CN101113979B - CBA reagent kit for detecting EB virus gp78 antibody and method for making same - Google Patents
CBA reagent kit for detecting EB virus gp78 antibody and method for making same Download PDFInfo
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Abstract
The invention discloses a CBA kit used for detecting anti-EB virus gp78 antibody, which includes coupling superbead, the superbead is made by coupling blank superbead and NeutrAvidin in a coupling way, the NeutrAvidin binds with the bioepiderm labeled EB virus gp78 antigen which is coupled with Biotin. The gp78 antibody CBA kit is suitable for crown screening, evaluation of the high incidence of nasopharyngeal carcinoma, diagnose and prognosis effect appraisal, observation, furthermore, the kit can be applied directly in nasopharyngeal carcinoma early diagnosis, judge prognosis of patients, etc. with the characteristics of rapid, objectivity and accurate.
Description
Technical field
The invention belongs to the medical biotechnology field, specifically, related to CBA kit of a kind of detection serum Epstein-Barr virus gp78 antibody that is used for flow cytometer and preparation method thereof.
Background technology
(Epstein-Barr virus EBV) belongs to gamma herpes viruses section to Epstein-Barr virus, and its basic structure comprises nuclear appearance thing, capsid and cyst membrane three parts.Capsid is the three-dimensional symmetries of 20 bodies, is made up of 162 shell particulates.Cyst membrane is made up of the nuclear membrane of infection cell, and the membrane glycoprotein of encoding viral is arranged on it, and the Epstein-Barr virus acceptor of identification on the lymphocyte arranged, and with function such as Fusion of Cells.Also has one deck albumen tunicle between this external cyst membrane and the capsid.Epstein-Barr virus only can be bred in bone-marrow-derived lymphocyte, can make its conversion, can go down to posterity for a long time.Had the genome of EBV by the cell of virus infections, and can produce various antigens, fixed have: EBV NA (EBNA), EA (EA), membranous antigen (MA), capsid antigen (VCA), lymphocyte identification membranous antigen (LYDMA).
Epstein-Barr virus extensively exists in the crowd, and it is pathogenic to have obvious regional feature.Main and nasopharyngeal carcinoma is closely related in China south, nasopharyngeal carcinoma patient's multiple Epstein-Barr virus antibody horizontal will be apparently higher than non-patient.Except that LYDMA, nasopharyngeal carcinoma patient EBNA, MA, VCA, EA all produce corresponding 1gG and IgA antibody, study these antigens and antibody thereof, to illustrating EBV and nasopharyngeal carcinoma relation and diagnosing all significant in early days.
It is the most frequently used means of present screening for nasopharyngeal cancer that immunoserology detects ebv infection.Indirect immunofluorescence (IFA) detects serum VCA/IgA at present, EA/IgA extensively adopts at clinical quilt.Yet these methods are not high to the diagnostic value of nasopharyngeal carcinoma, its susceptibility and specificity in the different experiments chamber result also inconsistent.The diagnosis of nasopharyngeal carcinoma finally relies on the pathologic finding of biopsy.But drawing materials, biopsy in reality, can receive many-sided restriction such as hospital's hardware, doctor's technical merit and patient's wish.Therefore, the researcher is seeking a kind of more accurate, responsive, quick, easy diagnostic method always both at home and abroad.
The streaming microballon analyzes that (Cytometric bead assay, CBA) technology is developed 30 years so far from being born and is perfect, and has promoted the development in Medical Biology field greatly.This technology has advantages such as measuring speed is fast, easy and simple to handle, good stability, sensitivity height; Be applied in the routine clinical inspection of multiple disease; But the kit that does not still have at present, anti EB virus membranous antigen gp78 antibody in a kind of detection serum of successful usefulness Flow Cytometry.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of CBA kit that is used for the detection Epstein-Barr virus gp78 antibody of flow cytometer.
A kind of CBA kit that detects Epstein-Barr virus gp78 antibody includes CBA coupling microballon, and this coupling microballon is that blank microballon of CBA and NeutrAvidin coupling form, and said NeutrAvidin has the Epstein-Barr virus gp78 antigen of biotin to combine with coupling.
Preferably, the Epstein-Barr virus gp78 antigen of biotin is arranged is the polypeptide with Biotin-Ahx-Thr Ser Thr SerHis Arg Pro His Arg Arg Pro Val Ser Lys Arg Pro Thr His Lys (TSTSHRPHRRPVSKRPTHK) series of synthetic in coupling.
Another object of the present invention provides the preparation method of mentioned reagent box.
The method of the CBA kit of a kind of preparation as above-mentioned detection Epstein-Barr virus gp78 antibody mainly may further comprise the steps:
(1) prepares microballon: with blank microballon vortex mixing; Take out the blank microballon of 60-80 μ l, wrap ultrasonic mixing with masking foil; Add 1.8-2.0 μ l1M DTT, the vortex mixing; Be put on the shaking table room temperature, 0.5-1.5hr again; Add 0.8-1.2ml coupling buffer, the vortex mixing; Centrifugal, abandon supernatant; Repeat with coupling buffer washing 2-4 time; Resuspended blank microballon is in 18-22 μ l coupling buffer;
(2) prepare the NeutrAvidin sample: take out the NeutrAvidin sample of 85-95 μ l1mg/ml, put into the Ep pipe that masking foil is wrapped; In the NeutrAvidin sample, add the 1.8-2.3 μ l sulfo-SMCC that at present joins; The vortex mixing; Being put on the shaking table room temperature, lucifuge, 0.5-1.5hr with Avidin example reaction liquid in above-mentioned;
(3) remove the composition that has neither part nor lot in reaction: in purification column, fill it up with coupling buffer, buffer is freely flowed out, repeat two to three times; Add ready NeutrAvidin sample in the step (2) to purification column therein after centrifugal; Centrifugal, remove the albumen of unmodified; Carry out next step immediately;
(4) coupling NeutrAvidin: joining in the ready blank microballon in the step (1) through the NeutrAvidin sample of modifying; The vortex mixing; Shaking table is hatched, room temperature, 1hr, lucifuge; Add 1.5-2.5 μ l NEM, the vortex mixing; Shaking table is hatched 10-20min; Add 0.8-1.3ml storage buffer; Centrifugal, abandon supernatant; Repeat with storage buffer washing three times; Be resuspended in 0.4-0.6ml storage buffer, this moment, final concentration was approximately 6 * 10
6Microballon/ml; 4 ℃ of preservations.
(5) combine polypeptide: the microballon that takes out the good NeutrAvidin of 70-80 μ l coupling is put in the EP pipe, and the coupling that adds 70-80 μ l has the Epstein-Barr virus gp78 antigen of biotin; The vortex mixing; Lucifuge, the room temperature shaking table is hatched 1-2hr, or 4 ℃, the shaking table incubated overnight; Centrifugal; Abandon supernatant; Add 450-500 μ l elution buffer, centrifugal; Repeat once with the elution buffer washing; Abandon supernatant, add 70-80 μ l stock solution, 4 ℃ of preservations behind the mixing.
Inventor of the present invention finds anti EB virus gp78 antigen-antibody level in the nasopharyngeal cancer patient serum under study for action apparently higher than healthy subjects, therefore detects the auxiliary characteristics that anti-gp78 antibody in the serum can be used as the diagnosis nasopharyngeal carcinoma.We are bright to encapsulate at the functional beads of the gp78 antigen polypeptide that utilizes chemosynthesis to BD company, can carry out serology quickly and easily and detect, and on flow cytometer, carry out quantitative test.Said gp78 antibody CBA detection kit is applicable to crowd's examination, assessment occurred frequently, diagnosis and the prognosis effect appraisal of nasopharyngeal carcinoma, observation; Can also directly apply to aspects such as early diagnosis nasopharyngeal carcinoma, judgement prognosis of patients situation, have advantages such as rapidity, objectivity and accuracy.It compared with prior art has following beneficial effect:
1. highly sensitive.The sensitivity of Flow Cytometry will be higher than technology such as conventional ELISA, has reduced the error that experimental technique itself is brought.
2. simple and fast.Detection of the present invention does not need cyclic washing, just can accomplish less than 2 hours.
3. be fit to general molecular biology reviewer and use, experimental result is read by corresponding analyser automatically, has got rid of subjectivity.
4. do not relate to poisonous, harmful reagent.
Description of drawings
Fig. 1 is the percentile chart that said kit detects nasopharyngeal carcinoma and the distribution of normal human serum gp78 antibody.
Fig. 2 is the ROC tracing analysis that said kit detects nasopharyngeal carcinoma and the distribution of normal human serum gp78 antibody.
Embodiment
The employed Epstein-Barr virus gp78 of present embodiment antigen is the polypeptide of chemosynthesis; This amino acid sequence of polypeptide is according to the resulting antigenic determinant of computer software analysis; It is high that its immunogenicity is strong, hydrophobicity and surface expose probability, amino terminal coupling six carbon atom and biotin in the building-up process.Peptide sequence is following: gp78 (BILF2): TK-19, Biotin-Ahx-TSTSHRPHRRPVSKRPTHK, (AHX=6 carbon).
Listed synthetic polypeptide is synthetic by Shenzhen writing brush space bioengineering company limited in the series of tables.
Goat anti human IgG, Fc
γSpecific R-PE (goat anti-human igg, Fc
γSection, 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;
Elution buffer: 1 * PBS, 0.05%Tween-20; Storage buffer: 1 * PBS, 1%BSA, 0.05%NaN
3
Encapsulating of polypeptide antigen:
The blank microballon (beads) of the employed CBA of present embodiment and employedly encapsulate damping fluid (coupling buffer), storage buffer (storage buffer) be U.S. BD (BECTON DICKINSON) Company products, it is following that it tests operation steps:
Prepare microballon: take out beads, vortex mixing 30s; Take out 75 μ l beads (about 500 reactions), wrap with masking foil; Ultrasonic mixing 1min; With deionized water configuration 1M DTT (dithiothreitol (DTT)), be divided into inner wrapping, get portion at every turn; Add 1.9 μ l1M DTT; Vortex mixing 5sec prevents that liquid splash from arriving the pipe top; Be put into beads on the shaking table room temperature, 1hr; (can prepare sulfo-SMCC) during this; Add 1ml coupling buffer (BD company), vortex mixing 5sec; Centrifugal, 900 * g, 3min; Abandon supernatant; Repeat with coupling buffer washing three times; Resuspended beads is in 20 μ l coupling buffer (BD company);
Prepare the NeutrAvidin sample: the NeutrAvidin sample (being kept among 1 * PBS pH7.2 ± 0.2) that takes out 90 μ l1mg/ml is put into the Ep pipe that masking foil is wrapped; Sulfo-SMCC (Sulfo-Succinimidyl4-(maleimidomethyl) Cyclohexane-1-Carboxylate) with deionized water configuration 2mg/ml annotates: join existing usefulness at present; In protein sample, add 2 μ l sulfo-SMCC; Vortex mixing 5sec prevents that liquid splash from arriving the pipe top; Be put into albumino reaction liquid on the shaking table room temperature, lucifuge, 1hr;
Removal has neither part nor lot in the composition of reaction: in the G-25 purification column, fill it up with coupling buffer (BD company), buffer is freely flowed out, repeat twice; Put into suitable test tube to purification column, centrifugal, 1,000 * g, 2min.Discard test tube; Be re-applied to purification column in the new test tube, add the protein sample of reaction; Centrifugal, 1,000 * g, 2min, this step has been removed the albumen of unmodified; Discard purification column; Carry out next step immediately;
Coupling NeutrAvidin: being relayed to from above-mentioned test tube in the beads pipe the step 1 through the protein sample of modifying; Vortex mixing 5sec prevents that liquid splash from arriving the pipe top; Albumen and beads shaking table are hatched, room temperature, 1hr, lucifuge; Add 2 μ l NEM (2mg/ml, N-ethyl maleic amide is dissolved in dimethyl sulfoxide (DMSO) (DMSO)), annotate: preferably be distributed into many parts, every part 20 μ l; Vortex mixing 5sec prevents that liquid splash from arriving the pipe top; Shaking table is hatched 15min; Add 1ml storagebuffer (BD company); Centrifugal, 900 * g, 3min; Abandon supernatant; Repeat with storage buffer washing three times; Be resuspended in 0.5ml storage buffer, this moment, final concentration was approximately 6 * 10
6Beads/ml; 4 ℃ of preservations (before first the using, preferably spending the night).
In conjunction with polypeptide: the microballon that takes out the good NeutrAvidin of 75 μ l couplings is put in the 1.5ml EP pipe, and the coupling that adds 75 μ l has the Epstein-Barr virus gp78 antigen polypeptide of biotin (10 μ g/ml, stock solution dilution); Vortex mixing 5sec; Lucifuge, the room temperature shaking table is hatched 1-2hr, or 4 ℃, the shaking table incubated overnight; 12,000rpm, 4min is centrifugal; Abandon supernatant; Add 500 μ l elution buffers, 12,000rpm, 4min is centrifugal; Repeat once with the elution buffer washing; Abandon supernatant, add 75 μ l stock solutions, 4 ℃ of preservations behind the mixing.
Serum sample detects
Detect antibody: before usefulness, all take reagent under the room temperature condition, and all operations process is all wanted lucifuge; The beads5sec that the vortex coupling is good; By needing 1000-2000 beads to calculate the beads consumption in each reaction tube, take out to be put in the Ep pipe; Add the stock solution dilution, final volume satisfies each reaction tube 50 μ l; Prepare 12 * 75mm test tube of respective number, each adds the good beads of 50 μ l dilution; Add 20 μ l storage buffer in the negative control test tube; Add 20 μ l and arrive test tubes with the good blood serum sample of 1:21 dilution (with the storage buffer dilution); Hatch 30min in the room temperature lucifuge behind the vortex mixing; Centrifugal, 1000 * g, 10min; Abandon supernatant; Add the antibody (diluting with 1:200) of 150 μ l PE marks, hatch 30min in the room temperature lucifuge behind the vortex mixing with PBS; Centrifugal, 1000 * g, 10min; Abandon supernatant, be resuspended in 150 μ l Washbuffer; Flow cytometer detects.
Testing result is analyzed
Choose through the 104 routine nasopharyngeal cancer patients and the 102 routine healthy subjects of age pairing and carry out the serum sample detection, behind the flow cytometer reading, analyze with SPSS13.0 statistics software.The t assay shows that gp78/IgA, gp78/IgG antibody horizontal have notable statistics difference (the P value is all less than 0.0001) in two groups of crowds, and its percentile chart is seen accompanying drawing 1.ROC tracing analysis result shows that the TG-AUC of gp78/IgA, gp78/IgG is respectively 0.82 and 0.76 (accompanying drawing 2), and gp78/IgA is a critical value with fluorescence intensity 4000, and its sensitivity and specificity are respectively 80%, 75%; Gp78/IgG is a critical value with fluorescence intensity 20000, and its sensitivity and specificity are respectively 70%, 79%.
Claims (2)
1. CBA kit that detects Epstein-Barr virus gp78 antibody; It is characterized in that: include CBA coupling microballon; This coupling microballon is that blank microballon of CBA and NeutrAvidin coupling form; And said NeutrAvidin has the Epstein-Barr virus gp78 antigen of biotin to combine with coupling, and this Epstein-Barr virus gp78 antigen is the polypeptide of being made up of Biotin-Ahx-Thr Ser Thr Ser His Arg Pro His Arg Arg Pro Val Ser Lys Arg Pro Thr His Lys of synthetic.
2. method for preparing the CBA kit of detection Epstein-Barr virus gp78 antibody as claimed in claim 1 is characterized in that: mainly may further comprise the steps:
(1) prepares microballon: with blank microballon vortex mixing; Take out the blank microballon of 60-80 μ l, wrap ultrasonic mixing with masking foil; Add 1.8-2.0 μ l 1M DTT, the vortex mixing; Be put on the shaking table room temperature, 0.5-1.5hr again; Add 0.8-1.2ml coupling buffer, the vortex mixing; Centrifugal, abandon supernatant; Repeat with coupling buffer washing 2-4 time; Resuspended blank microballon is in 18-22 μ l coupling buffer, and said coupling buffer is a U.S. BECTON DICKINSON Company products;
(2) prepare the NeutrAvidin sample: take out the NeutrAvidin sample of 85-95 μ l 1mg/ml, put into the Ep pipe that masking foil is wrapped; In the NeutrAvidin sample, adding the 1.8-2.3 μ l concentration of at present joining is the sulfo-SMCC of 2mg/ml; The vortex mixing; Be put on the shaking table room temperature, lucifuge, 0.5-1.5hr to above-mentioned NeutrAvidin example reaction liquid;
(3) remove the composition that has neither part nor lot in reaction: in purification column, fill it up with coupling buffer, coupling buffer is freely flowed out, repeat two to three times; Add ready NeutrAvidin sample in the step (2) to purification column therein after centrifugal; Centrifugal, remove the albumen of unmodified; Carry out next step immediately;
(4) coupling NeutrAvidin: joining in the ready blank microballon in the step (1) through the NeutrAvidin sample of modifying; The vortex mixing; Shaking table is hatched, room temperature, 1-2hr, lucifuge; Adding 1.5-2.5 μ l concentration is the methyl sulfoxide solution of the N-ethyl maleic amide of 2mg/ml, the vortex mixing; Shaking table is hatched 10-20min; Add 0.8-1.3ml storage buffer; Centrifugal, abandon supernatant; Repeat with storage buffer washing three times; Be resuspended in 0.4-0.6ml storage buffer, this moment, final concentration was 6 * 10
6Microballon/ml; 4 ℃ of preservations, said storagebuffer is a U.S. BECTON DICKINSON Company products;
(5) combine polypeptide: the microballon that takes out the good NeutrAvidin of 70-80 μ l coupling is put in the EP pipe, and the coupling that adds the 10 μ g/ml of 70-80 μ l has the Epstein-Barr virus gp78 antigen of biotin; The vortex mixing; Lucifuge, the room temperature shaking table is hatched 1-2hr, or 4 ℃, the shaking table incubated overnight; Centrifugal, abandon supernatant; Add 450-500 μ l elution buffer, centrifugal; Repeat once with the elution buffer washing; Abandon supernatant, add 70-80 μ l stock solution, 4 ℃ of preservations behind the mixing; Said elution buffer is 1 * PBS, 0.05% Tween-20.
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| CN1920562A (en) * | 2003-08-20 | 2007-02-28 | 香港神农有限公司 | Reagent kit for diagnosing EB virus ELISA and preparation thereof |
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| CN1920562A (en) * | 2003-08-20 | 2007-02-28 | 香港神农有限公司 | Reagent kit for diagnosing EB virus ELISA and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| Edward Morgan et al.Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.《Clinical Immunology》.2004,第110卷(第3期),252－266. * |
| Rudi Varro et al.Cytometric Bead Array to Measure Six Cytokines in Twenty-Five Microliters of Serum.《Clinical Chemistry》.2003,第49卷(第6期),1000-1002. * |
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