CN102426237B - ELISA-Array method for detection of encephalitis viruses, and special kit thereof - Google Patents
ELISA-Array method for detection of encephalitis viruses, and special kit thereof Download PDFInfo
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Abstract
The present invention discloses an ELISA-Array method for detection of encephalitis viruses, and a special kit thereof. The kit of the present invention comprises six capture antibodies, wherein the solution of each capture antibody is a mixing solution prepared by mixing the capture antibody and a spotting solution, the concentration of each capture antibody in the corresponding capture antibody solution is 0.05 mg/ml. Experimental results of the present invention show that: the six specific encephalitis virus monoclonal antibodies are prepared by the method of the present invention; with adopting the ELISA-Array technology platform, and optimizing the experimental conditions, the ELISA-Array technology capable of concurrent detection of the six encephalitis viruses is established, and can be adopted for detection of the six encephalitis-related viruses; compared to the common ELISA, the ELISA-Array method of the present invention has the following advantages that: the specificity of the ELISA-Array method is the same as the specificity of the common ELISA, the sensitivity is high, the highest sensitivity is more than 10 times the sensitivity of the common ELISA, and the ELISA-Array method has a high clinical application prospect.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of ELISA-Array method and dedicated kit thereof that detects encephalitis viroid.
Background technology
China is that encephalitis viroid endangers serious country, and correctly diagnosing pathogenic bacteria is bases of effectively controlling its propagation, is also to carry out the effectively key for the treatment of.The encephalitis viroid venereal disease substance that China is popular, mainly comprise: Japanese B encephalitis virus (Japanese Encephalitis virus, JEV), tick-brone encephalitis virus (Tick borne encephalitis virus, TBE), Eastern equine encephalitis virus (Eastern equine encephalitis virus, EEEV), sindbis virus (Sindbis Virus), dengue virus (Dengue virus, DV) etc., be the virus of arthropod-borne, wherein Japanese B encephalitis virus (Japanese Encephalliis virus, JEV), tick-brone encephalitis virus (Tick borne encephalitis virus, TBE), Eastern equine encephalitis virus (Eastern equine encephalitis virus, EEEV) can cause serious encephalitis symptom, and there is higher fatal rate, sindbis virus (Sindbis Virus) and dengue virus (Dengue virus, DV) clinical symptoms is comparatively slight, for above pathogen, should take clinically different treatment meanss and prevention and control measure, but above virus is arthropod-borne, all there is in early days similar symptom, therefore, set up method of early diagnosis accurately, prevention and control for encephalitis viroid disease have important value.
The at present existing multiple technical method that can simultaneously detect plurality of antigens, comprises two-phase gel electrophoresis, protein chip, mass spectrum, liquid phase suspending chip technology etc.But because complicated operation, cost are high, these technology are difficult to carry out clinical practice.
ELISA-Array technology is a kind of novel detection system, and it develops in traditional E LISA technical foundation, has gathered enzyme-linked immuno assay (ELISA) and microarray (Array) technology and the detection technique system of new generation that forms.Both protein chip technology high flux, high parallel feature had been acted on; This technology also keeps the maturation of ELISA method, the advantage that easy and simple to handle, cost is low simultaneously, for the ELISA-Array technology for detection of pathogen, domesticly there is no other unit research.
This technology is arranged in various antibody in microwell plate plate hole with the form of microarray by extensive full-automatic point sample equipment, can be used for detecting multiple pathogens simultaneously, and decades of times has improved the flux that ELISA detects.Because each micropore has been realized the multiple detection of a sample, in use, reduce clinical samples and detected the consumption of reagent.
In operative technique, this techniqueflow and traditional E LISA method are basic identical, and consumptive material used is 96 hole elisa plates, and instrument is enzyme connection automatic washing plate instrument, integrated enzyme reaction instrument, available volley of rifle fire application of sample, and in course of reaction, agents useful for same is also identical with traditional E LISA.Only colour developing and result reads process and traditional E LISA is variant.Replaced TMB (or DAB) substrate being used in traditional E LISA with chemical illuminating reagent, thereby further amplifying signal, with chip reading instrument test experience result, replaces enzyme connection detector.Therefore, applying unit can be continued to use most of existing ELISA pertinent instruments, and technician do not need very high specialty background and complicated training on operation, is easy to basic unit and detects acceptance and the popularization of unit.
At present, only there is both at home and abroad the research report of ELISA-Array technology for tumor markers, not yet for the detection of infectious disease.
Summary of the invention
An object of the present invention is to provide a kind of antibody group that detects encephalitis viroid.
Antibody group provided by the invention, comprises 6 kinds of capture antibodies;
Described 6 kinds of capture antibodies are respectively the antibody 4D5 of anti-Japanese B encephalitis virus, the antibody 2B5 of tick-borne encephalitis resisting virus, anti-sindbis virus's antibody 1F1, antibody 2B8, the antibody 4F9 of anti-dengue virus 4 types and the antibody 4E11 of anti-Eastern equine encephalitis virus of anti-dengue virus 2 types.
Described every kind of capture antibody all exists with the form of capture antibody solution;
Every kind of described capture antibody solution is all prepared as follows: the capture antibody solution that described capture antibody and sampling liquid are mixed to get, and the concentration of described capture antibody in the described capture antibody solution of correspondence is 0.025mg/ml-0.2mg/ml;
Described sampling liquid is prepared as follows: be that 0.01M, PH are 7.2 phosphate buffer mixing by glycerine, Triton-X100 and concentration, obtain sampling liquid, the concentration of described glycerine in described sampling liquid is 15%-25% (volumn concentration), and the concentration of described Triton-X100 in described sampling liquid is 0.001%-0.006% (volumn concentration).
Described capture antibody 2B8,4D5,2B5,4E111 and the 1F1 concentration in the described capture antibody solution of correspondence is 0.05mg/ml, and the concentration of described capture antibody 4F9 in the described capture antibody solution of correspondence is 0.1mg/ml;
The concentration of described glycerine in described sampling liquid is 20% (volumn concentration), and the concentration of described Triton-X100 in described sampling liquid is 0.004% (volumn concentration).
Described antibody group also comprises that 3 kinds are detected antibody: the antibody 2A10 of anti-japanese encephalitis virus, tick-brone encephalitis virus and/or dengue virus, anti-sindbis virus's antibody 1F1 and the antibody 4E11 of anti-Eastern equine encephalitis virus.
Tiring of described 2A10,1F1 and 4E11 is respectively (1: 320), (1: 80), (1: 80);
Every kind of described detection antibody all exists with the form of label mark detection antibody;
The label that described label mark detects in antibody is biotin.
During ELISA experiment detects, 3 strains detect antibody: biotin labeling 2A10, biotin labeling 1F1 and biotin labeling 4E11 tire, the antigen adopting is respectively tick-brone encephalitis virus inactivation antigen, sindbis virus's inactivation antigen, Eastern equine encephalitis virus inactivation antigen, and virus titer is respectively: 2 × 10
6pFU/ml, 2 × 10
7pFU/ml, 2.5 × 10
6pFU/ml, detects volume and is 50 μ L.
Described antibody group is made up of described 6 kinds of capture antibodies and described 3 kinds of detection antibody;
Every kind of antibody in described antibody group is independent packaging;
Every kind of antibody in described antibody group is monoclonal antibody;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue 2virus) and dengue virus 4 types (Dengue 4virus).
Another object of the present invention is to provide a kind of kit for detection of encephalitis viroid.
Kit provided by the invention, comprises described antibody group;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue 2virus) and dengue virus 4 types (Dengue 4virus).
The 3rd object of the present invention is to provide a kind of ELISA Plate that detects encephalitis viroid.
ELISA Plate provided by the invention, be prepared as follows: in each hole of ELISA Plate, add following 6 kinds of capture antibodies: the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus, the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus, anti-sindbis virus's monoclonal antibody 1F1, the monoclonal antibody 2B8 of anti-dengue virus 2 types, the monoclonal antibody 4F9 of anti-dengue virus 4 types and the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus, every kind of described capture antibody adds with the form of capture antibody solution;
In the solution of the monoclonal antibody 4D5 of described anti-Japanese B encephalitis virus, solvent is sampling liquid, and solute is the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus;
In the solution of the monoclonal antibody 2B5 of described tick-borne encephalitis resisting virus, solvent is sampling liquid, and solute is the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus;
In the solution of described anti-sindbis virus's monoclonal antibody 1F1, solvent is sampling liquid, and solute is anti-sindbis virus's monoclonal antibody 1F1, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of anti-sindbis virus's monoclonal antibody 1F1;
In the solution of the monoclonal antibody 2B8 of described anti-dengue virus 2 types, solvent is sampling liquid, and solute is the monoclonal antibody 2B8 of anti-dengue virus 2 types, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 2B8 of anti-dengue virus 2 types;
In the solution of the monoclonal antibody 4F9 of described anti-dengue virus 4 types, solvent is sampling liquid, and solute is the monoclonal antibody 4F9 of anti-dengue virus 4 types, and the concentration of solute in solution is 0.1mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4F9 of anti-dengue virus 4 types;
In the solution of the monoclonal antibody 4E11 of described anti-Eastern equine encephalitis virus, solvent is sampling liquid, and solute is the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus;
Described sampling liquid is prepared as follows: be that 0.01M, PH are 7.2 phosphate buffer mixing by glycerine, Triton-X100 and concentration, obtain sampling liquid, the concentration of described glycerine in described sampling liquid is 20% (volumn concentration), and the concentration of described Triton-X100 in described sampling liquid is 0.004% (volumn concentration).
The 4th object of the present invention is to provide a kind of kit that detects encephalitis viroid.
Kit provided by the invention, comprises described ELISA Plate and detects antibody mixed liquor;
The mixed liquor of described detection antibody is prepared as follows: the monoclonal antibody 4E11 of the monoclonal antibody 2A10 of anti-japanese encephalitis virus, tick-brone encephalitis virus and/or dengue virus, anti-sindbis virus's monoclonal antibody 1F1, anti-Eastern equine encephalitis virus is mixed with antibody diluent, obtain detecting the mixed liquor of antibody, described 2A10: 1F1: 4E11: the volume ratio of antibody diluent is 1: 4: 4: 320;
Tiring of described 2A10,1F1 and 4E11 is respectively (1: 320), (1: 80), (1: 80);
Described 2A10,1F1 and 4E11 all exist with the form of label labelled antibody;
Described label is specially biotin.
The application that described antibody group or described kit detect in encephalitis viroid product and/or detection encephalitis viroid in preparation is also the scope of protection of the invention;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue 2virus) and dengue virus 4 types (Dengue 4virus).
The 5th object of the present invention is to provide a kind of method that detects sample to be tested mesencephalitis viroid.
Method provided by the invention, comprises the steps:
1) 6 kinds of equal point samples of capture antibody in described antibody group or described kit, in point sample hole, are obtained to the point sample hole of containing capture antibody;
2) to step 1) add sample to be tested in the point sample hole that obtains, hatch, obtain point sample hole after application of sample;
3) to step 2) in point sample hole, add after the application of sample that obtains in described antibody group or described kit 3 kinds to detect the mixed liquor of antibody, hatch, obtain primary antibodie in conjunction with point sample hole;
4) to step 3) primary antibodie that obtains is in conjunction with the Avidin that adds HRP mark in point sample hole, hatches, and obtains two anti-binding point sample holes;
5) to step 4) Avidin of the HRP mark that obtains is in conjunction with adding chemical luminescence for liquid in point sample hole, scan detection, if detect, position at corresponding capture antibody is without interaction, sample do not contain or not candidate contain corresponding encephalitis viroid; If detect in corresponding capture antibody position and have interaction, determine and in sample, contain or candidate is contained corresponding encephalitis viroid.
Step 1) in, the volume ratio that adds of described 6 kinds of capture antibodies is 1: 1: 1: 1: 1: 1; Each capture antibody point sample amount is the every hole of 30nl/;
Step 2) in, described in the temperature of hatching be 37 DEG C, described in time of hatching be 2h; The addition of sample to be tested is 50ul/ hole;
Step 3) in, the mixed liquor of described detection antibody is prepared as follows: detect antibody by described 3 kinds and mix with antibody diluent, obtain 3 kinds of mixed liquors that detect antibody, described 2A10: 1F1: 4E11: the volume ratio of antibody diluent is 1: 4: 4: 320;
Described antibody diluent is to be that 0.01M, PH are 7.2 phosphate buffer containing the concentration of 5% calf serum;
Described temperature of hatching is 37 DEG C, described in time of hatching be 2h;
Step 4) in, described in the temperature of hatching be 37 DEG C, described in time of hatching be 1h;
In step 1) and step 2) between also comprise sealing, washing step.
Described sample to be tested is in vitro serum, cerebrospinal fluid or encephalitis viroid culture;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue 2virus) and dengue virus 4 types (Dengue 4virus).
Of the present invention experimental results show that, the present invention has prepared the monoclonal antibody specific of 6 kinds of encephalitis virusess, by utilizing this ELISA-Array technology platform, and carry out the optimization of experiment condition, set up the Array-ELISA technology that can simultaneously detect 6 kinds of encephalitis viroids, can be used for 6 kinds of encephalitis correlated virus (Japanese B encephalitis virus, tick-brone encephalitis virus, Eastern equine encephalitis virus, sindbis virus, dengue type 2 virus, dengue virus 4 types) detection, this technical method is compared with common ELISA, specificity is suitable, sensitivity is high, reach as high as more than 10 times, there is higher potential applicability in clinical practice.
Brief description of the drawings
Fig. 1 is the testing result of difference sample liquid and the different point sample concentration of 2B5 antibody
Fig. 2 is the testing result of 4D5,2B8,1F1,4E11, the different point sample concentration of 4F9 antibody
Fig. 3 is the ELISA-Array testing result of six kinds of encephalitis virusess
Fig. 4 is the ELISA-Array result that multiple encephalitis viruses detects simultaneously
Fig. 5 is the specific detection result of utilizing irrelevant antigen pair array 3
Fig. 6 is the testing result of 3 parts of TBEV infected patient serum
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The condition of embodiment 1, ELISA-Array is groped
One, the optimization of antibody spot sample condition
1, the preparation of monoclonal antibody and purifying
Monoclonal antibody 4D5,2B5,1F1,2B8,4F9,4E11 and 2A10 (are all documented in monoclonal antibody 2A10 and detect the application in flavivirus at protein chip technology, Sun Tingting, Li Yuchang, Liu Hong, Kang Xiaoping, Lin Fang, Zhu Qingyu, Yang Yinhui, Lu Cheng, Chinese Microbiology & Immunology magazine, the 8th phase of 30 volumes in 2010,775-778, the public all can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.Dengue virus monoclonal antiserum is learned property research, Liu Jinglan, Chen Wanrong, Cui Zhenmin, Xin Yanbin, Yang Baoan, Xu Pinfang, Zhu Qingyu, Yan Guozhen, Chinese Journal of Immunology, 02 phase in 1985,3-6 page, the public all can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.The preparation of dengue 2-type virus monoclonal antibody and qualification, Xin Yanbin, Yang Baoan, Xu Pinfang, Zhu Qingyu, Yan Guozhen, institute of Military Medical Science Institute periodical, the 10th the 1st phase of volume in 1986,59-62 page, the public all can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.) all prepare by hybridoma technology, and carry out purifying by affinity chromatography, obtain 7 kinds of antibody.Wherein, 4D5,2B5,1F1,2B8,4F9,4E11 is capture antibody.
The pathogen of every strain antibody institute target is as shown in Table 2 below, specific as follows:
4D5 is the monoclonal antibody of anti-Japanese B encephalitis virus (JEV); 2B5 is the monoclonal antibody of tick-borne encephalitis resisting virus (TBEV); 1F1 is the monoclonal antibody of anti-sindbis virus (SV); 2B8 is the monoclonal antibody of anti-dengue virus 2 types (DV-2); 4F9 is the monoclonal antibody of anti-dengue virus 4 types (DV-4); 4E11 is anti-Eastern equine encephalitis virus (EEEV) monoclonal antibody;
The conserved sequence of monoclonal antibody 2A10 target flavivirus, can be used as the common detection antibody of japanese encephalitis virus, tick-brone encephalitis virus and dengue virus, and 1F1 and 4E11 are both as capture antibody, again as detecting antibody.Utilize biotin labeling kit (biotin-NHS ester, Pierce, Germany, catalog number: 1854210) 3 strains are detected to antibody (2A10,1F1 and 4E11) carry out biotin labeling, labeling process operates to specifications and carries out, obtain 3 kinds and detect antibody: biotin labeling 2A10, biotin labeling 1F1 and biotin labeling 4E11, can be respectively after can specific detection being combined with viral antigen to this 3 strain antibody by common ELISA experiment and Avidin specific binding, the successful mark biotin of this 3 strain antibody is described.
During ELISA experiment detects, 3 strains detect antibody: biotin labeling 2A10, biotin labeling 1F1 and biotin labeling 4E11 tire, the antigen adopting is respectively tick-brone encephalitis virus inactivation antigen (preparation method sees below), sindbis virus's inactivation antigen (preparation method sees below), Eastern equine encephalitis virus inactivation antigen (preparation method sees below), and virus titer is respectively: 2 × 10
6pFU/ml, 2 × 10
7pFU/ml, 2.5 × 10
6pFU/ml, detects volume and is 50 μ L.Result is biotin labeling 2A10, and biotin labeling 1F1 and tiring of this 3 strain antibody of biotin labeling 4E11 are respectively: 1: 320,1: 80,1: 80.
2, Virus culture
(public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL Japanese B encephalitis virus (JEV), and the non-patent literature of recording this material is: Yang Yinhui, Zhu Xiaoguang, Zhang Yongguo, Kang Xiao equality.Utilize examination and the qualification of biochip technology to a strain unknown virus.China's laboratory medicine magazine, 2007, 30 (12): 1360-1363), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL dengue type 2 virus, the non-patent literature of recording this material is: Murthy HM, Clum S, Padmanabhan R.Dengue virus NS3 serine protease.Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects.J Biol Chem, 1999, 274 (9): 5573-5580.) and dengue virus 4 types (Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4virus, Laassri M, Bidzhieva B, Speicher J, Pletnev AG, Chumakov L., J.Med.Vorol., 2011May, 83 (5), 910-920, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.) inoculation C6/36 (purchased from ATCC, CRL-1660) cultivate;
(public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL sindbis virus, the non-patent literature of recording this material is: Xiong Hongyan, Liu Yujing etc., short wave ultraviolet and the research of crosslinked starch iodine to sindbis virus's deactivation in blood plasma, China's Disinfection magazine, the fourth phase in 1998), tick-brone encephalitis virus (TBEV, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, the non-patent literature of recording this material is: Hu Yuyang, Yang Yinhui, Liu Hong, Kang Xiaoping, Zhu Xiaoguang, take charge of bright silver, Zhu Qingyu, the foundation of russian spring-summer encephalitis virus (TBEV) real-time quantitative TaqMan PCR detection method, PLA's medical journal 2006, 31, 745-748) and EEEV (Eastern equine encephalitis virus EEEV strain SSP.North American Variant, GenBank accession number: X63135, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, the non-patent literature of recording this material is: Volchkov.V E, Volchkova.V.A.and Netesov SV, Complete nucleotide sequence of the Eastern equine encephalitis virus genome.Mol.Gen.Mikrobiol.Virusol.1991, 5, 8-15) inoculation BHK-21 cell is (purchased from ATCC, CAT.CCL 10) cultivate.
Incubation is all carried out in BSL-2 or BSL-3 laboratory, concrete cultivation is as follows: virus inoculation 3-4 days respectively, collecting cell culture supernatant after pathology appears in cell, carry out plaque test to determine virus titer (operation steps reference: medical experimental virology, 1985, Beijing, People's Medical Officer Press publishes), the virus titer of 6 kinds of culture supernatant is respectively: Japanese B encephalitis virus (JEV) culture 10
6pFU/ml, dengue type 2 virus culture 10
6pFU/ml, dengue virus 4 type cultures 1.5 × 10
6pFU/ml, sindbis virus's culture 2 × 10
7pFU/ml, TBEV tick-brone encephalitis virus culture 2 × 10
6pFU/ml, EEEV culture 10
7pFU/ml.
Spend the night and carry out inactivation of virus to adding in 5 kinds of Virus culture supernatants beta-propiolactone to final concentration to be 0.025%, 4 DEG C respectively.Within second day, place 1h in 37 DEG C, beta-propiolactone is divided and taken off, obtain respectively deactivation Japanese B encephalitis virus (JEV) culture, deactivation dengue type 2 virus culture, deactivation dengue virus 4 type cultures, deactivation sindbis virus culture (sindbis virus's inactivation antigen), deactivation TBEV tick-brone encephalitis virus culture (tick-brone encephalitis virus inactivation antigen), deactivation EEEV culture (Eastern equine encephalitis virus inactivation antigen).
3, the optimization of antibody spot sample condition
In point sample process, the result of use of difference sample liquid and point sample concentration is all assessed, in order to grope best point sample condition.Respectively above-mentioned 6 kinds of capture antibodies are diluted to point sample with 3 kinds of different sampling liquids: 1, (phosphate buffer of 0.01M, PH7.2's PBS (fills a prescription as follows: in 1L water, contain Na
2hPO
412H
2o 2.86g, NaCl8.5g, NaH
2pO
42H
2o 0.312g), 2, containing the PBS of 20% (volumn concentration) glycerine, 3, containing the PBS of 20% glycerine and 0.004% (volumn concentration) Triton-X100.
By observe and analysis result in the shape of each check point and signal intensity determine the point sample concentration of the suitableeest sampling liquid and antibody:
1) get respectively 3 kinds of different antibody spot sample liquid dilution capture antibodies, 2 times of serial dilutions, concentration is 0.0125,0.025,0.05,0.1 and 0.2mg/ml, totally 5 different point sample concentration;
2) utilize point sample instrument (BD6000; California, USA) by 6 kinds of equal point samples of capture antibody in point sample hole, 6 capture antibodies of each hole mid point, and the sample application array 1 in each hole is as shown in table 1A, puts in elisa plate (each capture antibody point sample amount is the every hole of 30nl/);
3) after point sample, seal with the PBS containing 3%BSA, put 1h in 37 DEG C of incubators, then wash 3 times with the PBS (PBST) of the Tween-20 containing 0.1%.Then elisa plate is dried, sealing, puts 4 DEG C of preservations stand-by;
4) adopt the pattern of double-antibody sandwich to detect.Be 5 × 10 by the TBEV viral cultures PBS dilution of the deactivation being obtained by above-mentioned steps 2
5pFU/ml, adds in ELISA-Array microwell plate, and 2h is hatched for 37 DEG C in 50ul/ hole.PBS-T washing three times;
5) add biotin labeling antibody mixed liquor (the biotin labeling 2A10 with antibody diluent dilution, biotin labeling 1F1, the volume ratio of biotin labeling 4E11 and antibody diluent is respectively 1: 4: 4: 320, antibody diluent is the PBS containing 5% calf serum), hatch 2h for 37 DEG C.PBS-T washing three times;
6) add the Avidin (health is ShiJi Co., Ltd's product, CW0124) of HRP mark of dilution in 1: 50,50ul/ hole, hatches 1h for 37 DEG C.PBS-T washing three times;
7) add chemical luminescence for liquid, 50ul/ hole, puts scanning in biological chip reading apparatus by microwell plate and detects.And carry out interpretation of result with Monster software.
Table 1A antibody spot sample array 1
| 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 |
Table 1B antibody spot sample array 2
| 1 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |
The pathogen of capture antibody institute target in table 2 antibody spot sample array 1 and array 2
As shown in Figure 1, wherein, A is the testing result figure as sampling liquid containing the PBS of 20% glycerine to testing result; B is the testing result figure as sampling liquid containing the PBS of 20% glycerine and 0.004%Triton-X100; C is the testing result figure using PBS as sampling liquid; D is the Analysis of test results of 3 kinds of difference sample liquids and different capture antibody concentration, the horizontal ordinate of D figure is variable concentrations capture antibody, ordinate is testing result signal intensity, the positive control antibodies sheep anti-mouse igg of P (Beijing Kang Wei ShiJi Co., Ltd product, catalog number (Cat.No.) is CW0147), in order to the system Quality Control point as detecting.
Can find out from figure A-C, first the form of point is observed, wherein PBS (figure C) and comparatively mellow and full, regular containing two kinds of sampling liquid institute point sample forms of PBS (figure B) of 20% glycerine and 0.004%Triton-X100, and have comparatively serious hangover containing the PBS sampling liquid institute point sample form of 20% glycerine, form irregular (figure A).
And then the testing result of 3 kinds of difference sample liquid institute point samples of comparison, there is notable difference in the result of use of 3 kinds of different sampling liquids, identical TBE virus detection limit, use 2B5 (specific antibody of the TBEV) signal intensity of PBS sampling liquid to be starkly lower than other two kinds, and the capture antibody form of putting containing the PBS sampling liquid of 20% glycerine is poor, easily produce hangover, be unfavorable for interpretation of result, and there is higher non-specific signal in the point sample antibody location of 4D5 and 2B8 in testing result, therefore the result of comprehensive point sample morphologic observation and detection signal strength, get the PBS that contains 20% glycerine+0.004%Triton-X100 as the suitableeest sampling liquid of this research.
Utilize antibody array 1, by detecting tick-brone encephalitis virus to determine suitable antibody spot sample concentration, in this experiment, 2B5 is special capture antibody, 2B8 and 4D5 are as negative control antibody, result is as shown in Fig. 1 D, can find out, antibody using the PBS of 20% glycerine+0.004%Triton-X100 as sampling liquid, the point sample concentration of 2B5 antibody is from 0.0125mg/ml to 0.2mg/ml, signal value strengthens with point sample Enrichment, to 0.05mg/ml, signal value reaches capacity, and the signal value of 2B8 and two negative control antibodies of 4D5 is all negative, therefore, get 0.05mg/ml as suitable capture antibody concentration.
And then utilize array 1 (table 1A) to detect DV-2 and JEV, and in order to grope the best point sample concentration of 2B8 and 4D5, detection method is the same, and the virus quantity of detection is JEV 2 × 10
5pFU/ML, DV-22 × 10
5pFU/ML, 50 μ l/ holes are detected, result is as shown in Fig. 2 A (testing results of the different point sample concentration of 2B8 antibody) and Fig. 2 B (testing results of the different point sample concentration of 4D5 antibody), in figure, represent the different point sample concentration of capture antibody with horizontal ordinate, with ordinate representation signal intensity, result shows that detection signal strength strengthens with capture antibody Enrichment, to 0.05mg/ml, signal reaches capacity, and therefore, 0.05mg/ml is the best point sample concentration of 2B8 and 4D5.
The best point sample concentration of utilizing array 2 (table 1B) to grope 4E11,1F1 and 4F9, detection method is the same, and the virus quantity of detection is respectively, EEEV 2.5 × 10
6pFU/ml, SV 5 × 10
6pFU/ml, DV-410
6pFU/ml, 50 μ l/ hole results are as shown in Fig. 2 C-2E, Fig. 2 C is the testing result of the different point sample concentration of 4E11, Fig. 2 D is the testing result of the different point sample concentration of 1F1, Fig. 2 E is the testing result of the different point sample concentration of 4F9, can find out, the best point sample concentration of 4F9 is 0.1mg/ml, and the best point sample concentration of 1F1 and 4E11 is 0.05mg/ml.
Two, the sensitivity of ELISA-Array technology for detection and specificity
Sensitivity and specificity are the important indicators of a kind of detection method quality of assessment.In order to determine detection sensitivity and the specificity of this ELISA-Array technology, to DV-2, DV-4, JEV, TBE, SV, the detection sensitivity of the pathogen such as EEEV is all measured.Antibody arranged array on detection microwell plate as shown in Table 3 and Table 4.
Table 3 antibody arranged array 3
| 1 | 2 | 3 |
| 4 | 5 | 6 |
| 7 |
The pathogen of table 4 antibody arranged array 3 and antibody institute target
A, ELISA-Array technology detecting method
1, utilize chip point sample instrument point ELISA microwell plate, respectively 6 kinds of capture antibodies (by 1 capture antibody obtaining of embodiment 1 step 1) being diluted respectively with the PBS containing 20% glycerine+0.004%Triton-X100 is concentration shown in table 4, employing utilizes microarray 3 (table 4) to carry out the detection of Virus Sample, and point sample method is with 3 of the step 1 of embodiment 1;
2, respectively by the viral cultures (JEV of the deactivation being obtained by 2 of embodiment 1 step 1, TBEV, SV, DV-2, DV-4, EEEV) 2 times of serial dilutions, every kind of virus is diluted 8 concentration altogether, adds respectively in ELISA-Array microwell plate, 50ul/ hole, hatches 2h for 37 DEG C.PBS-T washing three times;
3, add biotin labeling antibody (the biotin labeling 2A10 being obtained by 1 of embodiment 1 step 1 of antibody diluent dilution, the volume ratio of biotin labeling 1F1, biotin labeling 4E11 and antibody diluent is respectively: 1: 4: 4: 320, antibody diluent is the PBS containing 5% calf serum), hatch 2h for 37 DEG C.PBS-T washing three times;
4, the Avidin (health is ShiJi Co., Ltd's product, catalog number: CW0124) that adds the HRP mark of dilution in 1: 50,50ul/ hole, hatches 1h for 37 DEG C.PBS-T washing three times;
5, add chemical luminescence for liquid, 50ul/ hole, puts scanning in biological chip reading apparatus by microwell plate and detects.And carry out interpretation of result with Monster software.
B, common elisa technique detection method
1, be 2ug/ml by 6 kinds of capture antibody dilutions respectively with coating buffer (sodium carbonate-sodium bicarbonate buffer liquid, PH9.6), 50ul/ hole, 4 DEG C of coated spending the night;
2, PBS-T washs after 3 times, adds confining liquid (containing the PBS of 3%BSA), 200ul/ hole; Put in 37 DEG C of incubators and act on 1h;
3, by 2 times of serial dilutions of the viral cultures of deactivation (JEV, TBEV, SV, DV-2, DV-4, EEEV), 8 concentration of every kind of viral dilution, add respectively in ELISA-Array microwell plate, and 50ul/ hole, hatches 2h for 37 DEG C.PBS-T washing three times;
4, add biotin labeling antibody (the biotin labeling 2A10 being obtained by 1 of embodiment 1 step 1 of antibody diluent dilution, the volume ratio of biotin labeling 1F1, biotin labeling 4E11 and antibody diluent is respectively: 1: 4: 4: 320, antibody diluent is the PBS containing 5% calf serum), hatch 2h for 37 DEG C.PBS-T washing three times;
5, the Avidin (health is ShiJi Co., Ltd's product, catalog number: CW0124) that adds the HRP mark of dilution in 1: 50,50ul/ hole, hatches 1h for 37 DEG C.PBS-T washing three times;
6, add substrate nitrite ion, 50ul/ hole, lucifuge is placed 5-15min, then adds the sulfuric acid cessation reaction of 2M, 50ul/ hole;
7, enzyme connection detector detects OD450.
The comparison of C, sensitivity result
ELISA is current clinical the most frequently used detection method of carrying out pathogen antigen mensuration, and therefore, this research has been carried out common ELISA simultaneously and tested, and carries out the comparison of detection sensitivity, and the minimum virus quantity that two kinds of methods can detect is as shown in table 5.
The minimum virus quantity that two kinds of ELISA methods of table 5 can detect
Result shows, the sensitivity that detects except two kinds of methods of sindbis virus is about the same, all the other 5 kinds of viruses, the detection sensitivity of ELISA-Array is all than the high 5-10 of common ELISA doubly, this testing result shows, the detection sensitivity of ELISA-Array is significantly higher than common ELISA generally.
D, specific testing result
In ELISA-Array experiment (array 3), every kind of virus-specific result is as shown in Fig. 3 (ordinate is fluorescence intensity), and wherein, A is Japanese B encephalitis virus (JEV); B is tick-brone encephalitis virus (TBEV); C is sindbis virus (SV); D is Dengue-4 type viruses (DV-4); E is dengue type 2 virus (DV-2); F is Eastern equine encephalitis virus (EEEV), the positive control antibodies sheep anti-mouse igg of p in each figure, and in specific detection experiment, five kinds of viral antigens to be measured are gained after 4 times of dilutions of Virus culture supernatant.
From every kind of viral testing result figure, can find out, only there are the positive control antibody in the upper left corner and the specific antibody of virus to be measured to occur positive signal, the position of other capture antibodies is and occurs positive signal, illustrate that this antibody array testing result is special, for detection of five kinds of pathogen there is not each other cross reaction.
Specificity in order further to determine that this ELISA-Array method detects other irrelevant antigens simultaneously, utilize Vero cell (purchased from ATCC, CAT.CCL-81) culture supernatant, BHK-21 cell is (purchased from ATCC, CAT.CCL 10) culture supernatant, yellow fever virus (Yellow fevervirus, YFV, the one-step RT-PCR method of yellow fever virus detects, Peng Wenming, Deng Yongqiang, Yu Man, Fan Baochang, Zhu Qingyu, Qin Ede, microorganism magazine, the fourth phase in 2003, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.) culture, (datum hole Kenya virus is documented in Shi Huafang to datum hole Kenya viral cultures, Zhang Hailin, from stepping on cloud, Li Zhaoxiang etc., Yunnan is separated to datum hole Kenya virus first in patient body, China Amphixenosis magazine, first phase nineteen ninety, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.) react as negative antigen.Yellow fever virus, datum hole Kenya virus are all by inoculation BHK-21 acquisition that cell is cultivated.Virus culture supernatant adopts virus plaque method for measuring to carry out virulence titration, and the culture supernatant titre of yellow fever virus is 5 × 10
6pFU/ml, it is 2 × 10 that datum hole is agree subviral titre
6pFU/ml, two-strain is carrying out before ELISA-Array experiment detects all carrying out inactivation treatment (related content in specifically with embodiment 1) with beta-propiolactone, the same A of detection method.
As shown in Figure 4, in experiment, the working concentration of all negative control antigen is 1mg/ml to result, and all negative control testing results only have the position in the upper left corner 1 to have bright spot (positive), and other position does not have positive signal to occur.
All there is not positive test symbol at ELISA-Array and common ELISA in above-mentioned negative antigen, illustrates that ELISA-Array and common ELISA all have higher detection specificity.
The detection of embodiment 2, multiple virus mixture
In order to determine whether this ELISA-Array technology can be used for the detection of hybrid infection viruses, getting appropriate JEV, TBEV, DV2, EEEV, DV4 and SV Virus culture supernatant two or three virus mixes rear (in the detection of multiple virus mixture, every kind of viral use amount is the Virus culture supernatant of 4 times of dilutions), add in microwell plate and detect according to the ELISA-Array technology detecting method of the A of the step 2 of embodiment 1
As shown in Figure 5, A is DV-2 and EEEV to result; B is TBEV and DV2; C is TBEV and EEEV; D is JEV, TBEV and DV-4; E is DV-2, DV-4 and TBEV, use be array 3, as shown in table 3 and table 4.Can find out, all occur positive signal at the antibody of surveyed pathogen target, show that this technology can be used for the detection of multiple pathogens mixed infection.
The detection of embodiment 3, clinical sample inoculated into chick embryo culture
In order to verify the detection effect of ELISA-Array technology to clinical sample, get positive patients serum (Mudanjiang forestry central hospital that 3 TBEV infect, patient knows the inside story, these 3 parts of serum have been that TBEV infects the positive with immuno-fluorescence assay) inoculated into chick embryo allantoic cavity, put in 37 DEG C of incubators and cultivate 3, collect liquid in allantoic cavity, with after beta-propiolactone inactivation of viruses, obtain TBEV tick-brone encephalitis virus sample 1, 2, 3, carry out respectively real-time quantitative RT-PCR detection (upstream primer 5-GGG CGG TTC TTG TTC TCC-3, downstream primer 5-ACA CAT CAC CTC CTT GTCAGA CT-3, probe sequence 5-FAM-TGA GCC ACC ATC ACC CAG ACA CA-TAMRA-3, template is TBEV tick-brone encephalitis virus sample 1, 2, 3 RNA extract) and detect according to the ELISA-Array technology in the A of the step 2 of embodiment 1, with Vero cell (purchased from ATCC, CAT.CCL-81) the negative contrast of culture supernatant, with the positive contrast of inactivation antigen of TBEV.
Result as shown in Figure 6, wherein, the detection that A is common real-time quantitative RT-PCR; B is that ELISA-Array experiment detects, from Fig. 6 B, can find out, there is significant positive signal in the position at the specific antibody 2B5 of TBEV, and there is not signal in the detection antibody location of other virus, show that this sample is that TBEV infects sample, identical with RT-PCR testing result, illustrate that ELISA-Array technology detecting method testing result of the present invention is correct.
Claims (8)
1. detect an antibody group for encephalitis viroid, comprise 6 kinds of capture antibodies;
Described 6 kinds of capture antibodies are respectively the antibody 4D5 of anti-Japanese B encephalitis virus, the antibody 2B5 of tick-borne encephalitis resisting virus, anti-sindbis virus's antibody 1F1, antibody 2B8, the antibody 4F9 of anti-dengue virus 4 types and the antibody 4E11 of anti-Eastern equine encephalitis virus of anti-dengue virus 2 types;
Described every kind of capture antibody all exists with the form of capture antibody solution;
Every kind of described capture antibody solution is all prepared as follows: the capture antibody solution that capture antibody and sampling liquid are mixed to get, and the concentration of described capture antibody in the described capture antibody solution of correspondence is 0.025mg/ml-0.2mg/ml;
Described sampling liquid is prepared as follows: be that 0.01M, pH are 7.2 phosphate buffer mixing by glycerine, Triton-X100 and concentration, obtain sampling liquid, the volumn concentration concentration of described glycerine in described sampling liquid is 15%-25%, and the volumn concentration concentration of described Triton-X100 in described sampling liquid is 0.001%-0.006%.
2. antibody group according to claim 1, is characterized in that:
Described capture antibody 2B8,4D5,2B5,1F1 and the 4E11 concentration in the described capture antibody solution of correspondence is 0.05mg/ml, and the concentration of described capture antibody 4F9 in the described capture antibody solution of correspondence is 0.1mg/ml;
The volumn concentration concentration of described glycerine in described sampling liquid is 20%, and the volumn concentration concentration of described Triton-X100 in described sampling liquid is 0.004%.
3. antibody group according to claim 2, is characterized in that:
Described antibody group also comprises that 3 kinds are detected antibody: at least one viral antibody 2A10, anti-sindbis virus's antibody 1F1 and the antibody 4E11 of anti-Eastern equine encephalitis virus in anti-japanese encephalitis virus, tick-brone encephalitis virus and dengue virus.
4. antibody group according to claim 3, is characterized in that:
Tiring of described 2A10,1F1 and 4E11 is respectively 1:320,1:80,1:80;
Every kind of described detection antibody all exists with the form of label mark detection antibody;
The label that described label mark detects in antibody is biotin;
Described antibody group is made up of described 6 kinds of capture antibodies and described 3 kinds of detection antibody;
In described antibody group, every kind of antibody is independent packaging;
In described antibody group, every kind of antibody is monoclonal antibody;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue2virus) and dengue virus 4 types (Dengue4virus).
5. for detection of a kit for encephalitis viroid, comprise the antibody group described in claim 1-4;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue2virus) and dengue virus 4 types (Dengue4virus).
6. one kind is detected the ELISA Plate of encephalitis viroid, be prepared as follows: in each hole of ELISA Plate, add following 6 kinds of capture antibodies: the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus, the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus, anti-sindbis virus's monoclonal antibody 1F1, the monoclonal antibody 2B8 of anti-dengue virus 2 types, the monoclonal antibody 4F9 of anti-dengue virus 4 types and the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus, every kind of described capture antibody adds with the form of capture antibody solution;
In the solution of the monoclonal antibody 4D5 of described anti-Japanese B encephalitis virus, solvent is sampling liquid, and solute is the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4D5 of anti-Japanese B encephalitis virus;
In the solution of the monoclonal antibody 2B5 of described tick-borne encephalitis resisting virus, solvent is sampling liquid, and solute is the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 2B5 of tick-borne encephalitis resisting virus;
In the solution of described anti-sindbis virus's monoclonal antibody 1F1, solvent is sampling liquid, and solute is anti-sindbis virus's monoclonal antibody 1F1, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of anti-sindbis virus's monoclonal antibody 1F1;
In the solution of the monoclonal antibody 2B8 of described anti-dengue virus 2 types, solvent is sampling liquid, and solute is the monoclonal antibody 2B8 of anti-dengue virus 2 types, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 2B8 of anti-dengue virus 2 types;
In the solution of the monoclonal antibody 4F9 of described anti-dengue virus 4 types, solvent is sampling liquid, and solute is the monoclonal antibody 4F9 of anti-dengue virus 4 types, and the concentration of solute in solution is 0.1mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4F9 of anti-dengue virus 4 types;
In the solution of the monoclonal antibody 4E11 of described anti-Eastern equine encephalitis virus, solvent is sampling liquid, and solute is the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus, and the concentration of solute in solution is 0.05mg/ml; In each hole, add the solution 30nl of the monoclonal antibody 4E11 of anti-Eastern equine encephalitis virus;
Described sampling liquid is prepared as follows: be that 0.01M, pH are 7.2 phosphate buffer mixing by glycerine, Triton-X100 and concentration, obtain sampling liquid, the volumn concentration concentration of described glycerine in described sampling liquid is 20%, and the volumn concentration concentration of described Triton-X100 in described sampling liquid is 0.004%.
7. detect a kit for encephalitis viroid, comprise ELISA Plate claimed in claim 6 and detect antibody mixed liquor;
The mixed liquor of described detection antibody is prepared as follows: the monoclonal antibody 4E11 of at least one viral antibody 2A10, monoclonal antibody 1F1 of anti-sindbis virus, anti-Eastern equine encephalitis virus in anti-japanese encephalitis virus, tick-brone encephalitis virus and dengue virus is mixed with antibody diluent, obtain detecting the mixed liquor of antibody, described 2A10:1F1:4E11: the volume ratio of antibody diluent is 1:4:4:320;
Tiring of described 2A10,1F1 and 4E11 is respectively 1:320,1:80,1:80;
Described 2A10,1F1 and 4E11 all exist with the form of label labelled antibody;
Described label is specially biotin.
8. in claim 1-4, the kit described in arbitrary described antibody group or ELISA Plate claimed in claim 6 or claim 5 or 7 detects the application in encephalitis viroid product in preparation;
Described encephalitis viroid is at least one in following 6 kinds of encephalitis virusess: Japanese B encephalitis virus (Japanese Encephalitis virus), tick-brone encephalitis virus (Tick borne encephalitis virus), Eastern equine encephalitis virus (Eastern equine encephalitis virus), sindbis virus (Sindbis Virus), dengue type 2 virus (Dengue 2 virus) and dengue virus 4 types (Dengue 4 virus).
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| CN103575909B (en) * | 2013-11-05 | 2015-12-02 | 北京出入境检验检疫局检验检疫技术中心 | A kind of ELISA kit detecting eastern equine encephalomyelitis virus IgM antibody |
| CN106800600B (en) * | 2015-11-26 | 2020-06-05 | 中国人民解放军军事医学科学院微生物流行病研究所 | System for detecting tick-borne encephalitis virus infection |
| CN108508209A (en) * | 2018-02-12 | 2018-09-07 | 合肥知恩生物技术有限公司 | A kind of people's B encephalitis virus antibody solid phase competitive ELISA detection kit and preparation method thereof and detection method |
| CN108398550B (en) * | 2018-03-07 | 2022-07-26 | 深圳市伯劳特生物制品有限公司 | Composition, chip, preparation method of chip and detection device comprising chip |
| CN108535482A (en) * | 2018-04-11 | 2018-09-14 | 上海萨迦生物科技有限公司 | A kind of antibody chip kit for tumour early screening and diagnosis |
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