CN101650366A - Quick test paper for detecting enterovirus and method for preparing same - Google Patents
Quick test paper for detecting enterovirus and method for preparing same Download PDFInfo
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- CN101650366A CN101650366A CN200810147353A CN200810147353A CN101650366A CN 101650366 A CN101650366 A CN 101650366A CN 200810147353 A CN200810147353 A CN 200810147353A CN 200810147353 A CN200810147353 A CN 200810147353A CN 101650366 A CN101650366 A CN 101650366A
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Abstract
The invention discloses a quick test paper for detecting enterovirus and a method for preparing the same. The test paper detects enterovirus EV71 and Coxsackievirus A16 viruses in a sample by the immunochromatography adopting marking by colorful particles. The quick test strip is formed by sequentially overlapping and bonding a sample adsorption liquid layer, a colorful particle storing pad, a cellulose nitrate membrane and a water adsorption board on a bottom board, wherein the colorful particle storing pad is coated with a monoclonal antibody with a colorful particle mark; and the cellulosenitrate membrane is provided with a detection line sprayed by the monoclonal antibody of the EV71 and/or Coxsackievirus A16 and a control line sprayed by the polyclonal antibody of anti-mouse IgG. Thetest paper can quickly detects the EV71 and Coxsackievirus A16 viruses in the detected sample at the same time or respectively, finds the epidemic situation caused by the virus infection as early aspossible and has the advantages of being easily operated, quickly getting results, avoiding special operators and the like.
Description
Technical field
The present invention relates to the enteroviral quick detection test paper of a kind of detection with and manufacture method, especially detect and can cause the enterovirus EV71 of hand-foot-and-mouth disease and the quick detection test paper of Coxsackievirus A16 virus.
Background technology
Hand-foot-and-mouth disease (Hand foot mouth disease, HFMD) be global infectious disease, all there is this sick popular report in most of area, the world, it is the infectious disease that causes by enterovirus, pilosity is born in the infant below 5 years old, can cause fash, the ulcer at positions such as heating and hand, foot, oral cavity, individual patient can cause complication such as myocarditis, pulmonary edema, AME.The enterovirus that causes hand-foot-and-mouth disease has kind more than 20, and wherein Coxsackie virus (CoxAsckievirus) A16 type (CoxA16) and enterovirns type 71 (Enterovirus71.EV 71) are the most common.
Australia and the U.S., Sweden are the same, are one of countries that occurs the earliest EV 71 infection.It is popular that EV 71 all took place for 1972～1973 years, 1986 and Australia in 1999, and with central nervous system symptom (CNS), some patients also have serious Respiratory symptoms to critically ill patient mostly.20th century the mid-1970s, it is that the EV 71 of main clinical characteristics is popular that Bulgaria, Hungary break out in succession with CNS, only Bulgaria just surpasses 750 example morbidities, 149 people cause paralysis, 44 people's death.Britain broke out that to spread all over Wales, England together popular by the hand-foot-and-mouth disease that Cox A16 causes in the fourth quarter in 1994, the monitoring Sentinel point is observed 952 cases altogether, for this state has had the maximum since the record once popular, most 1～4 years old of patient, the most patients symptom is gentle.The epidemic disease data of this state since 1963 shows that be 2～3 years the interval that hand-foot-and-mouth disease is popular.The hand-foot-and-mouth disease that the also frequent generation of other country as Italy, France, Holland, Spain, Romania, Brazil, Canada, Germany is caused by various COxsackie, echovirus and EV 71.Japan is the more country of hand-foot-and-mouth disease morbidity, had repeatedly popular on a large scale in history, 1969～1970 years popular infects based on Cox A16,2 popular EV of being 71 in 1973 and 1978 cause, main clinic symptoms is a hand-foot-and-mouth disease, the state of an illness is generally gentle, but also observes the case of companion's aseptic meningitis simultaneously.Hand-foot-and-mouth disease was active once again in Japan in 1997～2000 years, and EV 71, Cox A16 all have separation, and the genotype of EV 71 strains is also with different in the past.In the later stage nineties 20th century, EV 71 begins to wreak havoc the East Asia Region.The Malaysian hand-foot-and-mouth disease that has taken place mainly to be caused by EV 71 was popular in 1997, had 2628 example morbidities 4～August, and 29 routine patient deaths are only just arranged 4～June.1.5 years old the dead's mean age, the course of disease only 2 days, 100% heating, 62% brothers' fash, 66% canker sore, the development of 28% illness is rapid, and 17% limb collapses from physical exhaustion, 17 routine chest films showed pulmonary edema.
Since March in year, fairly large hand-foot-and-mouth disease epidemic situation has also taken place in China's Anhui Province's Fuyang City.By May 1, Fuyang accumulative total reported hand-foot-and-mouth disease 3321 examples, and wherein 22 examples are dead; 978 examples are arranged just in hospitalization, severe cases 48 people wherein, 10 examples of being critically ill; Accepting treat-and-release 1209 people; 1112 people have been cured.EV71 infects and causes that the ratio of severe is higher than the other types enterovirus, and severe infant case fatality rate is higher, does not still have vaccine and specific treatment medicine at present.Therefore, can in time detect the EV71 virus and the Cox A16 virus of carrying in patient's body, help to find early and control epidemic situation,, save life patient and early treatment.
At present, detect enterovirus, the detection meanss such as viral separation, immunofluorescence technique, PCR that adopt both at home and abroad more.Isolation of virus is consuming time oversize and cost is higher; Immunofluorescence technique needs expensive fluorescent microscope and sample to be difficult for preserving; PCR method can not be distinguished the virus and the dead virus of thoughts contamination power, and said method all needs the professional to operate simultaneously.
Exactly because therefore the backwardness of existing detection method is developed easy to usely, detects sensitively, cheap testing product is the task of top priority.
The colored particle immunochromatographiassays assays provides a new detection approach for enteroviral detection.Immunity particulate technology is to utilize the solid phase particle of the synthetic certain particle size size of macromolecular material as carrier, bag by on have specificity affinity various immunologic active materials (antigen or antibody), be used for that immunology and other biological learn to detect and a technology of separating.Particulate as carrier is a raw material with certain macromolecule organic monomer normally, is prepared from through high molecular polymerization methods such as emulsion polymerization, suspension polymerization and irradiation polymerizations.Because preparation material and technology difference, particulate of a great variety, inert particulate such as polystyrene latex particulate, active particles such as Carboxylated Polystyrene particulate, magnetic particle and labeled microparticles four big based fine particles such as (with isotope, fluorescein or enzyme labeling) have now been made, nearly tens kinds of quantity.The particulate and the antigen (or antibody) that prepare are formed immune particulate through sensitization methods such as the plain bridging methods of physisorption, chemical coupling and biotin affinity.Be widely used in detection, isolation and purification, cell marking and the identification etc. of various soluble large molecule materials.In recent years, the particulate technology making nucleic acid molecular hybridization, DNA and RNA separate and research field such as PCR also demonstrates wide application prospect.
Emulsion particle is a kind of as immune particulate, adopt the synthetic high polymer latex beads, the polystyrene latex that is carboxylation is as carrier, antibody or antigen are formed immune latex diagnostic reagent by physisorption is immobilized in microsphere surface, can detect corresponding antigen or antibody by the latex agglutination experiment.The latex diagnostic reagent that is prepared into is used to diagnose multiple disease, has advantages such as easy, quick, sensitive, inexpensive and processing ease.
The colloidal gold immunochromatographimethod technology is a novel vitro diagnostic techniques that has grown up on monoclonal antibody technique, immunochromatography technique and collaurum developing technology basis since the nineties in 20th century, be according to the immune response principle, utilize the large aperture miillpore filter to be carrier, detect a kind of tachysynthesis analytical technology of test substance in the sample with collaurum as the solid phase labelling thing.
The present invention improves detection method on colored particle immunochromatography technique principle basis, set up employing colloidal gold method and emulsion process and detected enteroviral method.The improvement of detection method makes and detects more conveniently, and the result is more accurate, have quick, sensitive, easy and simple to handle, cost is low, need not characteristics such as professional's operation, really reaches the organic unity of complicated principle and ease of Use.
Summary of the invention
The present invention provides a kind of quick detection test paper that adopts the colored particle immunochromatographic method to detect EV71 and Cox A16 virus for solving above-mentioned problems of the prior art.It is easy to use, the result is accurate, cost is low, the transportation storage is easy, and need not professional's operation.The present invention also provides the method for preparing this test paper.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of detection EV71 and Cox A16 virus quick detection test paper are made up of base plate, water sucting plate, nitrocellulose filter, viral colored particle storage pad, sample liquid-adsorption layer; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with one or two detection lines and a polyclonal antibody control line, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with viral colored particle storage pad with water sucting plate respectively and are connected, on viral colored particle storage pad, be pressed with the sample liquid-adsorption layer, utilize the colored particle immunochromatographic method to detect virus.
Above-mentioned viral monoclonal antibodies can be EV71 viral monoclonal antibodies and/or Cox A16 viral monoclonal antibodies, also can be the mixing of the two.
Colored particle can be a kind of metal-sol particle, can comprise colloid gold particle, silver-colored particle, iron particle, magnetic-particle etc.; A kind of marking particle can comprise dye granule, latex particle, fluorescent grain etc.Wherein, the particle diameter of colloid gold particle size is a nanoscale, latex particle diameter 0.01～10 μ m; The adsorption mechanism of metal-sol particle is to utilize its electronegative character under alkali condition, with the positive charge group of protein molecule, by electrostatic attraction and combination; Latex particle is to utilize chemically combined mode to combine with protein molecule, forms immune diagnostic reagent.
The sample liquid-adsorption layer is made up of the trilaminate material stack, is followed successively by 10～25g/m
2Nonwoven layer, glass layer, 10～25g/m
2Nonwoven layer, above-mentioned substance all need be passed through surfactant damping fluid immersion treatment, and dry back is standby, and said apparatus also has the syphonic effect principle except that the capillarity principle is arranged, and accelerate the suction translational speed greatly.
EV71 virus or Cox A16 virus immunity mouse with deactivation, cell line injection mouse abdominal cavity, extract ascites and carry out purifying, in the hybridoma cell strain that filters out, obtain the EV71 viral monoclonal antibodies cell line and the Cox A16 viral monoclonal antibodies cell line of 2 plant height purity, pairing respectively, one strain is used for detection line bag quilt, and a strain can be used for the colored particle mark.
Test paper sample liquid-adsorption layer is put into sample to be tested (liquid level must not surpass the MAX line), because the capillarity sample will move to water sucting plate along test strips, when moving to colored particle storage pad, EV71 virus in the sample and/or Cox A16 viral antigen respectively with EV71 or Cox A16 viral monoclonal antibodies label probe generation specific bond, when moving to detection line, EV71 and/or Cox A16 viral antigen combine with corresponding viral monoclonal antibodies in the detection line again in the sample, therefore its colored particle is stranded on the detection line, and the detection line place shows red positive; If do not have EV71 virus or CoxA16 virus in the opposite sample to be tested, corresponding viral monoclonal antibodies label probe just not can with corresponding viral monoclonal antibody generation specific bond on the detection line, there is not colored particle to be detained, promptly only there is a red control line negative, this test paper double antibodies sandwich method principle that Here it is adopts.According to this principle, two lines are positive, and line is negative to draw judgement.
The control line that is provided with on the nitrocellulose filter is formed by sheep anti mouse polyclonal antibody bag, when EV71 and/or the Cox A16 viral monoclonal antibodies when moving to sheep anti mouse polyclonal antibody control line of sample by the colored particle mark, no matter have or not EV71 and/or Cox A16 virus in the sample, the capital combines delay with the sheep anti mouse polyclonal antibody that has configured, and it is red that control line is shown.Therefore control line does not have colour band and produces that then the representative operation is wrong, during detection the sample liquid level surpass the MAX line or test paper expired.
Owing to adopt technique scheme, EV71 provided by the present invention and/or Cox A16 virus quick detection test paper have such beneficial effect, promptly convenient quick, removable, be convenient to enteroviral field screening work, and high specificity, highly sensitive, need not the technical skill personnel operation, and readability as a result.
Description of drawings
The structural representation of Fig. 1 one embodiment of the present invention
The testing result synoptic diagram of Fig. 2 one embodiment of the present invention
The structural representation of Fig. 3 another embodiment of the invention
The testing result synoptic diagram of Fig. 4 another embodiment of the invention
Reference numeral
Base plate 1; Nitrocellulose filter 2; Control line 4; Detection line 3; Colored particle storage pad 5; Sample liquid-adsorption layer 6; Water sucting plate 7.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is further explained:
To shown in Figure 4, a kind of hand-foot-and-mouth disease quick detection test paper comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6 as Fig. 1; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a detection line 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate 7, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with viral colored particle storage pad with water sucting plate respectively and are connected, on viral colored particle storage pad, be pressed with the sample liquid-adsorption layer, utilize the colored particle immunochromatographic method to detect EV71 and/or Cox A16 virus.
Embodiment 1: the preparation of anti-EV71 viral monoclonal antibodies
1. the preparation of hybridoma
(1) SP2/0 myeloma cell cultivated 48-72 hour in containing 10% calf serum DMEM nutrient solution, treated the cell well-grown, prepared to merge.
(2) antigen immune: hypodermic injection after the complete freund adjuvant emulsification of EV71 antigen 1 0ug and equivalent, carry out fundamental immunity.Merged preceding 3 days, and in mice spleen, injected above-mentioned EV71 antigen 1 0ug respectively with the abdominal cavity.
(3) preparation immune spleen cell: last booster immunization is put to death mouse after 3 days, the aseptic splenic lymphocyte of getting.
(4) Fusion of Cells
Fusion agent: PEG (4000); Nutrient solution: 10% calf serum DMEM.The lymphocyte of the BALB/C mouse of SP2/O myeloma cell and immunity is by the ratio fusion of 1: 10 or 1: 5.
(5) detection of antibodies
Hybridoma promptly begins with ELISA method detection specificity antibody after 96 orifice plates are cultivated a week.-4 ℃ of bags that spend the night after washing next day, are added culture supernatant 100ul by EV71 antigen 1 ug/ml, add the sheep anti-mouse igg antibody reaction of horseradish peroxidase-labeled, screen positive hole.
(6) cloning and enlarged culture: cloning and enlarged culture according to a conventional method.According to conventional method screening cell strain of monoclonal antibody, obtain the EV71 viral monoclonal antibodies cell line of two plant height purity, pairing.
2. MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) can adopt routine techniques manufacture order clonal antibody, for example, mouse ascites method manufacture order clonal antibody, or external free serum culture method.
Embodiment 2:
A kind of EV71 virus colloidal gold test as shown in Figure 1, comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, and wherein, colored particle storage pad 5 is preferably EV71 viral monoclonal antibodies gold mark pad; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a detection line 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with EV71 viral monoclonal antibodies gold mark pad with water sucting plate respectively and are connected, on EV71 viral monoclonal antibodies gold mark pad, be pressed with the sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect EV71 virus.
The test paper method for making:
1. two kinds of EV71 monoclonal antibodies that above-mentioned two strain cell lines are produced, a kind of detection line bag quilt that is used for, a kind of colloid gold label that is used for.
The preparation of collaurum and with the combining of EV71 viral monoclonal antibodies
(1) get distilled water and add an amount of gold chloride magnetic agitation and be warmed to 90 ℃, add an amount of citrate three sodium and continue heated and stirred to seething with excitement 3～10 minutes, preferred 5 minutes, it was standby to keep in Dark Place after the cooling.
(2) collaurum-antibody conjugates preparation and purifying
Tri-distilled water dissolved chlorine auric acid is heated to boiling, to final concentration be 0.01%, every 100ml adds 10% trisodium citrate aqueous solution 1.5ml.Boil 10min again and be orange red, cooling back 0.2mol/L K
2CO
3Transfer to pH8.5, according to the EV71 IgG antibody of 1-3mg antibody/100ml collaurum adding purifying, preferred 2mg antibody/100ml collaurum adds bovine serum albumin(BSA) 250mg/100ml collaurum then, stir 10min fast, add 10%NaCl, making the concentration of NaCl is 1%, shakes up, respectively with 2000,4000, the 10000r/min gradient centrifugation, 10min/ time, what obtain at last that sediment is preliminary purification advances the anti-EV71 bond of mark, is dissolved in storage liquid.Get collaurum-antibody conjugates solution, evenly be sprayed on the plain film of glass fibre, be placed on the smooth plastic plate, freezing then 1 hour, to put on the freeze drier freeze-drying and spend the night, slitting to the bone dry is preserved in the dry environment.
3. the preparation of film: the anti-EV71 monoclonal anti body and function 0.01MPBS of embodiment 1 preparation is diluted to 3.5mg/ml.Be sprayed on the nitrocellulose filter with the speed of Membrane jetter, form detection line above antibody 1ul/cm.
The sheep anti-mouse igg polyclonal antibody is diluted to 2mg/ml with 0.01MPBS.With Membrane jetter the speed of above antibody with 1ul/cm is sprayed on the nitrocellulose filter, forms control line.
After the nitrocellulose filter that is fixed with antibody dried, place 25-37 ℃ of confining liquid to soak 60 minutes.Nitrocellulose filter after the sealing was placed 37 ℃ of baking boxs dry 30 minutes.Abundant drying for standby.
4. test strips is pressed the known technology combination, as shown in Figure 1.
5. using method and result judge: during use, test paper sample liquid-adsorption layer is put into sample to be tested (for example, blood, plasma sample, just sample dilution, saliva etc.), observations in the time of 5 minutes.Be higher than detected level if detect the EV71 virus concentration, it is positive that two red lines appear in the view window place, as shown in Figure 2; Be lower than boundary value, it is negative a red line to occur; Control line does not have colour band, and to produce the representative operation wrong or test paper is expired.
Embodiment 3:
A kind of EV71 virus latex detects test paper, comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, and wherein, colored particle storage pad 5 is preferably EV71 monoclonal antibody emulsion particle storage pad; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a detection line 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with EV71 monoclonal antibody colored particle storage pad with water sucting plate respectively and are connected, on EV71 monoclonal antibody colored particle storage pad, be pressed with the sample liquid-adsorption layer, utilize the latex chromatography to detect EV71 virus.
The test paper method for making:
1. two kinds of EV71 monoclonal antibodies that above-mentioned two strain cell lines are produced, a kind of detection line bag quilt that is used for, a kind of emulsion particle mark that is used for.
2. the preparation of emulsion particle:
1) gets latex solution 100ul, add borate buffer solution 900ul, high speed centrifugation 10min;
2) abandon supernatant, add the borate buffer solution washing, once more high speed centrifugation 10min;
3) abandon supernatant, add borate buffer solution suspension precipitation, add EV71 monoclonal antibody 20ug, volume is settled to 1ml;
4) in " 3 " gained solution, add 10ul EDC (15mg/ml), hatch 4h, high speed centrifugation 10min;
5) abandon supernatant, suspend with confining liquid (5%BSA) and precipitate, sealing is spent the night.
3. the preparation of film: machine system film, utilize computer control transmission speed, guarantee that the antibody amount of bag quilt on the per unit film equates.
Detection line: another strain EV71 viral monoclonal antibodies
Control line: sheep anti mouse polyclonal antibody
4. test strips is pressed the known technology combination.
5. using method and result judge: during use, test paper sample liquid-adsorption layer is put into sample to be tested, observations in the time of 5 minutes.Be higher than detected level if detect the EV71 virus concentration, it is positive that two red lines appear in the view window place; Be lower than boundary value, it is negative a red line to occur; Control line does not have colour band, and to produce the representative operation wrong or test paper is expired.
The preparation of embodiment 4 anti-Cox A16 viral monoclonal antibodies
The preparation method is with embodiment 1.Wherein, the immunity amount of immune mouse is: hypodermic injection Cox A16 antigen 1 0ug, add complete freund adjuvant, and carry out fundamental immunity.Merged preceding 3 days, and in mice spleen, injected above-mentioned EV71 antigen 1 0ug respectively with the abdominal cavity.
A kind of Cox A16 virus colloidal gold test comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, and wherein colored particle storage pad is preferably Cox A16 monoclonal antibody gold mark pad; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a detection line 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with Cox A16 monoclonal antibody gold mark pad with water sucting plate respectively and are connected, on Cox A16 viral monoclonal antibodies gold mark pad, be pressed with the sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect Cox A16 virus.
Test paper method for making and using method are identical with embodiment 2.
A kind of Cox A16 virus latex detects test paper, comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, and wherein to be preferably Cox A16 monoclonal antibody latex be example storage pad to colored particle storage pad; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a detection line 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with CoxA16 virus colored particle storage pad with water sucting plate respectively and are connected, on Cox A16 virus colored particle storage pad, be pressed with the sample liquid-adsorption layer, utilize the latex chromatography to detect Cox A16 virus.
Test paper method for making and using method are identical with embodiment 3.
A kind of EV71 and the viral combined colloidal gold test of Cox A16, as shown in Figure 3, comprise base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, wherein, colored particle storage pad 5 is preferably the gold mark pad that contains EV71 monoclonal antibody and Cox A16 monoclonal antibody, the content ratio of two kinds of monoclonal antibodies is 1: 4 to 4: 1, preferred 1: 1; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with two detection lines 3 and a polyclonal antibody control line 4, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with viral monoclonal antibodies gold mark pad with water sucting plate respectively and are connected, on viral monoclonal antibodies gold mark pad, be pressed with the sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect EV71 virus and Cox A16 virus.
The test paper method for making:
The preparation of collaurum and with the combining of EV71 viral monoclonal antibodies
(1) get distilled water and add an amount of gold chloride magnetic agitation and be warmed to 90 ℃, add an amount of citrate three sodium and continue heated and stirred to seething with excitement 3～10 minutes, preferred 5 minutes, it was standby to keep in Dark Place after the cooling.
(2) collaurum-antibody conjugates preparation and purifying
Tri-distilled water dissolved chlorine auric acid is heated to boiling, to final concentration be 0.01%, every 100ml adds 10% trisodium citrate aqueous solution 1.5ml.Boil 10min again and be orange red, cooling back 0.2mol/L K
2CO
3Transfer to pH8.5, a strain Cox A16 monoclonal antibody 2mg of purifying among a strain EV71 IgG antibody 2mg of purifying and the embodiment 4 among the following adding of the quick stirring embodiment 1, the protein 25 0mg that adds cow's serum, stir 10min fast, add 10%NaCl, the concentration that makes NaCl is 1%, shake up, respectively with 2000,4000, the 10000r/min gradient centrifugation, 10min/ time, obtain anti-EV71 of golden mark and Cox A16 associating bond that sediment is preliminary purification at last.Get collaurum-antibody conjugates solution, evenly be sprayed on the plain film of glass fibre, be placed on the smooth plastic plate, freezing then 1 hour, to put on the freeze drier freeze-drying and spend the night, slitting to the bone dry is preserved in the dry environment.
3. the preparation of film: the anti-EV71 monoclonal anti of another strain body and function 0.01MPBS of embodiment 1 preparation is diluted to 3.5mg/ml.Be sprayed on the nitrocellulose filter with the speed of Membrane jetter, form detection line above antibody 1ul/cm.
Another strain Cox A16 monoclonal anti body and function 0.01M PBS of embodiment 3 preparations is diluted to 3.5mg/ml.With Membrane jetter the speed of this antibody with 1ul/cm is sprayed on the nitrocellulose filter, forms an other detection line.
The sheep anti-mouse igg polyclonal antibody is diluted to 2mg/ml with 0.01MPBS.With Membrane jetter the speed of above antibody with 1ul/cm is sprayed on the nitrocellulose filter, forms control line.
After the nitrocellulose filter that is fixed with antibody dried, place 25-37 ℃ of confining liquid to soak 60 minutes.
Nitrocellulose filter after the sealing was placed 37 ℃ of baking boxs dry 30 minutes.Abundant drying for standby.
4. test strips is pressed the known technology combination.
5. using method and result judge: during use, test paper sample liquid-adsorption layer is put into sample to be tested, observations in the time of 5 minutes.Be higher than detected level if detect EV71 or Cox A16 virus concentration, red line appears in the position that the view window place is sprayed with corresponding viral monoclonal antibodies, and red line also appears in the control line place simultaneously, two detection lines and a control promptly occur earlier, and then the result is positive, as shown in Figure 4; Be lower than boundary value, it is negative a red line to occur; Control line does not have colour band, and to produce the representative operation wrong or test paper is expired.
Embodiment 8
According to embodiment 7 described EV71 and the viral combined colloidal gold test of Cox A16, in the preparation process of film, also can be with after EV71 monoclonal antibody and the mixing of Cox A16 monoclonal antibody, spray film with Membrane jetter with the speed of 1ul/cm after being diluted to 3.5mg/ml with 0.01M PBS again, form one and mix detection line.Wherein, the mixing ratio of EV71 monoclonal antibody and Cox A16 monoclonal antibody is 1: 4 to 4: 1, preferred 1: 1.
During use, test paper sample liquid-adsorption layer is put into sample to be tested, observations in the time of 5 minutes.If EV71 or CoxA16 virus concentration any or two kinds wherein are higher than detected level in the sample, the view window place is sprayed with the position of mixing monoclonal antibody and red line occurs, and red line also appears in the control line place simultaneously, and then the result is positive; Be lower than boundary value, it is negative a red control line to occur; Control line does not have colour band, and to produce the representative operation wrong or test paper is expired.
Embodiment 9
EV71 among the embodiment 7 or 8 and the viral combined colloidal gold test of Cox A16, also can be according to the preparation method of monoclonal antibody gold mark pad in embodiment 2 and 3, prepare EV71 monoclonal antibody gold mark pad and Cox A16 monoclonal antibody gold mark pad respectively, be superposed to two-layer use then, to replace containing the mixing gold mark pad of EV71 monoclonal antibody and Cox A16 monoclonal antibody, this kind method is more easy in process of production.
In addition, also the colloid gold label method in embodiment 7,8 or 9 can be replaced with the emulsion particle mark, make the viral combined latex chromatography detecting test paper of EV71 and Cox A16.Labeling method is with embodiment 3.
Claims (10)
1. one kind is detected enteroviral quick detection test paper, it comprises backboard, water sucting plate, sample liquid-adsorption layer, nitrocellulose filter, colored particle storage pad, it is characterized in that: described nitrocellulose filter is provided with at least one monoclonal antibody detection line and polyclonal antibody control line, contains the attached colored particle of monoclonal antibody bag in the colored particle storage pad; Said monoclonal antibody is selected from least a in following:
The enterovirus EV 71 mouse monoclonal antibody;
Coxsackie virus Coxsackievirus A16 mouse monoclonal antibody.
2. detect enteroviral quick detection test paper described in the claim 1, it is characterized in that: described colored particle can be a kind of colloid gold particle, collargol particle, CI particle, magnetic-particle, dye granule, latex particle or fluorescent grain.
3. detect enteroviral quick detection test paper described in the claim 2, it is characterized in that: the preferred colloid gold particle of described colored particle, its particle diameter are nanoscale.
4. detect enteroviral quick detection test paper described in the claim 2, it is characterized in that: the preferred latex particle of described colored particle, its particle diameter are 0.01～10 μ m.
5. detect enteroviral quick detection test paper described in the claim 1, it is characterized in that: contain EV71 viral monoclonal antibodies and the attached colored particle of Coxsackievirus A16 viral monoclonal antibodies hybrid packet in the described colored particle storage pad.
6. detect enteroviral quick detection test paper described in the claim 5, it is characterized in that: described nitrocellulose filter is provided with two detection lines, detection line for EV71 viral monoclonal antibodies spraying wherein, another be the detection line that Coxsackievirus A16 viral monoclonal antibodies sprays.
7. detect enteroviral quick detection test paper described in the claim 5, it is characterized in that: described nitrocellulose filter is provided with a detection line that is mixed spraying by EV71 viral monoclonal antibodies and Coxsackievirus A16 viral monoclonal antibodies.
8. one kind prepares the method that detects enteroviral quick detection test paper described in the claim 1, it is characterized in that: may further comprise the steps:
(1) MONOCLONAL ANTIBODIES SPECIFIC FOR: the mouse spleen lymphocyte that SP2/0 myeloma cell and antigen immune are crossed merges, form hybridoma, after screening, obtain two strains respectively and can produce the cell line of enterovirus EV71 monoclonal antibody of high-purity, pairing and the cell line of coxsackie virus (Coxsackievirus) the A16 monoclonal antibody that two strains can produce high-purity, pairing.
(2) use the colored particle labeled monoclonal antibody;
(3) preparation of film: another strain EV71 monoclonal antibody or the Sa Qi A16 of the section monoclonal anti body and function 0.01MPBS of preparation in the above-mentioned steps (1) are diluted to 3.5mg/ml, with Membrane jetter the speed of above antibody with 1ul/cm is sprayed on the nitrocellulose filter, forms detection line; Polyclonal antibody is diluted to 2mg/ml, is sprayed on nitric acid with the speed of 1ul/cm with Membrane jetter; After film dried, soaked 60 minutes in 25-37 ℃ of confining liquid, it is fully dry, standby to take out the back;
(4) the colored particle labelled antibody with preparation in the step (2) is coated on the glass fibre membrane, forms colored particle storage pad.
(5) the stickup sample liquid-adsorption layer of overlap joint, colored particle are stored pad, nitrocellulose filter, water sucting plate mutually in turn on base plate.
9. the preparation described in the claim 8 detects the method for enteroviral quick detection test paper, it is characterized in that: adopt colloid gold label antibody in the step (2), its preparation method is: use 0.2mol/L K
2CO
3Transfer to collaurum pH to 8.5, stir the EV71 monoclonal antibody or the Sa Qi A16 of the section monoclonal antibody 1-3mg/100ml collaurum that add purifying down, add bovine serum albumin 250mg then, stir the back and add NaCl, making its concentration is 1%, and it is centrifugal to shake up the back, is dissolved in the storage liquid precipitation standby.
10. the preparation described in the claim 8 detects the method for enteroviral quick detection test paper, it is characterized in that: adopt the latex particle labelled antibody in the step (2), its preparation method is: get latex solution 100ul, add borate buffer solution 900ul, remove supernatant after centrifugal, wash with borate buffer solution, centrifugal, after removing supernatant, add borate buffer solution suspension precipitation again, add EV71 monoclonal antibody or the Sa Qi A16 of section monoclonal antibody 20ug, volume is settled to 1ml, adds the EDC of 10ul 15mg/ml again in gained solution, breed after 4 hours centrifugal, abandon supernatant, suspend with 5%BSA and precipitate, sealing is spent the night.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0514489B1 (en) * | 1990-02-07 | 1998-01-07 | Hygeia Sciences, Inc. | Device and method for conducting immunoassays |
| CN1584599A (en) * | 2004-06-15 | 2005-02-23 | 浙江大学 | Reagent strip for determining antibody and antigen of swine circular virus II |
| CN1700005A (en) * | 2005-04-18 | 2005-11-23 | 重庆工学院 | Avian influenza immunogold diagnosis test strip and test card |
| CN1904615A (en) * | 2005-07-28 | 2007-01-31 | 北京庄笛浩禾生物医学科技有限公司 | Influenza A,B virus antigen colloidal gold combined detection test paper |
| CN101017168A (en) * | 2007-02-15 | 2007-08-15 | 王继华 | Fluorescence rubber latex quantitative chromatography indicator paper and manufacture method thereof |
| CN101187665A (en) * | 2007-12-27 | 2008-05-28 | 北京博晖创新光电技术股份有限公司 | Enterovirus immunofluorescence chromatographic assay test paper and its preparation method |
| WO2008071340A1 (en) * | 2006-12-11 | 2008-06-19 | The Jordanian Pharmaceutical Manufacturing Co. | Antigen detection method involving an oligonucleotide enhanced colloidal gold signal |
| CN201087837Y (en) * | 2007-03-30 | 2008-07-16 | 万华普曼生物工程有限公司 | Human myohemoglobin/creatine kinase isoenzyme/myocardium calcium protein I diagnosis test paper |
| CN201096785Y (en) * | 2007-03-30 | 2008-08-06 | 万华普曼生物工程有限公司 | Nitrogen-terminal brain natriuretic peptide/C-reaction albumen/troponin I diagnosis testing paper |
-
2008
- 2008-08-11 CN CN200810147353.9A patent/CN101650366B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0514489B1 (en) * | 1990-02-07 | 1998-01-07 | Hygeia Sciences, Inc. | Device and method for conducting immunoassays |
| CN1584599A (en) * | 2004-06-15 | 2005-02-23 | 浙江大学 | Reagent strip for determining antibody and antigen of swine circular virus II |
| CN1700005A (en) * | 2005-04-18 | 2005-11-23 | 重庆工学院 | Avian influenza immunogold diagnosis test strip and test card |
| CN1904615A (en) * | 2005-07-28 | 2007-01-31 | 北京庄笛浩禾生物医学科技有限公司 | Influenza A,B virus antigen colloidal gold combined detection test paper |
| WO2008071340A1 (en) * | 2006-12-11 | 2008-06-19 | The Jordanian Pharmaceutical Manufacturing Co. | Antigen detection method involving an oligonucleotide enhanced colloidal gold signal |
| CN101017168A (en) * | 2007-02-15 | 2007-08-15 | 王继华 | Fluorescence rubber latex quantitative chromatography indicator paper and manufacture method thereof |
| CN201087837Y (en) * | 2007-03-30 | 2008-07-16 | 万华普曼生物工程有限公司 | Human myohemoglobin/creatine kinase isoenzyme/myocardium calcium protein I diagnosis test paper |
| CN201096785Y (en) * | 2007-03-30 | 2008-08-06 | 万华普曼生物工程有限公司 | Nitrogen-terminal brain natriuretic peptide/C-reaction albumen/troponin I diagnosis testing paper |
| CN101187665A (en) * | 2007-12-27 | 2008-05-28 | 北京博晖创新光电技术股份有限公司 | Enterovirus immunofluorescence chromatographic assay test paper and its preparation method |
Cited By (23)
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|---|---|---|---|---|
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| CN102353780A (en) * | 2010-06-04 | 2012-02-15 | 北京庄笛浩禾生物医学科技有限公司 | Coxsackie virus A16 (CA16) detection testing strip |
| CN102253210B (en) * | 2010-06-04 | 2013-08-21 | 北京庄笛浩禾生物医学科技有限公司 | Test strip for detecting enterovirus 71 (EV71) IgM antibody |
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