CN101551393B - An immunodiagnostic kit for detecting type IV dengue virus NS1 antigen - Google Patents
An immunodiagnostic kit for detecting type IV dengue virus NS1 antigen Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses an immunodiagnosis kit for detecting specific IV-type dengue virus antigen, comprising a microporous reaction plate coated with monoclonal antibody 2E8A5, sample treatment fluid, monoclonal antibody 7A6A1 combined with markers, positive control, negative control, wash concentrate, color developing solution and stop solution. The kit can detect IV-type dengue virus antigen specifically, does not cross-react with other three types of serotype dengue virus antigens and detects that the supernatant sensitivity of the IV-type dengue virus is 64 times of that of the commercial kit of the same type, namely, the Pan-E Dengue Early ELISA test kit, thus greatly improving the sensitivity of detection of clinical serum samples and simultaneously reducing omission rate in clinical applications.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of medicinal preparation, especially for the kit of diagnosis IV type dengue virus.
Background technology
Dengue fever is a kind of tropical infectious disease, propagates through Aedes aegypti and aedes albopictus, is divided into 4 serotypes according to the antigenicity difference of E albumen, is respectively I type, II type, III type and IV type (being called for short DV1, DV2, DV3 and DV4).The dengue virus infection of any type all can cause a series of clinical symptoms, shows as subclinical infection, heating, dengue fever or even more serious dengue hemorrhagic fever and dengue shock syndrome.In recent years, because the prophylactico-therapeutic measures of the development of international tourism and communicable disease is improper, dengue fever is widely current in the torrid zone and semi-tropical more than 100 countries and regions, and particularly Southeast Asia one band is popular very serious.At present, have 2,500,000,000 people to receive the threat of dengue virus infection in the world approximately, annual generation dengue virus infection patient surpasses 100,000,000 people, and has 500,000 people to develop into dengue hemorrhagic fever or dengue shock syndrome, causes about 30000 people dead.The primary infection dengue virus can produce lifelong immanoprotection action for the subinfection again of homologous virus; But the infection once more for other serotype lacks the cross immunity protective effect; Owing to there is the virus infections enhancement effect (ADE) of antibody dependence, special-shaped dengue virus subinfection again is to cause the dengue fever patient that the primary hazards of dengue hemorrhagic fever take place simultaneously.And in stepping on the leather epidemic-stricken area, often have the alternately popular of four type dengue virus, and increased the danger of the other dengue virus repeated infection of different serotypes, more increased the possibility that dengue hemorrhagic fever takes place.
Because the existence of ADE has increased the difficulty of stepping on the leather vaccine development, at present, the leather vaccine of stepping on protectiveness is not succeeded in developing as yet, clinically to the dengue virus infection medicine of lack of specific also.Practice shows, make a definite diagnosis the M & M that can reduce dengue hemorrhagic fever greatly in early days, so the diagnosis of early infection is depended in the treatment of dengue fever to a great extent.But the special clinical manifestation of the early stage shortage of most dengue virus infection persons only has influenza-like symptoms such as heating, shiver with cold, is difficult to and other fever diseases and the differentiation of Hemorrhagic fever disease, must depend on breadboard diagnosis.
The laboratory diagnostic method of dengue fever mainly comprises viral separation, antibody and detection of nucleic acids at present.Wherein the virus separation is the goldstandard that dengue virus infection diagnosis and serotype are identified, but this method is time-consuming and higher for breadboard conditional request.Although the detection method of viral nucleic acid is sensitiveer, quicker than traditional isolation of virus; But the molecular diagnosis operation is loaded down with trivial details relatively; Technical merit is had relatively high expectations; And be prone to take place to pollute cause false positive results, because changing, sequences such as the variation of strain, potential sudden change also possibly cause false negative result simultaneously.And antibody test reagent can not be used for early diagnosis; And because between 4 serotype dengue virus and and other Tobamovirus between have serological cross reaction, the crowd who particularly inoculates japanese encephalitis virus, yellow fever virus vaccine is prone to take place antibody false positive reaction result.Therefore, set up that a kind of can specificity to distinguish the early diagnosis kit that four serotypes of dengue virus infect imperative.
The non-structural protein 1 of dengue virus (nonstructural protein 1; NS1) be a kind of conservative relatively glycoprotein; Two kinds of forms of membranous type and secreting type are arranged, in infection cell, highly express, have very strong antigenicity; Therefore and discover the NS1 CAg that in dengue fever patient's early stage blood, has high concentration, detect the generation that circulation NS1 antigen among the acute stage patients serum can be used for early diagnosis dengue virus infection or early warning dengue hemorrhagic fever (DHF).The method that detects dengue virus through the antigen capture ELISA method based on dengue virus NS 1 can be divided into two types; One type is on the basis of polyclonal antibody, to set up; There is very big-difference in these class methods in the antiserum of different batches, be difficult to repetition and realize breadboard standardization.The monoclonal antibody of the another kind of NS1 of utilization detects dengue virus; Like commercial kit Pan-E Dengue Early ELISA test kit (Panbio; Queensland, Australia) with Platelia Dengue NS1 Ag kits (Bio-Rad, France).Pan-EDengue Early ELISA test kit contains the monoclonal antibody of anti-I～IV type dengue virus NS 1, realizes the early diagnosis of dengue virus infection through the ELISA method, and its susceptibility, specificity and stability all are enhanced.But the inventor further discovers; This kit is different to the susceptibility of the dengue virus of I～IV type; Be respectively 195PFU/mL, 195PFU/mL, 1563PFU/mL, 25000PFU/mL; And the viral level in the blood samples of patients sample of early infection dengue virus is relatively low, and the omission phenomenon might appear in the infection that detects III type and IV type dengue virus with this kit.Moreover; These two kinds of kits can not be distinguished the dengue virus of four kinds of serotypes; Because the caused clinical symptoms of dengue virus infection of different serotypes is different with therapeutic scheme; The clinician must confirm the dengue virus infection of which kind of serotype as early as possible, so that take therapy measure fast.Therefore, in time confirming the type of dengue virus infection, is the prerequisite that reduces the dengue virus infection mortality ratio, also is the effective measures of control dengue virus diffusion, and on the market commercial kit all can't reach this purpose at present.
Summary of the invention
Technical matters to be solved by this invention is the deficiency that overcomes prior art; A kind of have high sensitivity and specific IV type dengue virus NS 1 antigen immunodiagnosis kit are provided; It can directly detect the DV4 NS1 antigen in the blood preparation, realizes the early stage classification diagnosis of dengue virus infection.
In order to solve above technical matters; The present invention provides a kind of immunodiagnosis kit of the IV of detection type dengue virus NS 1 antigen; This kit is based on the detection kit of double-antibodies sandwich ELISA; Form by the micro reaction plate that encapsulates capture antibody, sample preparation liquid, the detection antibody that combines with label, positive control, negative control thing, concentrated washing lotion, colour developing liquid and stop buffer; It is characterized in that described capture antibody is monoclonal antibody 2E8A5, is that the hybridoma cell strain 2E8A5 secretion of CCTCC-C200855 obtains by preserving number; Described detection antibody is monoclonal antibody 7A6A1, is that the hybridoma cell strain 7A6A1 secretion of CCTCC-C200854 obtains by preserving number.
In the kit of the present invention; Described monoclonal antibody 2E8A5 and monoclonal antibody 7A6A1 all are immunoglobulin (Ig)s of IgG1 subclass; Wherein monoclonal antibody 2E8A5 can be simultaneously and the NS1 protein combination of IV and III type dengue virus, is that the hybridoma cell strain 2E8A5 of CCTCC-C200855 secretes by preserving number; Monoclonal antibody 7A6A1 can specificity combine IV type dengue virus, is the hybridoma cell strain 7A6A1 secretion of CCTCC-C200854 by preserving number.Described hybridoma cell strain 2E8A5 and 7A6A1 are with the DV4NS1 albumen of reorganization and natural NS1 albumen cross immunity Balb/c mouse; Merge with mouse boosting cell after the immunity and commercial murine myeloma cell NS-1 then, obtain with the HAT screening of medium at last.
In the kit of the present invention, but described label is meant nonactive position and the material of quantitative test, the for example enzyme that can be marked at antibody; The liquid that develops the color accordingly then contains the material that can react and produce change color with said label, the for example substrate of enzyme.These all are the general knowledge of this area; Those skilled in the art can be according to these general knowledge selection marquee thing and corresponding colour developing liquid easily; Like horseradish peroxidase and substrate hydrogen peroxide urea thereof and tetramethyl benzidine (being called for short TMB) is exactly label and the combination of colour developing liquid commonly used in the ELISA test, is applicable to the present invention too.Described sample preparation liquid, concentrated washing lotion and stop buffer all are the common agents in the double-antibodies sandwich ELISA; Described positive control is meant IV type dengue virus NS 1 albumen, and described negative control thing is the blank thing that does not contain IV type dengue virus NS 1 albumen.
Kit of the present invention is a kind of detection kit based on double-antibodies sandwich ELISA; Wherein said capture antibody 2E8A5 is a kind of monoclonal antibody with cross reactivity; The indirect immunofluorescence testing result shows that this monoclonal antibody can combine III type and IV type dengue virus simultaneously; Screen from the monoclonal antibody of one group of anti-IV type dengue virus NS 1 albumen but detect antibody 7A6A1, can specificity combine IV type dengue virus, therefore; This kit can detect IV type dengue virus accurately and rapidly; And with other I type, II type and III type dengue virus and japanese encephalitis virus, the equal no cross reaction of yellow fever virus, and IV type dengue virus had very high susceptibility, the susceptibility that detects IV type dengue virus is 64 times of commercial kit Pan-E Dengue EarlyELISA test kit; Greatly reduce the probability of omission, help the early diagnosis and therapy of dengue virus; In addition, kit of the present invention can detect IV type dengue virus specifically, helps dengue virus infection is made early stage classification diagnosis.Simultaneously, this kit cost is low, easy and simple to handle, simultaneously the fast detecting gross sample; Main agents all provides with the working fluid form, and is easy and simple to handle; Characteristics with high sensitivity, high specific; Also be the kit of at present first in the world ability specific detection to IV type dengue virus antigen.
Embodiment:
Further illustrate kit of the present invention through concrete example and laboratory report below:
1, preparation method and the qualification result of described monoclonal antibody 2E8A5 and 7A6A1:
(1) preparation of immunizing antigen
The immunogene that the present invention is used to prepare monoclonal antibody is genetic recombination DV4NS1 albumen and the natural viral antigen of deactivation.Genetic recombination DV4 NS1 albumen is with the preparation of a kind of engineered strain of the DV4 of carrying NS1 gene; Its preparation is undertaken by conventional method; Obtain NS1 antigen through carry out purifying with the method for nickel-triglycollamic acid metal affinity chromatography, detailed preparation method can consult and use handbook.Behind the NS1 protein purification, with Coomassie brilliant blue (Coomassie) analysis of protein reagent (PIERCE, Cat, No.ED62976) quantitative.Western blot shows that to purified recombinant Identification of Fusion Protein result specific reaction band appears in mouse anti his MAb at the about 45KDa of molecular weight place, and is consistent with the DV4 NS1 molecular weight size of prediction.The natural viral antigen of deactivation is to obtain from the virus host cell (C6/36, a kind of aedes albopictus cell) that DV4 infects.
(2) immune mouse
Get female BALB/c mouse in 4-6 age in week; Adopt for the first time Freund's complete adjuvant and the emulsification of equal-volume DV4 NS1 antigen mixing; Every subcutaneous multi-point injection 30 μ g of mouse; Per 10 days later on DV4NS1 antigen with incomplete Freund and natural DV4 antigen or reorganization replaces immunity after totally 4 times, injects DV4 NS1 antigen 1 00 μ g and carries out booster immunization in merging preceding 3 days every mouse peritoneals.
(3) preparation of hybridoma and evaluation
Behind the booster immunization 3 days, mouse spleen is got in sterile working, and the murine myeloma cell strain NS-1 that processes splenocyte suspension and exponential phase was by 10: 1 mixed, and (PEG, MW4000 Sigma) merge under the effect at 45% polyglycol.Concrete grammar is following: in 37 ℃ of water-baths; In 1min, slowly add 1.0ml PEG, the limit edged shakes up gently, stops merging respectively at adding 1ml, 2ml, 3ml, 4ml and 5ml serum-free RPMI-1640 nutrient culture media in 1min, 2min, 3min, 4min and the 5min; Add 10ml at last and contain the RPMI-1640 nutrient culture media of 15% hyclone; The centrifugal 5min of room temperature 800rpm abandons supernatant, and the RPMI-1640 nutrient culture media that contains 15% hyclone with 60ml has hanged cell gently.On 6 96 well culture plates of this cell suspension adding, at 37 ℃, 5%CO
2CO2gas incubator in cultivate.In fused cell, add 120 μ l, 2 * HAT nutrient culture media next day.Changed liquid once with 1 * HAT nutrient culture media in per 3 days later on, when the clone grow to account for the hole floorage 1/10 the time, get culture supernatant and carry out pair screening with NS1 albumen and DV4 virus through indirect elisa method.Positive colony goes to enlarged culture in 24 well culture plates, still carries out cloning for the clone of strong positive with limiting dilution assay through ELISA and immunofluorescence (DV4 infection cell antigen sheet) repetition measurement.Cloning 2～3 times to positive rate reaches 100%; The rearmounted liquid nitrogen of two cell line amplification cultivation selecting secretory antibody to tire high is preserved; Be designated as hybridoma cell strain 2E8A5 and hybridoma cell strain 7A6A1; And on November 24th, 2008 in China typical culture collection center (CCTCC) preservation, its preservation registration number is respectively CCTCC-C200855 and CCTCC-C200854.
(4) anti-DV4NS1 protein monoclonal antibody subclass detects
The employing indirect ELISA method detects, and method is following: with the antigen coated microwell plate of DV4, hatch with the Hybridoma Cell Culture supernatant sealing back; Different with the anti-mouse of rabbit of the HRP mark of 1: 1000 times of dilution respectively again subclass SIGs reactions are comprising the anti-mouse IgG1 of rabbit (U.S. ZYMED LABORATORIES, INC; Catalog number (Cat.No.) 61-0120), the anti-mouse IgG2a of rabbit (the same, catalog number (Cat.No.) 61-0220); The anti-mouse IgG2b of rabbit (the same, catalog number (Cat.No.) 61-0320), anti-mouse IgG3 is (the same for rabbit; Catalog number (Cat.No.) 61-0420), the anti-mouse IgM of rabbit (the same, catalog number (Cat.No.) 61-6820).Testing result shows that the culture supernatant of above-mentioned two strain of hybridoma is the IgG1 positive, explains that promptly the monoclonal antibody of this two strain of hybridoma secretion is the IgG1 positive.
(5) preparation of monoclonal antibody ascites and purifying
The ascites preparation: induce legal system in the employing body and be equipped with monoclonal antibody among the present invention, promptly the inoculation hybridoma prepares ascites in the mouse body.Concrete grammar is following: injection 0.5ml incomplete Freund (Sigma company) in every mouse peritoneal can be grown oncocyte with the ascites tumor form at intraperitoneal.After about 1～2 week, with 2 * 10
6The hybridoma of individual exponential phase is suspended in serum-free RPMI 1640 nutrient culture media, injects mouse peritoneal.After about 1～2 week, put ascites with No. 7 syringe needles, the ascites supernatant is collected in centrifugal back, and adding Sodium azide to final concentration immediately is 0.1%, deposit in 4 ℃ subsequent use.
The monoclonal antibody purifying: adopt the antibody in sad-ammonium sulfate precipitation method purifying ascites, method of operating is following: ascites is with 2 times of 60mM, pH5.0 acetate buffer solution dilutions, and it is sad in 30 minutes, dropwise slowly to add while stirring under the room temperature, and to add 33 μ l sad for ascites before every milliliter of dilution; A large amount of depositions occur, 4 ℃ left standstill 2 hours, 10000g, and 4 ℃ are centrifugal 30 minutes; Get supernatant, add the pH7.4 100mM phosphate buffer of 1/10 volume, and with 1N NaOH adjust pH to 7.4; Ice bath stirs and slowly adds ammonium sulfate down, is 45% saturation degree for every milliliters of liquid adds 0.277 ammonium sulfate, 4 ℃ of hold over night; Centrifugal 30 minutes of 10000g, 4 ℃ abandon supernatant, and deposition is dissolved in an amount of 10mM phosphate buffer; With same liquid, 4 ℃ of dialysed overnight are changed liquid three times.With Coomassie brilliant blue (Coomassie) analysis of protein reagent (PIERCE, Cat, No.ED62976) quantitative.The glycerine that antibody after the mensuration concentration adds final concentration 50% is in-80 ℃ of preservations.
(6) specificity of monoclonal antibody is identified
A. indirect elisa method is identified the specificity of monoclonal antibody
Respectively with four serotypes reorganization DV NS1 antigens and the natural antigen coated microwell plate of DV, detect according to the indirect elisa method of routine.Promptly in the microwell plate that encapsulates, add the Hybridoma Cell Culture supernatant of this patent invention, hatch 1h for 37 ℃, add the HRP mark goat anti-mouse igg (Sigma of dilution in 1: 1000; Inc); 30min is hatched for 37 ℃ in 100 μ l/ holes, adds TMB colour developing liquid chamber temperature lucifuge colour developing 10min, adds 1M H
2SO
4Cessation reaction is surveyed 450nm light absorption value (A
450).Table 1 result shows the monoclonal antibody 2E8A5 of this patent invention and the specific monoclonal antibody that 7A6A1 is DV4; With the equal no cross reaction of other three types dengue virus.
Table 1 DV4NS1 monoclonal antibody and reorganization DV4 NS1 antigen and the antigen reactive indirect ELISA result of natural DV4
B. IIF is identified the specificity of monoclonal antibody
Infect the C6/36 cell with DV1, DV2, DV3 and DV4 respectively, when pathology appears in 2/3 cell, collecting cell; 1 * PBS with precooling washes cell two times, then cell is dripped on the slide of aseptic drying, after the drying; Be prepared into smear, fully dry, dry up after fixing 10 minutes with cold acetone; Hatch with the goat anti-mouse igg of positive hybridoma cell culture supernatant and FITC mark respectively, and the C6/36 cellular antigens sheet of setting up uninfecting virus is as negative control, at last with after 0.25% the Azo-Blue dyeing; Fluorescent microscope is observed fluoroscopic image down; Carry out the result with intensity of fluorescence with the dyeing form and judge, detect antibody intensity and count the positive with (+～++ ++), antibody intensity (±) and (-) count feminine gender.Show the intercrossing monoclonal antibody that the monoclonal antibody 2E8A5 of this patent invention and III type and IV type dengue virus combine simultaneously like table 2 result; And 7A6A1 combines with the DV4 antigentic specificity, and with other three equal no cross reactions of serotype DV.
The immunofluorescence testing result of table 2DV4 NS1 monoclonal antibody
C. Western blotting is identified the specificity of monoclonal antibody
With the DV4 NS1 albumen of deactivation DV4 nutrient solution or reorganization,, sample is added in the 10%SDS-polyacrylamide gel with one times of 2 * SDS sample loading buffer dilution; Electrophoretic separation protein makes at the protein transduction of separating on the gel through electroelution to print on the nitrocellulose membrane, and transfer film sealed 24 hours in 4 ℃ with the 10mM PBS that contains 7% skimmed milk and 3%BSA; Transfer film is contained in the special reaction plate, adds respectively in the Hybridoma Cell Culture supernatant, room temperature reaction 1 hour; Behind the 10mM PBS washing film that contains 0.5%Tween20; The HRP mark sheep anti-mouse igg that adds 1: 500 times of dilution, room temperature reaction 1 hour is behind same cleansing solution washing film; After the DAB colour developing, use the deionized water color development stopping.The result shows that monoclonal antibody 2E8A5 of the present invention is the visible very strong protein bound bands of 45 kilodaltons with reorganization DV4NS1 albumen and deactivation DV4 nutrient solution at molecular weight, and is consistent with predicted molecular weight; And specific monoclonal antibody 7A6A1 does not all produce reaction with reorganization DV4NS1 albumen and deactivation DV4 nutrient solution, and the anti-identification of possible merchandiser comformational epitope is relevant.
2, the composition of kit of the present invention and preparation
(1) preparation of agents useful for same
A. sample preparation liquid: be made up of sample preparation liquid A and sample preparation liquid B, wherein A is 1.5M glycine solution (PH2.8), and B is a 1.5M Tris-Hcl solution (PH9.7);
B. concentrate washing lotion: contain 20 * PBS of 2%Tween-20, promptly contain 4.56g NaH2PO4,58.02gNa2HPO4.12H in the 1L solution
2O, 175.3g NaCl behind 15 pounds of 20min autoclavings, adds 20ml Tween-20 and stirs 20 times of dilutions during use;
C. the natural NS1 antigen 1 of positive control: DV4: 1000 dilutions;
D. negative control: contain the 10mM PH7.4 PBS of 0.1%Tween-20, the preparation method is following: with 4.56g NaH
2PO4,58.02g Na
2HPO4.12H
2O and 175.3g NaCl water dissolving and quantitatively to 1 liter, 15 pounds of 20min autoclavings add 0.1%Tween-20 after diluting 20 times;
E. liquid develops the color: be made up of colour developing liquid A and B, get the two equivalent mixing during use and use.The preparation method of liquid A, B of wherein developing the color is following:
0.89g citric acid and 0.16g EDTA disodium are dissolved in the 1000ml water, and 115 ℃ of high pressure 30min add TMB 0.25g after reducing to 90 ℃, must show liquid A after shaking up, and preserve in 4 ℃ of black outs;
9.33g citric acid and 14.6g EDTA disodium are dissolved in the 1000ml water, behind 115 ℃ of high pressure 30min, add 0.75% hydrogen peroxide urea 12.8ml after reducing to 90 ℃, must show liquid B after shaking up, preserve in 4 ℃ of black outs;
F. stop buffer: 1M H
2SO
4
(2) preparation method of the said micro reaction plate that encapsulates monoclonal antibody 2E8A5 is following: monoclonal antibody 2E8A5 of the present invention is diluted to 10 μ g/ml with the phosphate buffer of 10mMpH7.6, encapsulates polystyrene 96 hole microwell plates with 150 μ l/ holes, spend the night in 4 ℃.After bat was done, every hole added the confining liquid of 0.25% casein (Sigma) in 300 μ l/ holes, spent the night with the sealing nonspecific binding site in 4 ℃.Dry lath, vacuum drying 12～24h, subsequent use with the vacuum-packed 4 ℃ of preservations of aluminum foil bag.
(3) preparation method of the monoclonal antibody 7A6A1 of said horseradish peroxidase-labeled is following:
Adopt improvement sodium periodate method, method of operating is following: 5mg HRP stirring and dissolving in the 1mL distilled water, is added 0.2mL and newly joins 0.1M sodium periodate lucifuge stirring at room 30min, put in the 1mM pH4.4 sodium-acetate buffer 4 ℃ of dialysed overnight; Add next day after 0.2M pH9.5 carbonate buffer solution makes pH value reach 9.0～9.5, mix with the antibody 10mg that regulates pH to 9.5 in advance, stirred gently 2～3 hours in the room temperature lucifuge, add 100 μ l then and newly join the 4mg/mL sodium borohydride, 4 ℃ of lucifuges are gently stirred and are spent the night; The antibody-solutions that will spend the night next day is with 5～10 times of 1 * PBS dilutions, and ice bath stirs and dropwise adds equal-volume saturated ammonium sulfate (ammonium sulfate is transferred pH to 7.4 with preceding elder generation with ammoniacal liquor) down, and putting 4 ℃ of lucifuges spends the night; 4 ℃ of centrifugal 30min of 12000rpm abandon supernatant, and deposition is dissolved in an amount of 1 * PBS damping fluid, and puts 4 ℃ of dialysed overnight among 1 * PBS, changes liquid three times.Collect 2%BSA glycerine-PBS protective agent that bond adds final concentration 50%, with 1000 times of phosphate buffer dilutions, be working fluid at last.
3, use kit of the present invention and detect the IV type dengue virus NS 1 antigen in the blood serum sample
(1) detection method:
Get sample to be measured 50 μ l, add sample preparation liquid A 50 μ l, 37 ℃ of effect 1h add sample treating fluid B 50 μ l mixings again behind the mixing; Get 100 μ l and add in the polystyrene 96 hole trace test plates that 2E8A5 encapsulates, establish negative control and positive control simultaneously, 37 ℃ of incubation 1h wash lath after 20 times of dilutions of concentrated cleaning solution; After washing plate 6 times, add the monoclonal antibody 7A6A1 of the HRP mark of dilution in 1: 1000,100 μ l/ holes, 37 ℃ of incubation 30min; The same wash plate 8 times after, add colour developing liquid (colour developing liquid A and B mixed in equal amounts show with join at present), 100 μ l/ holes; Behind the room temperature lucifuge colour developing 10min, add stop buffer, 100 μ l/ holes, cessation reaction.
(2) result judges: with the blank well zeroing, measure absorbance (A value) in the 450nm wavelength.Positive control mean value>=0.50, negative control mean value≤0.10, experiment is set up.Sample A value >=negative control A value mean value * 2.1 then is judged to the positive, otherwise negative.
(3) kit of the present invention detects the specificity and the sensitivity analysis of correlated virus
Confirm that through DV1, DV2, DV3, DV4 being carried out the plaque experiment virus titer is respectively 2.6 * 10
5PFU/mL, 6.88 * 10
4PFU/mL, 2.37 * 10
5PFU/mL, 7.84 * 10
5PFU/mL.Adopt DV1, DV2, DV3, the DV4 of the method detection deactivation of above-mentioned foundation, from 1 * 10
5The a plurality of gradients of beginning doubling dilution detect; Commercial kit " pan-E DENGUE EARLY the ELISA " (Panbio of while to detect four serotype DV NS1 antigens; Australia) carry out synchronous detection relatively, operation steps is carried out according to the kit instructions.The testing result of kit of the present invention shows that detection sensitivity to the DV4 culture supernatant is up to 391PFU/ml; With all the other 3 kinds of serotype dengue virus nutrient solution no cross reactions, and with the virus of other Flavivirus such as encephalitis B virus and yellow fever virus cross reaction does not take place all.Explain that the double-antibody sandwich antigen capture ELISA detection of setting up has the dengue virus serotype specificity.As shown in table 3.Commercial pan-E DENGUE EARLY ELISA testing result shows, and is different to the detection sensitivity of the DV culture supernatant of four serotypes, sees table 4 result for details.Commercial kit is 25000PFU/ml to the detection sensitivity of DV4 culture supernatant, detects the sensitivity of DV4 culture supernatant far below kit of the present invention.
Though the interpretation mode as a result of kit of the present invention and commercially available reagent box pan-E DENGUE EARLY ELISA's is different; Be embodied in and assert that positive standard is different; But in following specificity experiment and clinical trial; Kit of the present invention detects by above-mentioned interpretation mode and false positive all do not occur, and the positive criteria that kit of the present invention is described is reliably, proves further that also kit of the present invention has than better sensitivity of prior art and specificity simultaneously.
Table 3 kit of the present invention detects four serotype DV culture supernatant results
Table is annotated
a: the result of this kit judges as follows: 2.1 times of sample detection value>=negative control mean value (be calculated as 0.095 * 2.1=0.2) positive, on the contrary negative.
Table 4 import reagent box pan-E DENGUE EARLY ELISA detects four serotype DV culture supernatant results
Table is annotated: the result of this kit judges as follows: displayed value＜9.0 are negative, and displayed value 9.0-11.0 is a probable positive, show that＞11.0 is positive.
4, clinical testing
At first clinical serum specimen is carried out the complex dissociation that pre-service forms the antibody in NS1 and the serum with 1.5M glycocoll (PH2.8); Thereby discharge free NS1 antigen, use the back method of 1.5M Tris-Hcl (PH9.7) neutralization to detect again with above-mentioned foundation.
Kit of the present invention detects the specificity of normal human serum sample:
Kit of the present invention has detected 500 routine normal human serums, confirms the critical value of this method with this testing result.Detected value is analyzed; Calculating mean value is 0.125; Standard deviation is 0.025, adds that with mean value 5 standard deviations are as the detection critical value of this method promptly: 0.125+0.025 * 5=0.25, with more than or equal to critical value as judging the detected value positive criteria; 500 routine normal human serums are all negative, and the specificity that can confirm this method is 100%.
Claims (2)
1. the immunodiagnosis kit of a specific detection IV type dengue virus NS 1 antigen; This kit is based on the detection kit of double-antibodies sandwich ELISA; Form by the micro reaction plate that is used to encapsulate capture antibody, sample preparation liquid, the detection antibody that combines with label, positive control, negative control thing, concentrated washing lotion, colour developing liquid and stop buffer; It is characterized in that described capture antibody is 2E8A5, is that the hybridoma cell strain 2E8A5 secretion of CCTCC NO:C200855 obtains by preserving number; Described detection antibody is 7A6A1, is that the hybridoma cell strain 7A6A1 secretion of CCTCC NO:C200854 obtains by preserving number.
2. kit as claimed in claim 1 is characterized in that described label is a horseradish peroxidase.
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| CN102898517B (en) * | 2012-08-16 | 2014-06-04 | 南方医科大学珠江医院 | Anti-West Nile virus non-structural protein 1 antibodies and application thereof |
| CN103869066B (en) * | 2014-03-28 | 2017-01-04 | 山东农业大学 | A kind of tembusu virus double-antibody sandwich elisa detection method |
| CN105548540A (en) * | 2015-12-29 | 2016-05-04 | 中国医学科学院医学生物学研究所 | Common detection kit for I-IV type dengue fever viruses |
| WO2017139587A2 (en) * | 2016-02-11 | 2017-08-17 | Massachusetts Institute Of Technology | Anti-dengue virus ns1 protein monoclonal antibodies |
| KR102218561B1 (en) * | 2019-01-28 | 2021-02-19 | 충북대학교 산학협력단 | Method of Detecting Dengue Virus Using Monoclonal Antibody Pair |
| CN118126164B (en) * | 2023-07-28 | 2024-10-01 | 南方医科大学 | Anti-DENV NS1 protein monoclonal antibody 8C3 and application thereof |
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