p40 (BdRF1), dUTPase (BLLF3), gp78 (BILF2), P33 (BARF1), EA-D p17 (BMLF1-BSLF2), gp140 (BALF1-1), P23 (BLRF2), P38 (BFRF1), each 1 of BFLF2.Above-mentioned 2 kinds of Epstein-Barr virus, 5 kinds of gene engineering antigens or 32 peptide species are wrapped respectively by different polychrome microballoons, then the coupling microballoon is carried out various combination, carry out multiple Epstein-Barr virus serology and detect (comprise all IgA, IgG antibody, and the IgM detection of antibodies of 2 kinds of natural components and 5 kinds of gene antigens) above 39 kinds of antigens.Wherein antigen-antibody detects has natural component lysate/IgA, lysate/IgG, lysate/IgM, a VCA/IgA to what nasopharyngeal carcinoma had a diagnostic value; Genetic engineering EA-D/IgA, EA-D/IgG, EBNA1/IgA, EBNA1fragment/IgA, p18/IgA, p18/IgM, p23/IgA, p23/IgM; Polypeptide class p47/54/IgA, p47/54/IgG, EBNA1/IgA, EBNA1/IgG, p18/IgA, gp78/IgA, gp78/IgG, p143/IgG, EBNA2/IgG, EBNA-LP/IgG, dUTPase/IgG, Rta/IgG.
The present inventor finds that under study for action the EB virus antibody profile of nasopharyngeal cancer patient is also not quite identical, therefore single index has detected certain limitation, just can remedy this defective and a plurality of anti EB virus antigen-antibody indexs in the serum are carried out check and analysis.Of the present invention on level to the Epstein-Barr virus genome analysis, filter out the index that diagnostic value is arranged, and set up the polychrome microballoon liquid-phase chip of the antibody of the multiple anti EB virus antigen of joint-detection.Described polychrome microballoon liquid phase chip reagent box is applicable to crowd's examination, frequently-occurring assessment, diagnosis and the prognosis effect appraisal of nasopharyngeal carcinoma, observation, can also directly apply to aspects such as early diagnosis nasopharyngeal carcinoma, judgement patient prognosis situation, have advantages such as rapidity, objectivity and accuracy.It compared with prior art has following beneficial effect:
1 flux is big.Can detect tens of kinds of Epstein-Barr virus antibody in the same sample simultaneously.
2. required sample size is few.The present invention only needs the serum sample of 1 μ l just can finish the detection of multiple anti EB virus antigen-antibody.
3. simple and fast.Detection of the present invention does not need cyclic washing, just can finish less than 2 hours.
4. be fit to general molecular biology reviewer and use, do not require abundant professional knowledge.Experimental result is read automatically by corresponding analyser, has got rid of subjectivity.
5. do not relate to poisonous, harmful reagent.
Embodiment
Present embodiment uses natural or gene engineering antigen can obtain by purchase, wherein, EB antigen mesochite antigen gp125 obtains by monoclonal antibody and P3H3 cell pyrolysis liquid affinitive layer purification, shell antigen p18 (1-119aa), be the NO.35 in the series of tables, p23 (being the NO.36 in the series of tables), NO.37 in the nuclear antigen EBNA-1 total length series of tables and nuclear antigen EBNA-1 (1-90,408-498aa) (being the NO.34 in the series of tables), EA-D (EBV early antigen D (EA-D) recombinant, be the NO.33 in the series of tables) be that purifying obtains behind expression in escherichia coli, the total antigen of Epstein-Barr virus is to obtain from the P3H3 cell pyrolysis liquid, synthesize 32 polypeptide in addition, comprised 2 of alkaline DNA enzymes, 2 of thymus gland kinases, 2 of archaeal dna polymerases, 2 of Zebra (BZLF1), Gp350/220 (BLLF1), P54/47 (BMRF1), EBNA1 (BKRF1), gp110/gp125 (BALF4), P18 (BFRF3), gN (BLRF1), EBNA2 (BYRF1), EBNA-3a (BLRF3), EBNA-3c (BERF3), EBNA-LP, RNA (ribonucleic acid) reductase (BORF2), p138 (BALF2), Rta (BRLF1), p150/160 (BcLF1), p143 (BNRF1), p40 (BdRF1), dUTPase (BLLF3), gp78 (BILF2), P33 (BARF1), EA-D p17 (BMLF1-BSLF2), gp140 (BALF1-1), P23 (BLRF2), Gp115 (BGLF1), P38 (BFRF1), each 1 of BFLF2.The employed polypeptide of present embodiment is that immunogenicity is strong, hydrophobicity and surface expose the high amino acid sequence of probability; These polypeptide can carry out coupling with biotin, Avidin, fluorescer etc.The amino terminal of every peptide species is coupling six carbon atom and biotin respectively.Synthetic peptide sequence is as follows:
alkaline DNase(BGLF5)：TP-17，Biotin-Ahx-TAPWVPSGLFADDESTP
alkaline DNase(BGLF5)：LP-20，Biotin-Ahx-LRSPETEAFVRNLDRPPQMP
thymidine kinase(BXLF1)：DD-18，Biotin-Ahx-DWAGLSKVISDFERGNRD
thymidine kinase(BXLF1)：RY-18，Biotin-Ahx-RKCQEDESPENERHENFY
DNA polymerase(BALF5)：FR-18，Biotin-Ahx-FLRPNKGLLKKPDKEYLR
DNA polymerase(BALF5)：DF-20，Biotin-Ahx-DNNSGAALSVLQNFTARPPF
Zebra(BZLF1)：MV-19，Biotin-Ahx-MDPNSTSEDVKFTPDPYQV
Zebra(BZLF1)：AE-17，Biotin-Ahx-AAPARRTRKPQQPESLE
P54/47(BMRF1)：RQ-20，Biotin-Ahx-RKRTSSEARQKQKHPKKVKQ
EBNA1(BKRF1)：GS-18，Biotin-Ahx-GSGPRHRDGVRRPQKRPS
gp110/gp 125(BALF4)：AA-19，Biotin-Ahx-ARDRFPGLRRRRYHDPETA
P18(BFRF3)：GQ-17，Biotin-Ahx-GGGQPHDTAPRGARKKQ
gN(BLRF1)：TS-18，Biotin-Ahx-TEAQDQFYSYTCNADTFS
Gp350/220(BLLF1)：HK-20，Biotin-Ahx-HHAEMQNPVYLIPETVPYIK
EBNA2(BYRF1)：LT-19，Biotin-Ahx-LTHQSTPNDPDSPEPRSPT
EBNA-3a(BLRF3)：MV-19，Biotin-Ahx-MDKDRPGPPALDDNMEEEV
EBNA-3c(BERF3)：AY-19，Biotin-Ahx-APQAPYQGYQEPPAPQAPY
EBNA-LP：RR-20，Biotin-Ahx-RRHRSPSPTRGGQEPRRVRR
RNA (ribonucleic acid) reductase (BORF2): RA-21, Biotin-Ahx-REEQNERSPAEQMPPRPMEPA
p138(BALF2)：EL-18，EDDGQRPDDEPRYTYWQL
Rta(BRLF1)：YD-22，Biotin-Ahx-YSPKDEQRDIAEVLDHLKTNRD
p150/160(BcLF1)：AN-20，Biotin-Ahx-APRDRRETYSLQHRRPNHMN
p143(BNRF1)：LE-18，Biotin-Ahx-LRGTNNDPRPQRQERARE
p40(BdRF1)：GL-22，Biotin-Ahx-GPREDTNPQQPTTEGHHRGKKL
dUTPase(BLLF3)：LI-18，Biotin-Ahx-LQNQRRYNSTLRPSELKI
gp78(BILF2)：TK-19，Biotin-Ahx-TSTSHRPHRRPVSKRPTHK
P33(BARF1)：CV-13，Biotin-Ahx-CVGKNDKEEAHGV
EA-Dp17(BMLF1-BSLF2)：RF-22，Biotin-Ahx-RSTRKQARQERSQRPLPNKPWF
gp140(BALF1-1)：PD-21，Biotin-Ahx-PTPPNDEERESNEEPPPPYED
P23(BLRF2)：TK-25，Biotin-Ahx-TRPRESNDPNATRRARSRSRGREAK
BFLF2：RA-23，Biotin-Ahx-RQQRPADPALRRLMHPHHRNYTA
P38(BFRF1)：PH-17，Biotin-Ahx-PIRRHRTRETRRMRGSH
Ahx=6 carbon, more than listed synthetic polypeptide (NO.1 in the series of tables～NO.32) is synthetic by Shenzhen writing brush space bioengineering company limited.
Native antigen
1.EBV antigen (P3H3 cell lysate is the total antigen lysate of Epstein-Barr virus) is available from Biodesign;
EBV viral capsid antigen (VCA gp125 is the anti-VCA of shell) is available from Biodesign;
Gene engineering antigen
EBV early antigen D (EA-D) recombinant is available from Biodesign;
EBV p23 recombinant is available from Biodesign;
EBV nuclear antigen-1 (EBNA-1) recombinant is available from Biodesign;
(1-90 is 408-498aa) available from ProSpec-Tany TechnoGEne Ltd for Recombinant EBV mosaic EBNA-1;
Recombinant EBV p18 (1-119aa) is available from ProSpec-Tany TechnoGEne Ltd;
Goat anti human IgG, Fc γSpecific R-PE (goat anti-human igg, Fc γSection, the R-PE mark) available from JacksonImmunoResearch;
Donkey anti-hu IgM, Fc 5uFragment R-PE (the anti-people IgM of donkey, Fc 5uSection, the R-PE mark) available from JacksonImmunoResearch;
R-Phycoerythrin-AffiniPure Goat Anti-Human Serum IgA, alpha Chain Specific (goat-anti people IgA, α chain, R-PE mark) is available from Jackson ImmunoResearch.
The preparation of polychrome microballoon liquid-phase chip:
The coupling hydroxyl that the employed polychrome microballoon of present embodiment is a Luminex company and the polychrome microballoon of Avidin, every kind of natural or gene engineering antigen bag is by in different coupling hydroxyl microballoons, and the polypeptide of chemosynthesis (1-32 in the series of tables) bag is had the microballoon of Avidin in coupling.Its experiment operation steps is as follows:
Bag is by natural or gene engineering antigen: take out 0.5ml and (contain 1.25 * 10 6Individual microballoon) the polychrome microballoon of coupling hydroxyl is put into the smooth Ep pipe of 1.5ml import, the centrifugal 12000rpm of room temperature, 4min; Abandon supernatant, be resuspended in 80 μ l activation buffer (10mM NaH 2PO 4, pH6.3) in; Add 10 μ l 50mg/ml S-NHS, 10 μ l 50mg/ml EDC, mixing, the room temperature lucifuge is hatched 20min; Centrifugal, 12000rpm, 4min; Abandon supernatant, with coupling buffer (10mM NaH 2PO 4, 150mM NaCl pH6.3) washes 2 times, each 200 μ l, 12000rpm, 4min; A kind of antigen (any in above-mentioned 39 kinds of antigens) the 250 μ l that add the correspondence of final concentration 50-200 μ g/ml, mixing, room temperature lucifuge reaction 2hr; Centrifugal, 12000rpm, 4min abandons supernatant; Wash 2 times with wash buffer (pH 7.4 for PBS, 0.05%Tween-20) 500 μ l, 12000rpm, 4min is centrifugal; Add blocking buffer (PBS, 1%BSA, 0.05%NaN 3, pH 7.4) and 200 μ l, room temperature, shaking table shakes 30min; Centrifugal, 12000rpm, 4min abandons supernatant; Add storage buffer (PBS, 1%BSA, 0.05%NaN 3, pH 7.4) and 500 μ l, 4 ℃ of preservations are standby;
Bag is synthesized polypeptide: the polychrome microballoon that takes out 0.3ml coupling Avidin is put into the smooth Ep pipe of 1.5ml import; The polypeptide that adds 300 μ l, 10 μ g/ml, 4 ℃ of lucifuges are spent the night; 12000rpm, 4min abandons supernatant; 1000 μ l wash 3 times with elution buffer, and are centrifugal, 12000rpm, 4min; Add storage buffer 500 μ l; Get 10 μ l and be applied on the tally, count at microscopically; 4 ℃ of preservations are standby.
It is as follows to detect serum Epstein-Barr virus antibody step:
Serum sample is with containing 1 * PBS damping fluid of 1%BSA with 20: 1 dilute serum samples; Each detection hole of Millipore plate adds the serum sample 20 μ l after the dilution; It is 1000-1500/50 μ l that every kind of polychrome microballoon that is coated with antigen or synthetic polypeptide is diluted to polychrome microballoon number with storage buffer; Every hole adds 50 μ l and dilutes good polychrome microballoon; 30min is hatched in the room temperature shading; With wash buffer wash-out 3 times; The anti-people IgA or IgG or the IgM that add the PE mark, room temperature, 30min is left standstill in shading; The multi-functional liquid phase chip analyzer of Luminex reading numerical values.
Data analysis
Each sample surveys twice, averages, and utilizes statistical analysis software that the fluorescent value of testing result is carried out statistical significant difference and ROC tracing analysis, its result such as table 1.The ROC area under curve illustrates that between 0.5-0.7 diagnosis effect is relatively poor, and diagnosis effect is medium between 0.7-0.9, and is better greater than 0.9 diagnosis effect.We choose the ROC area under curve more than 0.7, the conduct that all can reach more than 70% of sensitivity and specificity detects index, if the ROC area under curve is greater than 0.7, then it can be used as the index of diagnosis nasopharyngeal carcinoma, and then its sensitivity and specificity estimated, find out best point of crossing as its critical value.
In the application of described polychrome microballoon liquid phase chip reagent box, each index result of tested serum can critical reference value corresponding with it make comparisons, in order to auxiliary diagnosis of nasopharyngeal carcinoma.But the equal independent assortment of the index that all diagnostic values of the present invention are high is used for different testing goals, the pairing discriminant function difference of various combination.For example, for the fluorescence intensity mean value of EA-D/IgA and EBNA-1/IgA, but substitution discriminatory analysis function, Y1=0.000 * EA-D/IgA+0.001 * EBNA1/IgA-4.323; Y2=3.475 * 10-5EA-D/IgA+0.000 * EBNA1/IgA-1.221.If Y1＞Y2 then doubts to nasopharyngeal carcinoma, need further check; If Y2＞Y1 then is a healthy person.
Above-mentioned 39 kinds of antigens have all been carried out nasopharyngeal cancer patient to present embodiment and healthy volunteer's serology IgA, IgG detect, and utilizes statistical analysis software that the fluorescent value of testing result is carried out statistical significant difference and ROC tracing analysis, its result such as table 1 and table 2.The ROC area under curve illustrates that between 0.5-0.7 diagnosis effect is relatively poor, and diagnosis effect is medium between 0.7-0.9, and is better greater than 0.9 diagnosis effect.We choose the ROC area under curve more than 0.7, the conduct that all can reach more than 70% of sensitivity and specificity detects index, comprise: natural component or genetic engineering class, EA-D/IgA, EA-D/IgG, EBNA-1/IgA, EBNA-1 fragment/IgA, VCA/IgA, lysate/IgA, lysate/IgG, lysate/IgM, p18/IgA, p23/IgA; The polypeptide class, p47/54/IgA, p47/54/IgG, EBNA-1/IgA, p18/IgA, gp78/IgA, gp78/IgG, dUTPase/IgG, EBNA-LP/IgG, EBNA-2/IgG, p143/IgG, Rta/IgG.Can be used as totally 11 kinds of the IgA of diagnosis index, totally 10 kinds of IgG, 3 kinds of IgM.
Table 1. liquid-phase chip technology detects multiple anti EB virus antigen-antibody statistical analysis in nasopharyngeal carcinoma and healthy people
Ag/Ab Std.
Area Error 95％confidence interval P value
EA-D/IgA EA-D/IgG EA-D/IgM 0.876 0.876 0.600 0.0248 0.0258 0.0415 0.8275 to 0.9246 0.8251 to 0.9262 0.5189 to 0.6817 ＜0.0001 ＜0.0001 0.0189
p47/54 peptide/IgA p47/54 peptide/IgG EBNA-1/IgA EBNA-IgG EBNA-1/IgM EBNA1 fragment/IgA EBNA1 fragment/IgG EBNA1 fragment/IgM EBNA1 peptide/IgA EBNA1 peptide/IgG VCA/IgA VCA/IgG VCA/IgM gp125 peptide/IgA gp125 peptide/IgG Lysate/IgA Lysate/IgG lysate/IgM p18/IgA p18/IgG p18/IgM P18 peptide/IgA P18 peptide/IgG p23/IgA p23/IgG p23/IgM p23 peptide/IgA p23 peptide/IgG Gp78/IgA gp78/IgG 0.704 0.709 0.860 0.688 0.686 0.883 0.654 0.589 0.842 0.740 0.749 0.557 0.687 0.661 0.628 0.915 0.841 0.829 0.816 0.517 0.745 0.811 0.547 0.706 0.585 0.702 0.633 0.608 0.817 0.789 0.0548 0.0536 0.0283 0.0394 0.0398 0.0244 0.0411 0.0419 0.0426 0.0531 0.0358 0.0429 0.0391 0.0568 0.0600 0.0223 0.0300 0.0306 0.0314 0.0428 0.0360 0.0446 0.0627 0.0378 0.0421 0.0381 0.0411 0.0419 0.0309 0.0334 0.5961 to 0.8111 0.6036 to 0.8136 0.8048 to 0.9158 0.6105 to 0.7648 0.6084 to 0.7644 0.8351 to 0.9306 0.5734 to 0.7344 0.5069 to 0.6710 0.7584 to 0.9253 0.6360 to 0.8441 0.6785 to 0.8190 0.4726 to 0.6410 0.6106 to 0.7640 0.5496 to 0.7724 0.5103 to 0.7454 0.8713 to 0.9589 0.7820 to 0.8996 0.7692 to 0.8893 0.7544 to 0.8776 0.4329 to 0.6005 0.6740 to 0.8153 0.7232 to 0.8980 0.4244 to 0.6703 0.6313 to 0.7796 0.5027 to 0.6679 0.6269 to 0.7763 0.5522 to 0.7133 0.5263 to 0.6905 0.7568 to 0.8780 0.7239 to 0.8548 0.0008 0.0006 ＜0.0001 ＜0.0001 ＜0.0001 ＜0.0001 0.0003 0.0374 ＜0.0001 ＜0.0001 ＜0.0001 0.1836 ＜0.0001 0.0079 0.0349 ＜0.0001 ＜0.0001 ＜0.0001 ＜0.0001 0.6960 ＜0.0001 ＜0.0001 0.4345 ＜0.0001 0.0459 ＜0.0001 0.0018 0.0120 ＜0.0001 ＜0.0001
EA-R/IgA EA-R/IgG DNA pol1/IgA DNA pol1/IgG DNA pol2/IgA DNA pol2/IgG DNase/IgA DNase/IgG DNase 2/IgA DNase 2/IgG dUTPase/IgA dUTPase/IgG EBNA-LP/IgA EBNA-LP/IgG EBNA2/IgA EBNA2/IgG EBNA-3a/IgA EBNA-3a/IgG EBNA-3c/IgA EBNA-3c/IgG gp350/IgA gp350/IgG gp140/IgA gp140/IgG gN/IgA gN/IgG BFLF2/IgA BFLF2/IgG p17/IgA p17/IgG 0.556 0.602 0.549 0.511 0.636 0.673 0.550 0.508 0.523 0.520 0.610 0.760 0.688 0.731 0.673 0.711 0.576 0.633 0.589 0.525 0.670 0.581 0.526 0.543 0.607 0.507 0.510 0.544 0.559 0.632 0.0529 0.0517 0.0425 0.0429 0.0513 0.0490 0.0425 0.0429 0.0428 0.0432 0.0411 0.0355 0.0383 0.0364 0.0389 0.0374 0.0524 0.0505 0.0520 0.0534 0.0566 0.0615 0.0428 0.0432 0.0592 0.0625 0.0429 0.0431 0.0427 0.0417 0.4525 to 0.6598 0.5009 to 0.7038 0.4655 to 0.6321 0.4265 to 0.5945 0.5355 to 0.7365 0.5772 to 0.7693 0.4665 to 0.6331 0.4236 to 0.5919 0.4391 to 0.6068 0.4354 to 0.6050 0.5295 to 0.6906 0.6902 to 0.8294 0.6126 to 0.7626 0.6600 to 0.8025 0.5965 to 0.7490 0.6378 to 0.7846 0.4731 to 0.6785 0.5340 to 0.7319 0.4874 to 0.6914 0.4204 to 0.6299 0.5587 to 0.7808 0.4600 to 0.7010 0.4424 to 0.6100 0.4582 to 0.6275 0.4908 to 0.7228 0.3848 to 0.6299 0.4258 to 0.5941 0.4600 to 0.6288 0.4756 to 0.6431 0.5505 to 0.7140 0.2888 0.0534 0.2531 0.8063 0.0103 0.0011 0.2440 0.8570 0.5887 0.6400 0.0088 ＜0.0001 ＜0.0001 ＜0.0001 ＜0.0001 ＜0.0001 0.1525 0.0121 0.0914 0.6346 0.0051 0.1840 0.5372 0.3209 0.0780 0.9036 0.8148 0.3037 0.1634 0.0022
p138/IgA p138/IgG p143/IgA p143/IgG p150/IgA p150/IgG p33/IgA p33/IgG p38/IgA p38/IgG p40/IgA p40/IgG Rta/IgA Rta/IgG TK1/IgA TK1/IgG Zebra 1/IgA Zebra 1/IgG Zebra 2/IgA Zebra 2/IgG 0.568 0.684 0.689 0.785 0.554 0.643 0.671 0.675 0.570 0.564 0.540 0.562 0.673 0.777 0.580 0.557 0.659 0.607 0.526 0.630 0.0535 0.0489 0.0382 0.0333 0.0531 0.0507 0.0392 0.0392 0.0425 0.0428 0.0528 0.0532 0.0393 0.0345 0.0420 0.0425 0.0399 0.0420 0.0426 0.0413 0.4635 to 0.6734 0.5883 to 0.7801 0.6140 to 0.7638 0.7200 to 0.8504 0.4497 to 0.6577 0.5440 to 0.7427 0.5945 to 0.7482 0.5976 to 0.7514 0.4868 to 0.6536 0.4800 to 0.6479 0.4361 to 0.6432 0.4575 to 0.6662 0.5959 to 0.7498 0.7090 to 0.8441 0.4976 to 0.6622 0.4741 to 0.6406 0.5804 to 0.7369 0.5251 to 0.6896 0.4428 to 0.6100 0.5493 to 0.7111 0.1965 0.0005 ＜0.0001 ＜0.0001 0.3108 0.0068 ＜0.0001 ＜0.0001 0.0994 0.1384 0.4543 0.2427 ＜0.0001 ＜0.0001 0.0615 0.1795 0.0002 0.0120 0.5336 0.0026
Table 2.EB virus antigen-antibody is as critical value, susceptibility, the specificity of diagnosis nasopharyngeal carcinoma index
Ag/Ab cut-off(IF) sensitivity specificity
Lysate/IgA Lysate/IgG lysate/IgM EA-D/IgA EA-D/IgG p47/54 peptide/IgA 300 1800 400 400 2000 600 82.3％ 80.2％ 78.4％ 74.7％ 78.9％ 72.7％ 92.0％ 73.9％ 71.9％ 87.5％ 83.0％ 60.4％
p47/54 peptide/IgG EBNA-1/IgA EBNA1 fragment/IgA EBNA1 peptide/IgA EBNA1 peptide/IgG VCA/IgA p18/IgA p18/IgM P18 peptide/IgA p23/IgA p23/IgM Gp78/IgA gp78/IgG dUTPase/IgG EBNA-LP/IgG EBNA2/IgG p143/IgG Rta/IgG 2000 6000 600 800 5500 500 200 600 1200 350 600 450 5500 1500 1000 2000 1000 1300 60.0％ 85.4％ 81.8％ 81.8％ 72.7％ 70.8％ 75.0％ 68.2％ 83.3％ 62.5％ 60.2％ 76.0％ 71.3％ 76.6％ 71.3％ 60.6％ 71.2％ 70.2％ 70.8％ 76.1％ 79.2％ 81.3％ 64.6％ 69.3％ 77.1％ 64.6％ 70.5％ 66.7％ 69.8％ 71.3％ 75.0％ 70.8％ 61.5％ 70.8％ 70.8％ 71.8％
The present invention utilizes the high flux characteristics of liquid-phase chip technology, detected the distribution of multiple anti EB virus antigen-antibody in nasopharyngeal cancer patient and healthy people, screen the antigen-antibody that diagnostic value is arranged, and set up the kit that detects multiple Epstein-Barr virus antigen/antibody simultaneously.Kit can improve discovery rate morning of nasopharyngeal carcinoma in to nasopharyngeal carcinoma people at highest risk examination, make the patient in time take the treatment measure, and then improves its survival rate and quality of life.
Series of tables
＜110〉Zhongshan Univ. Cancer Cure Center
＜120〉liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of detection and preparation method thereof
<160>37
<170>PatentIn version 3.1
<210>1
<211>17
<212>PRT
＜213〉Epstein-Barr virus
<400>1
Thr Ala Pro Trp Val Pro Ser Gly Leu Phe Ala Asp Asp Glu Ser Thr Pro
1 5 10 15
<210>2
<211>20
<212>PRT
＜213〉Epstein-Barr virus
<400>2
Leu Arg Ser Pro Glu Thr Glu Ala Phe Val Arg Asn Leu Asp Arg Pro Pro Gln Met Pro
1 5 10 15 20
<210>3
<211>18
<212>PRT
＜213〉Epstein-Barr virus
<400>3
Asp Trp Ala Gly Leu Ser Lys Val Ile Ser Asp Phe Glu Arg Gly Asn Arg Asp
1 5 10 15
<210>4
<211>18
<212>PRT
＜213〉Epstein-Barr virus
<400>4
Arg Lys Cys Gln Glu Asp Glu Ser Pro Glu Asn Glu Arg His Glu Asn Phe Tyr
1 5 10 15
<210>5
<211>18
<212>PRT
＜213〉Epstein-Barr virus
<400>5
Phe Leu Arg Pro Asn Lys Gly Leu Leu Lys Lys Pro Asp Lys Glu Tyr Leu Arg
1 5 10 15
<210>6
<211>20
<212>PRT
＜213〉Epstein-Barr virus
<400>6
Asp Asn Asn Ser Gly Ala Ala Leu Ser Val Leu Gln Asn Phe Thr Ala Arg Pro Pro Phe
1 5 10 15 20
<210>7
<211>19
<212>PRT
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Met Asp Pro Asn Ser Thr Ser Glu Asp Val Lys Phe Thr Pro Asp Pro Tyr Gln Val
1 5 10 15
<210>8
<211>17
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Ala Ala Pro Ala Arg Arg Thr Arg Lys Pro Gln Gln Pro Glu Ser Leu Glu
1 5 10 15
<210>9
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Arg Lys Arg Thr Ser Ser Glu Ala Arg Gln Lys Gln Lys His Pro Lys Lys Val Lys Gln
1 5 10 15 20
<210>10
<211>18
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Gly Ser Gly Pro Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser
1 5 10 15
<210>11
<211>19
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<400>11
Ala Arg Asp Arg Phe Pro Gly Leu Arg Arg Arg Arg Tyr His Asp Pro Glu Thr Ala
1 5 10 15
<210>12
<211>17
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<400>12
Gly Gly Gly Gln Pro His Asp Thr Ala Pro Arg Gly Ala Arg Lys Lys Gln
1 5 10 15
<210>13
<211>18
<212>PRT
＜213〉Epstein-Barr virus
<400>13
Thr Glu Ala Gln Asp Gln Phe Tyr Ser Tyr Thr Cys Asn Ala Asp Thr Phe Ser
1 5 10 15
<210>14
<211>20
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<400>14
His His Ala Glu Met Gln Asn Pro Val Tyr Leu Ile Pro Glu Thr Val Pro Tyr Ile Lys
1 5 10 15 20
<210>15
<211>19
<212>PRT
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<400>15
Leu Thr His Gln Ser Thr Pro Asn Asp Pro Asp Ser Pro Glu Pro Arg Ser Pro Thr
1 5 10 15
<210>16
<211>19
<212>PRT
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<400>16
Met Asp Lys Asp Arg Pro Gly Pro Pro Ala Leu Asp Asp Asn Met Glu Glu Glu Val
1 5 10 15
<210>17
<211>19
<212>PRT
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<400>17
Ala Pro Gln Ala Pro Tyr Gln Gly Tyr Gln Glu Pro Pro Ala Pro Gln Ala Pro Tyr
1 5 10 15
<210>18
<211>20
<212>PRT
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<400>18
Arg Arg His Arg Ser Pro Ser Pro Thr Arg Gly Gly Gln Glu Pro Arg Arg Val Arg Arg
1 5 10 15 20
<210>19
<211>21
<212>PRT
＜213〉Epstein-Barr virus
<400>19
Arg Glu Glu Gln Asn Glu Arg Ser Pro Ala Glu Gln Met Pro Pro Arg Pro Met Glu Pro
1 5 10 15 20
Ala
<210>20
<211>18
<212>PRT
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<400>20
Glu Asp Asp Gly Gln Arg Pro Asp Asp Glu Pro Arg Tyr Thr Tyr Trp Gln Leu
1 5 10 15
<210>21
<211>22
<212>PRT
＜213〉Epstein-Barr virus
<400>21
Tyr Ser Pro Lys Asp Glu Gln Arg Asp Ile Ala Glu Val Leu Asp His Leu Lys Thr Asn
1 5 10 15 20
Arg Asp
<210>22
<211>20
<212>PRT
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<400>22
Ala Pro Arg Asp Arg Arg Glu Thr Tyr Ser Leu Gln His Arg Arg Pro Asn His Met Asn
1 5 10 15 20
<210>23
<211>18
<212>PRT
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<400>23
Leu Arg Gly Thr Asn Asn Asp Pro Arg Pro Gln Arg Gln Glu Arg Ala Arg Glu
1 5 10 15
<210>24
<211>22
<212>PRT
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<400>24
Gly Pro Arg Glu Asp Thr Asn Pro Gln Gln Pro Thr Thr Glu Gly His His Arg Gly Lys
1 5 10 15 20
Lys Leu
<210>25
<211>18
<212>PRT
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<400>25
Leu Gln Asn Gln Arg Arg Tyr Asn Ser Thr Leu Arg Pro Ser Glu Leu Lys Ile
1 5 10 15
<210>26
<211>19
<212>PRT
＜213〉Epstein-Barr virus
<400>26
Thr Ser Thr Ser His Arg Pro His Arg Arg Pro Val Ser Lys Arg Pro Thr His Lys
1 5 10 15
<210>27
<211>13
<212>PRT
＜213〉Epstein-Barr virus
<400>27
Cys Val Gly Lys Asn Asp Lys Glu Glu Ala His Gly Val
1 5 10
<210>28
<211>22
<212>PRT
＜213〉Epstein-Barr virus
<400>28
Arg Ser Thr Arg Lys Gln Ala Arg Gln Glu Arg Ser Gln Arg Pro Leu Pro Asn Lys Pro
1 5 10 15 20
Trp Phe
<210>29
<211>21
<212>PRT
＜213〉Epstein-Barr virus
<400>29
Pro Thr Pro Pro Asn Asp Glu Glu Arg Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu
1 5 10 15 20
Asp
<210>30
<211>25
<212>PRT
＜213〉Epstein-Barr virus
<400>30
Thr Arg Pro Arg Glu Ser Asn Asp Pro Asn Ala Thr Arg Arg Ala Arg Ser Arg Ser Arg
1 5 10 15 20
Gly Arg Glu Ala Lys
25
<210>31
<211>23
<212>PRT
＜213〉Epstein-Barr virus
<400>31
Arg Gln Gln Arg Pro Ala Asp Pro Ala Leu Arg Arg Leu Met His Pro His His Arg Asn
1 5 10 15 20
Tyr Thr Ala
<210>32
<211>17
<212>PRT
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<400>32
Pro Ile Arg Arg His Arg Thr Arg Glu Thr Arg Arg Met Arg Gly Ser His
1 5 10 15
<210>33
<211>404
<212>PRT
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<400>33
Met Glu Thr Thr Gln Thr Leu Arg Phe Lys Thr Lys Ala Leu Ala Val Leu Ser Lys Cys
1 5 10 15 20
Tyr Asp His Ala Gln Thr His Leu Lys Gly Gly Val Leu Gln Val Asn Leu Leu Ser Val
25 30 35 40
Asn Tyr Gly Gly Pro Arg Leu Ala Ala Val Ala Asn Ala Gly Thr Ala Gly Leu Ile Ser
45 50 50 60
Phe Glu Val Ser Pro Asp Ala Val Ala Glu Trp Gln Asn His Gln Ser Pro Glu Glu Ala
65 70 75 80
Pro Ala Ala Val Ser Phe Arg Asn Leu Ala Tyr Gly Arg Thr Cys Val Leu Gly Lys Glu
85 90 95 100
Leu Phe Gly Ser Ala Val Glu Gln Ala 5er Leu Gln Phe Tyr Lys Arg Pro Gln Gly Gly
105 110 115 120
Ser Arg Pro Glu Phe Val Lys Leu Thr Met Glu Tyr Asp Asp Lys Val Ser Lys Ser His
125 130 135 140
His Thr Cys Ala Leu Met Pro Tyr Met Pro Pro Ala Ser Asp Arg Leu Arg Asn Glu Gln
145 150 155 160
Met Ile Gly Gln Val Leu Leu Met Pro Lys Thr Ala Ser Ser Leu Gln Lys Trp Ala Arg
165 170 175 180
Gln Gln Gly Ser Gly Gly Val Lys Val Thr Leu Asn Pro Asp Leu Tyr Val Thr Thr Tyr
185 190 195 200
Thr Ser Gly Glu Ala Cys Leu Thr Leu Asp Tyr Lys Pro Leu Ser Val Gly Pro Tyr Glu
205 210 215 220
Ala Phe Thr Gly Pro Val Ala Lys Ala Gln Asp Val Gly Ala Val Glu Ala His Val Val
225 230 235 240
Cys Ser Val Ala Ala Asp Ser Leu Ala Ala Ala Leu Ser Leu Cys Arg Ile Pro Ala Val
245 250 255 260
Ser Val Pro Ile Leu Arg Phe Tyr Arg Ser Gly Ile Ile Ala Val Val Ala Gly Leu Leu
265 270 275 280
Thr Ser Ala Gly Asp Leu Pro Leu Asp Leu Ser Val Ile Leu Phe Asn His Ala Ser Glu
285 290 295 300
Glu Ala Ala Ala Ser Thr Ala Ser Glu Pro Glu Asp Lys Ser Pro Arg Val Gln Pro Leu
305 310 315 320
Gly Thr Gly Leu Gln Gln Arg Pro Arg His Thr Val Ser Pro Ser Pro Ser Pro Pro Pro
325 330 335 340
Pro Pro Arg Thr Pro Thr Trp Glu Ser Pro Ala Arg Pro Glu Thr Pro Ser Pro Ala Ile
345 350 355 360
Pro Ser His Ser Ser Asn Thr Ala Leu Glu Arg Pro Leu Ala Val Gln Leu Ala Arg Lys
365 370 375 380
Arg Thr Ser Ser Glu Ala Arg Gln Lys Gln Lys His Pro Lys Lys Val Lys Gln Ala Phe
385 390 395 400
Asn Pro Leu Ile
<210>34
<211>181
<212>PRT
＜213〉Epstein-Barr virus
<400>34
Gln Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu Lys Gly Asp Thr
1 5 10 15 20
Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln Arg Arg Gly Gly Asp Asn His Gly
25 30 35 40
Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser
45 50 50 60
Gly Ser Gly Pro Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile
65 70 75 80
Gly Cys Lys Gly Thr His Gly Gly Thr Gly Pro Val Gly Glu Ala Asp Tyr Phe Glu Tyr
85 90 95 100
His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly Ala Ile Glu Gln Gly
105 110 115 120
Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gln Gly Asp Gly Gly
125 130 135 140
Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys
145 150 155 160
Phe Glu Asn Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg Thr
165 170 175 180
Thr
<210>35
<211>119
<212>PRT
＜213〉Epstein-Barr virus
<400>35
Met Ala Arg Arg Leu Pro Lys Pro Thr Leu Gln Gly Arg Leu Glu Ala Asp Phe Pro Asp
1 5 10 15 20
Ser Pro Leu Leu Pro Lys Phe Gln Glu Leu Asn Gln Asn Asn Leu Pro Asn Asp Val Phe
25 30 35 40
Arg Glu Ala Gln Arg Ser Tyr Leu Val Phe Leu Thr Ser Gln Phe Cys Tyr Glu Glu Tyr
45 50 50 60
Val Gln Arg Thr Phe Gly Val Pro Arg Arg Gln Arg Ala Ile Asp Lys Arg Gln Arg Ala
65 70 75 80
Ser Val Ala Gly Ala Gly Ala His Ala His Leu Gly Gly Ser Ser Ala Thr Pro Val Gln
85 90 95 100
Gln Ala Gln Ala Ala Ala Ser Ala Gly Thr Gly Ala Leu Ala Ser Ser Ala Pro Ser
105 110 115
<210>36
<211>162
<212>PRT
＜213〉Epstein-Barr virus
<400>36
Met Ser Ala Pro Arg Lys Val Arg Leu Pro Ser Val Lys Ala Val Asp Met Ser Met Glu
1 5 10 15 20
Asp Met Ala Ala Arg Leu Ala Arg Leu Glu Ser Glu Asn Lys Ala Leu Lys Gln Gln Val
25 30 35 40
Leu Arg Gly Gly Ala Cys Ala Ser Ser Thr Ser Val Pro Ser Ala Pro Val Pro Pro Pro
45 50 50 60
Glu Pro Leu Thr Ala Arg Gln Arg Glu Val Met Ile Thr Gln Ala Thr Gly Arg Leu Ala
65 70 75 80
Ser Gln Ala Met Lys Lys Ile Glu Asp Lys Val Arg Lys Ser Val Asp Gly Val Thr Thr
85 90 95 100
Arg Asn Glu Met Glu Asn Ile Leu Gln Asn Leu Thr Leu Arg Ile Gln Val Ser Met Leu
105 110 115 120
Gly Ala Lys Gly Gln Pro Ser Pro Gly Glu Gly Thr Arg Pro Arg Glu Ser Asn Asp Pro
125 130 135 140
Asn Ala Thr Arg Arg Ala Arg Ser Arg Ser Arg Gly Arg Glu Ala Lys Lys Val Gln Ile
145 150 155 160
Ser Asp
<210>37
<211>641
<212>PRT
＜213〉Epstein-Barr virus
<400>37
Gln Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu Lys Gly Asp Thr
1 5 10 15 20
Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln Arg Arg Gly Gly Asp Asn His Gly
25 30 35 40
Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser
45 50 50 60
Gly Ser Gly Pro Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile
65 70 75 80
Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala
85 90 95 100
Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly
105 110 115 120
Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly
125 130 135 140
Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly
145 150 155 160
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Gly
165 170 175 180
Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly
185 190 195 200
Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala
205 210 215 220
Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala
225 230 235 240
Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly
245 250 255 260
Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala
265 270 275 280
Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala
285 290 295 300
Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly
305 310 315 320
Gly Ala Gly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly
325 330 335 340
Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Arg Gly Arg Gly Arg Glu Arg
345 350 355 360
Ala Arg Gly Gly Ser Arg Glu Arg Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg
365 370 375 380
Pro Arg Ser Pro Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro
385 390 395 400
Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu Tyr His Gln Glu
405 410 415 420
Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly Ala Ile Glu Gln Gly Pro Ala Asp
425 430 435 440
Asp Pro Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg Arg Lys
445 450 455 460
Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn
465 470 475 480
Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg Thr Thr Asp Glu
485 490 495 500
Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu Tyr Asn Leu
505 510 515 520
Arg Arg Gly Thr Ala Leu Ala Ile Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro
525 530 535 540
Phe Gly Met Ala Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys
545 550 555 560
Tyr Phe Met Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys Asp Ala Ile Lys
565 570 575 580
Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn Ile Arg Val Thr Val Cys Ser Phe
585 590 595 600
Asp Asp Gly Val Asp Leu Pro Pro Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu
605 610 615 620
Gly Asp Asp Gly Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln
625 630 635 640
Glu

Claims (5)

1. liquid phase chip reagent box that detects multiple anti EB virus antigen-antibody, it is characterized in that, mainly include: at least a polychrome microballoon that is coated with the coupling Avidin of synthetic polypeptide, and/or be coated with coupling hydroxyl polychrome microballoon natural and/or gene engineering antigen more than at least a; Wherein, native antigen is total antigen lysate of Epstein-Barr virus or shell antigen VCA; Gene engineering antigen is the amino acid series of any among NO.33～NO.37 in the series of tables; Described synthetic polypeptide is to have in the series of tables any amino acid series among NO.1～NO.32.
2. according to the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of the described detection of claim 1, it is characterized in that the amino terminal of every kind of synthetic polypeptide is coupling six carbon atom and biotin respectively.
3. according to the liquid phase chip reagent box of claim 1 or the multiple anti EB virus antigen-antibody of 2 described detections, it is characterized in that, include: the coupling that is coated with the synthetic polypeptide of NO.9 in the series of tables, NO.10, NO.12, NO.15, NO.18, NO.21, NO.23, NO.26 or NO.30 respectively has the polychrome microballoon of Avidin; Be coated with the polychrome microballoon of the coupling hydroxyl of the amino acid series of NO.33～NO.37 in the total antigen lysate of Epstein-Barr virus shell antigen VCA or the series of tables respectively.
4. a method for preparing the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of the described detection of claim 1 is characterized in that, mainly may further comprise the steps:
(1) bag is by natural or gene engineering antigen: will contain 1.1 * 10 altogether 6Individual-1.3 * 10 6The polychrome microballoon of the 0.5ml coupling hydroxyl of individual microballoon is put into the Ep pipe, and room temperature is centrifugal; Abandon supernatant, be resuspended among the activation buffer; Add 10 μ l 50mg/mlS-NHS, 10 μ l 50mg/ml EDC, mixing, the room temperature lucifuge is hatched 30 ± 15min; Centrifugal; Abandon supernatant, with coupling buffer washing, each 100-300 μ l is centrifugal; Add 250 μ l in the described natural or gene engineering antigen of arbitrary final concentration 50-200 μ g/ml, mixing, room temperature lucifuge reaction 1--3hr; Centrifugal, abandon supernatant; Wash 1-3 time with wash buffer, 12000rpm, 4min is centrifugal; Add blocking buffer 200 μ l, the microballoon final concentration is 2000-2500/μ l,, room temperature, shaking table shakes 30 ± 15min; Centrifugal, abandon supernatant; Obtain natural or the gene engineering antigen bag by in different coupling hydroxyl microballoons; And/or
(2) bag is synthesized polypeptide: the polychrome microballoon that takes out 0.1-0.4ml coupling Avidin is put into the Ep pipe; The final concentration that adds arbitrary isopyknic storage buffer dilution is the synthetic polypeptide of 8-12 μ g/ml, and 4 ℃ of lucifuges are spent the night; Centrifugal, abandon supernatant; Wash 1-5 time with elution buffer, centrifugal; Add storage buffer 200-500 μ l, the microballoon final concentration is 1200-1500/μ l; The coupling that obtains polypeptide bag quilt has the microballoon of Avidin.
5. the preparation method of the liquid phase chip reagent box of the multiple anti EB virus antigen-antibody of detection according to claim 4 is characterized in that: mainly may further comprise the steps:
(1) bag is by natural or gene engineering antigen: will contain 1.1 * 10 altogether 6Individual-1.3 * 10 6The polychrome microballoon of the 0.5ml coupling hydroxyl of individual polychrome microballoon is put into the Ep pipe, and room temperature is centrifugal; Abandon supernatant, be resuspended among the activation buffer; Add 10 μ l 50mg/mlS-NHS, 10 μ l 50mg/ml EDC, mixing, the room temperature lucifuge is hatched 30 ± 15min; Centrifugal; Abandon supernatant, with coupling buffer washing, each 100-300 μ l is centrifugal; Add 250 μ l in the described natural or gene engineering antigen of arbitrary final concentration 20-200 μ g/ml, mixing, room temperature lucifuge reaction 1--3hr; Centrifugal, abandon supernatant; Wash 1-3 time with wash buffer, 12000rpm, 4min is centrifugal; Add blocking buffer 200 μ l, polychrome microballoon final concentration is 2000-2500/μ l, room temperature, and shaking table shakes 30 ± 15min; Centrifugal, abandon supernatant; Obtain natural or the gene engineering antigen bag by in different coupling hydroxyl microballoons; And/or
(2) bag is synthesized polypeptide: the polychrome microballoon that takes out 0.1-0.4ml coupling Avidin is put into the Ep pipe; The final concentration that adds arbitrary isopyknic storage buffer dilution is the synthetic polypeptide of 10 μ g/ml, and 4 ℃ of lucifuges are spent the night; Centrifugal, abandon supernatant; Wash 1-5 time with elution buffer, centrifugal; Add storage buffer 200-500 μ l, the final concentration of polychrome microballoon is 1200-1500/μ l, and the coupling that obtains polypeptide bag quilt has the microballoon of Avidin.
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