CN101650366B - 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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- CN101650366B CN101650366B CN200810147353.9A CN200810147353A CN101650366B CN 101650366 B CN101650366 B CN 101650366B CN 200810147353 A CN200810147353 A CN 200810147353A CN 101650366 B CN101650366 B CN 101650366B
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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 cellulose nitrate 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. The test 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 as possible 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 most areas, the world, it is the infectious disease being caused by enterovirus, the multiple infant who is born in below 5 years old, can cause fash, the ulcer at the positions such as heating and hand, foot, oral cavity, indivedual patients can cause the 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 occurring the earliest EV 71 infection.It was popular all to there is EV 71 in 1972～1973 years, 1986 and Australia in 1999, and critically ill patient is mostly with central nervous system symptom (CNS), and some patients also have serious Respiratory symptoms.In 20 century 70 mid-terms, Bulgaria, Hungary break out that to take the EV 71 that CNS is main clinical characteristics popular in succession, and only Bulgaria just surpasses 750 example morbidities, and 149 people cause paralysis, and 44 people are dead.Britain has broken out that to spread all over together the hand-foot-and-mouth disease that Wales, England causes by Cox A16 popular in the fourth quarter in 1994, monitoring Sentinel point is observed 952 cases altogether, Wei Gai state has had the maximum since record once popular, most 1～4 years old of patient, and most patients symptom is gentle.The epidemic disease data demonstration of this state since 1963, be 2～3 years the interval that hand-foot-and-mouth disease is popular.Also often there is the hand-foot-and-mouth disease being caused by various COxsackie, echovirus and EV 71 in other country as Italy, France, Holland, Spain, Romania, Brazil, Canada, Germany.Japan is the hand-foot-and-mouth disease more country of falling ill, had in history repeatedly popular on a large scale, 1969～1970 years popular be take Cox A16 and infected as main, 2 popular EV of being 71 of 1973 and 1978 cause, main clinic symptoms is hand-foot-and-mouth disease, the state of an illness is generally gentleer, but also observes the case of companion's aseptic meningitis simultaneously.Within 1997～2000 years, hand-foot-and-mouth disease is active once again in Japan, and EV 71, Cox A16 all have separation, and the genotype of EV 71 strains is also from the past different.In the later stage nineties 20th century, EV 71 starts to wreak havoc East Asia Region.It is popular to have there is the hand-foot-and-mouth disease that mainly caused by EV 71 in Malaysia in 1997, has 2628 example morbidities 4～August, only just has 29 routine patient deaths 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, rapidly, 17% limb collapses from physical exhaustion, 17 routine chest films showed pulmonary edema in 28% illness development.
Since March in year, also there is fairly large hand-foot-and-mouth disease epidemic situation 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; There are 978 examples 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.The ratio that EV71 infection causes severe is higher than other types enterovirus, and children with serious disease case fatality rate is higher, there is no at present vaccine and specific treatment medicine.Therefore, can detect in time EV71 virus and the Cox A16 virus of in patient body, carrying, contribute to find early and control epidemic situation, to patient and early treatment, save life.
At present, detect enterovirus, both at home and abroad the detection meanss such as virus separation, immunofluorescence technique, PCR that adopt 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 virus and the dead virus of thoughts contamination power, and said method all needs professional to operate simultaneously.
Therefore, exactly because the backwardness of existing detection method, develops easy to usely, detecting sensitive, cheap testing product is the task of top priority.
Colored particle immunochromatographiassays assays provides a new detection approach for enteroviral detection.Immunity particulate technology is to utilize the solid phase particle of synthesis of polymer material certain particle size size as carrier, the various immunologic active materials (antigen or antibody) on coated with specificity affinity, learn and detect and a separated technology for immunology and other biological.As the particulate of carrier, normally take certain macromolecule organic monomer is raw material, through high molecular polymerization methods such as emulsion polymerization, suspension polymerization and irradiation polymerizations, is prepared from.Different owing to preparing material and technique, particulate of a great variety, now made inert particulate if polystyrene colloidal lactoconium, active particles are as the four large based fine particles such as Carboxylated Polystyrene particulate, magnetic particle and labeled microparticles (by isotope, fluorescein or enzyme labeling), nearly tens kinds of quantity.The particulate preparing and antigen (or antibody) are formed to immune particulate through sensitization methods such as physisorption, chemical coupling and biotin affinity element bridging methods.Be widely used in detection, isolation and purification, cell marking and the identification etc. of various soluble large molecule materials.In recent years, particulate technology also demonstrates wide application prospect at making nucleic acid molecular hybridization, DNA with the research fields such as separated and PCR of RNA.
Emulsion particle is a kind of as immune particulate, adopt synthetic high molecular emulsion microballoon, be that the polystyrene latex of carboxylation is as carrier, antibody or antigen are formed to immune latex diagnostic reagent by physisorption is immobilized in microsphere surface, by latex agglutination, test and can detect corresponding antigen or antibody.The latex diagnostic reagent being prepared into, for diagnosing various diseases, has the advantages such as easy, quick, sensitive, inexpensive and processing ease.
Colloidal gold immunochromatographimethod technology is the novel vitro diagnostic techniques having grown up on monoclonal antibody technique, immunochromatography technique and collaurum developing technology basis since the nineties in 20th century, according to immune response principle, utilize large aperture miillpore filter for carrier, the collaurum of usining detects a kind of tachysynthesis analytical technology of test substance in sample as solid phase labelling thing.
The present invention, on colored particle immunochromatography technique principle basis, improves detection method, has set up employing colloidal gold method and emulsion process and has detected enteroviral method.The improvement of detection method, makes to detect more convenient, and result is more accurate, have quick, sensitive, easy and simple to handle, cost is low, without features such as professional's operations, really reach the organic unity of complicated principle and ease of Use.
Summary of the invention
The present invention, for solving above-mentioned problems of the prior art, provides a kind of quick detection test paper that adopts colored particle immunochromatographic method to detect EV71 and Cox A16 virus.It is easy to use, result is accurate, cost is low, transportation storage is easy, and operates without professional.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:
Detect EV71 and a Cox A16 virus quick detection test paper, by base plate, water sucting plate, nitrocellulose filter, viral colored particle storage pad, sample liquid-adsorption layer, formed; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with one or two detection lines and a polyclonal antibody control line, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and viral colored particle storage respectively in nitrocellulose filter two ends, on viral colored particle storage pad, be pressed with sample liquid-adsorption layer, utilize 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, can be also 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 marking particle, can comprise dye granule, latex particle, fluorescent grain etc.Wherein, the particle diameter of colloid gold particle size is 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 be combined with protein molecule, forms immune diagnostic reagent.
Sample liquid-adsorption layer is comprised of trilaminate material stack, is followed successively by 10～25g/m
2nonwoven layer, glass layer, 10～25g/m
2nonwoven layer, above-mentioned substance all needs through surfactant damping fluid immersion treatment, and dry rear standby, said apparatus, apart from outside capillarity principle, also has syphonic effect principle, greatly accelerates water suction translational speed.
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 filtering out, obtain respectively EV71 viral monoclonal antibodies cell line and the Cox A16 viral monoclonal antibodies cell line of 2 plant height purity, pairing, one strain is coated for detection of line, and a strain can be used for colored particle mark.
Test paper sample liquid-adsorption layer is put into sample to be tested (liquid level must not surpass MAX line), because capillarity sample will move to water sucting plate along test strips, when moving to colored particle storage pad, EV71 virus in 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, in sample, EV71 and/or Cox A16 viral antigen combine with corresponding viral monoclonal antibodies in detection line again, therefore its colored particle is stranded on detection line, detection line place shows red positive, if there is no EV71 virus or CoxA16 virus in contrary sample to be tested, corresponding viral monoclonal antibodies label probe just not can with corresponding viral monoclonal antibody generation specific bond on detection line, do not have colored particle to be detained, 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 draws judgement.
The control line arranging on nitrocellulose filter is to form by sheep anti mouse polyclonal antibody is coated, when the EV71 of sample by colored particle mark and/or Cox A16 viral monoclonal antibodies move to sheep anti mouse polyclonal antibody control line, no matter in sample, have or not EV71 and/or Cox A16 virus, delay is combined in capital with the sheep anti mouse polyclonal antibody having set, control line is shown red.Therefore control line produces and represents that operation is wrong without colour band, and during detection, sample liquid level is expired over MAX line or test paper.
Owing to adopting technique scheme, EV71 provided by the present invention and/or Cox A16 virus quick detection test paper have such beneficial effect, be fast and easy, removable, be convenient to enteroviral field screening work, and high specificity, highly sensitive, need not technical skill personnel operate, and result readability.
Accompanying drawing explanation
The structural representation of Fig. 1 one embodiment of the present invention
The testing result schematic diagram of Fig. 2 one embodiment of the present invention
The structural representation of Fig. 3 another embodiment of the invention
The testing result schematic 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:
If Fig. 1 is to as shown in Fig. 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; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with a detection line 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate 7, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and viral colored particle storage respectively in nitrocellulose filter two ends, on viral colored particle storage pad, be pressed with sample liquid-adsorption layer, utilize 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 cultivates 48-72 hour in containing 10% calf serum DMEM nutrient solution, treats that Growth of Cells is good, prepares to merge.
(2) antigen immune: hypodermic injection after the complete freund adjuvant emulsification of EV71 antigen 1 0ug and equivalent, carry out fundamental immunity.Merge first 3 days, in mice spleen, inject respectively above-mentioned EV71 antigen 1 0ug with abdominal cavity.
(3) prepare 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 SP2/O myeloma cell and immune BALB/C mouse merges in the ratio of 1: 10 or 1: 5.
(5) detection of antibody
Hybridoma is cultivated after one week at 96 orifice plates, starts with ELISA method detection specificity antibody.-4 ℃ of coated EV71 antigen 1 ug/ml that spend the night, after washing next day, add culture supernatant 100ul, add the sheep anti-mouse igg antibody reaction of horseradish peroxidase-labeled, screen positive hole.
(6) cloning and expansion are cultivated: cloning and expansion are cultivated 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. the preparation of monoclonal antibody
(1) can adopt routine techniques manufacture order clonal antibody, for example, mouse ascites method manufacture order clonal antibody, or external Serium-free Culture.
Embodiment 2:
An 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; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with a detection line 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and EV71 viral monoclonal antibodies gold mark respectively in nitrocellulose filter two ends, on EV71 viral monoclonal antibodies gold mark pad, be pressed with sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect EV71 virus.
Test paper method for making:
1. two kinds of EV71 monoclonal antibodies that above-mentioned two strain cell lines produced, a kind of coated for detection of line, a kind of for colloid gold label.
The preparation of collaurum and with the combination of EV71 viral monoclonal antibodies
(1) get distilled water and add appropriate gold chloride magnetic agitation and be warmed to 90 ℃, add appropriate citrate three sodium to continue heating and be stirred to boiling 3～10 minutes, preferably 5 minutes, keep in Dark Place after cooling standby.
(2) preparation of collaurum-antibody conjugates and purifying
Tri-distilled water dissolved chlorine auric acid, be heated to boiling, to final concentration be 0.01%, every 100ml adds 10% trisodium citrate aqueous solution 1.5ml.Boil again 10min and be orange red, the cooling rear 0.2mol/L K that uses
2cO
3be adjusted to pH8.5, the EV71 IgG antibody that adds purifying according to 1-3mg antibody/100ml collaurum, preferably 2mg antibody/100ml collaurum, then adds bovine serum albumin(BSA) 250mg/100ml collaurum, rapid stirring 10min, add 10%NaCl, making the concentration of NaCl is 1%, shakes up, respectively with 2000,4000,10000r/min gradient centrifugation, 10min/ time, what finally obtain that sediment is preliminary purification enters the anti-EV71 bond of mark, is dissolved in storage liquid.Get collaurum-antibody conjugates solution, be evenly sprayed on glass fibre element film, be placed on smooth plastic plate, then freezing 1 hour, put into freeze-drying on freeze drier and spend the night, to slitting after bone dry, in dry environment, preserve.
3. the preparation of film: the anti-EV71 monoclonal antibody of embodiment 1 preparation is diluted to 3.5mg/ml with 0.01MPBS.With Membrane jetter, the speed of above antibody 1ul/cm is sprayed on nitrocellulose filter, forms detection line.
Sheep anti-mouse igg polyclonal antibody is diluted to 2mg/ml with 0.01MPBS.With Membrane jetter, by above antibody, the speed with 1ul/cm is sprayed on nitrocellulose filter, formation control line.
After the nitrocellulose filter that is fixed with antibody is dried, be placed in 25-37 ℃ of confining liquid and soak 60 minutes.Nitrocellulose filter after sealing is placed in to 37 ℃ of baking boxs dry 30 minutes.Abundant drying for standby.
4. test strips is pressed known technology combination, as shown in Figure 1.
5. using method and result judgement: 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.If detect EV71 virus concentration higher than detected level, view window place occurs that two red lines are positive, as shown in Figure 2; Lower than boundary value, occur that a red line is negative; Control line produces and represents that operation is wrong or test paper is expired without colour band.
Embodiment 3:
An EV71 virus latex 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; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with a detection line 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and EV71 monoclonal antibody colored particle storage respectively in nitrocellulose filter two ends, on EV71 monoclonal antibody colored particle storage pad, be pressed with sample liquid-adsorption layer, utilize latex chromatography to detect EV71 virus.
Test paper method for making:
1. two kinds of EV71 monoclonal antibodies that above-mentioned two strain cell lines produced, a kind of coated for detection of line, a kind of for emulsion particle mark.
2. the preparation of emulsion particle:
1) get latex solution 100ul, add borate buffer solution 900ul, high speed centrifugation 10min;
2) abandon supernatant, add borate buffer solution washing, again high speed centrifugation 10min;
3) abandon supernatant, add borate buffer solution to suspend and precipitate, 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, with confining liquid (5%BSA), suspend and precipitate, sealing is spent the night.
3. the preparation of film: machine masking, utilize computer to control transmission speed, guarantee that antibody amount coated on per unit film equates.
Detection line: another strain EV71 viral monoclonal antibodies
Control line: sheep anti mouse polyclonal antibody
4. test strips is pressed known technology combination.
5. using method and result judgement: during use, test paper sample liquid-adsorption layer is put into sample to be tested, observations in the time of 5 minutes.If detect EV71 virus concentration higher than detected level, view window place occurs that two red lines are positive; Lower than boundary value, occur that a red line is negative; Control line produces and represents that operation is wrong or test paper is expired without colour band.
The preparation of the anti-Cox A16 of embodiment 4 viral monoclonal antibodies
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.Merge first 3 days, in mice spleen, inject respectively above-mentioned EV71 antigen 1 0ug with abdominal cavity.
A 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; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with a detection line 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and Cox A16 monoclonal antibody gold mark respectively in nitrocellulose filter two ends, on Cox A16 viral monoclonal antibodies gold mark pad, be pressed with 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 Cox A16 virus latex Test paper, comprises base plate 1, water sucting plate 7, nitrocellulose filter 2, colored particle storage pad 5, sample liquid-adsorption layer 6, and to be wherein preferably Cox A16 monoclonal antibody latex be example storage pad to colored particle storage pad; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with a detection line 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and CoxA16 virus colored particle storage respectively in nitrocellulose filter two ends, on Cox A16 virus colored particle storage pad, be pressed with sample liquid-adsorption layer, utilize 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, preferably 1: 1; Base plate middle part is nitrocellulose filter, on nitrocellulose filter, be provided with two detection lines 3 and a polyclonal antibody control line 4, termination, base plate one end is water sucting plate, other end termination is sample liquid-adsorption layer, mutual overlapping connection padded with water sucting plate and viral monoclonal antibodies gold mark respectively in nitrocellulose filter two ends, on viral monoclonal antibodies gold mark pad, be pressed with sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect EV71 virus and Cox A16 virus.
Test paper method for making:
The preparation of collaurum and with the combination of EV71 viral monoclonal antibodies
(1) get distilled water and add appropriate gold chloride magnetic agitation and be warmed to 90 ℃, add appropriate citrate three sodium to continue heating and be stirred to boiling 3～10 minutes, preferably 5 minutes, keep in Dark Place after cooling standby.
(2) preparation of collaurum-antibody conjugates and purifying
Tri-distilled water dissolved chlorine auric acid, be heated to boiling, to final concentration be 0.01%, every 100ml adds 10% trisodium citrate aqueous solution 1.5ml.Boil again 10min and be orange red, the cooling rear 0.2mol/L K that uses
2cO
3be adjusted to pH8.5, a strain Cox A16 monoclonal antibody 2mg who adds purifying in a strain EV71 IgG antibody 2mg of purifying in embodiment 1 and embodiment 4 under rapid stirring, the protein 25 0mg that adds cow's serum, rapid stirring 10min, adds 10%NaCl, and the concentration that makes NaCl is 1%, shake up, respectively with 2000,4000,10000r/min gradient centrifugation, 10min/ time, finally obtain the anti-EV71 of golden mark and Cox A16 associating bond that sediment is preliminary purification.Get collaurum-antibody conjugates solution, be evenly sprayed on glass fibre element film, be placed on smooth plastic plate, then freezing 1 hour, put into freeze-drying on freeze drier and spend the night, to slitting after bone dry, in dry environment, preserve.
3. the preparation of film: the anti-EV71 monoclonal antibody of another strain of embodiment 1 preparation is diluted to 3.5mg/ml with 0.01MPBS.With Membrane jetter, the speed of above antibody 1ul/cm is sprayed on nitrocellulose filter, forms detection line.
Another strain Cox A16 monoclonal antibody of embodiment 3 preparations is diluted to 3.5mg/ml with 0.01M PBS.With Membrane jetter, by this antibody, the speed with 1ul/cm is sprayed on nitrocellulose filter, forms an other detection line.
Sheep anti-mouse igg polyclonal antibody is diluted to 2mg/ml with 0.01MPBS.With Membrane jetter, by above antibody, the speed with 1ul/cm is sprayed on nitrocellulose filter, formation control line.
After the nitrocellulose filter that is fixed with antibody is dried, be placed in 25-37 ℃ of confining liquid and soak 60 minutes.
Nitrocellulose filter after sealing is placed in to 37 ℃ of baking boxs dry 30 minutes.Abundant drying for standby.
4. test strips is pressed known technology combination.
5. using method and result judgement: during use, test paper sample liquid-adsorption layer is put into sample to be tested, observations in the time of 5 minutes.If detect EV71 or Cox A16 virus concentration higher than detected level, there is red line in the position that view window place is sprayed with corresponding viral monoclonal antibodies, and red line also appears in control line place simultaneously, occurs two detection lines and a control first, and result is positive, as shown in Figure 4; Lower than boundary value, occur that a red line is negative; Control line produces and represents that operation is wrong or test paper is expired without colour band.
Embodiment 8
According to the EV71 described in embodiment 7 and the viral combined colloidal gold test of Cox A16, in the preparation process of film, also can be by after EV71 monoclonal antibody and the mixing of Cox A16 monoclonal antibody, after being diluted to 3.5mg/ml with 0.01M PBS again, with Membrane jetter, with the speed of 1ul/cm, spray film, 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, preferably 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 the concentration of wherein any of EV71 or CoxA16 virus or two kinds is higher than detected level in sample, there is red line in the position that view window place is sprayed with mixture monoclonal antibody, and red line also appears in control line place simultaneously, and result is positive; Lower than boundary value, occur that a red control line is negative; Control line produces and represents that operation is wrong or test paper is expired without colour band.
Embodiment 9
EV71 in 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 respectively EV71 monoclonal antibody gold mark pad and Cox A16 monoclonal antibody gold mark pad, then be superposed to two-layer use, with the mixing gold mark pad that replaces containing EV71 monoclonal antibody and Cox A16 monoclonal antibody, this kind of method is more easy in process of production.
In addition, also the colloidal gold labeling method in embodiment 7,8 or 9 can be replaced with to emulsion particle mark, make the viral combined latex chromatography detecting test paper of EV71 and Cox A16.Labeling method is with embodiment 3.
Claims (5)
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, in colored particle storage pad, contain the attached colored particle of monoclonal antibody bag; Said monoclonal antibody is selected from least one in following:
EV71 viral monoclonal antibodies;
Cox A16 viral monoclonal antibodies;
And described colored particle is colloid gold particle or latex particle, the particle diameter of wherein said colloid gold particle is nanoscale, and the particle diameter of described latex particle is 0.01～10 μ m.
2. described in claim 1, detect enteroviral quick detection test paper, it is characterized in that: in described colored particle storage pad, contain EV71 viral monoclonal antibodies and the attached colored particle of Cox A16 viral monoclonal antibodies mixing bag.
3. described in claim 2, detect enteroviral quick detection test paper, it is held to levy and is: described nitrocellulose filter is provided with two detection lines, the detection line that is EV71 viral monoclonal antibodies spraying wherein, another be the detection line that Cox A16 viral monoclonal antibodies sprays.
4. described in claim 2, detect enteroviral quick detection test paper, it is characterized in that: described nitrocellulose filter is provided with a detection line that is mixed spraying by EV71 viral monoclonal antibodies and Cox A16 viral monoclonal antibodies.
5. prepare a method that detects enteroviral quick detection test paper described in claim 1, it is characterized in that: comprise the following steps:
(1) preparation of monoclonal antibody: the mouse spleen lymphocyte that SP2/0 myeloma cell and antigen immune are crossed merges, form hybridoma, after screening, obtain respectively the cell line of EV71 viral monoclonal antibodies and the cell line of the Cox A16 viral monoclonal antibodies that two strains can produce high-purity, pairing that two strains can produce high-purity, pairing;
(2) use colored particle labeled monoclonal antibody, wherein:
Adopt colloidal gold labeled monoclonal antibody, its preparation method is: use 0.2mol/L K
2cO
3be adjusted to collaurum pH to 8.5, the EV71 viral monoclonal antibodies or the Cox A16 viral monoclonal antibodies 1-3mg/100ml collaurum that under stirring, add purifying, then add bovine serum albumin 250mg, after stirring, add NaCl, making its concentration is 1%, shake up rear centrifugally, precipitation is dissolved in storage liquid standby; Or
Adopt latex particle labelled antibody, its preparation method is: get latex solution 100 μ l, add borate buffer solution 900 μ l, after centrifugal, remove supernatant, with borate buffer solution washing, centrifugal, go after supernatant, then add the borate buffer solution precipitation that suspends, add EV71 viral monoclonal antibodies or Cox A16 viral monoclonal antibodies 20 μ g, volume is settled to 1ml, again to the EDC that adds 10 μ l15mg/ml in gained solution, breed after 4 hours centrifugally, abandon supernatant, with 5%BSA, suspend and precipitate, sealing is spent the night;
(3) preparation of film: another strain EV71 viral monoclonal antibodies or the Cox A16 viral monoclonal antibodies of preparation in above-mentioned steps (1) are diluted to 3.5mg/ml with 0.01M PBS, with Membrane jetter, by above antibody, the speed with 1 μ l/cm is sprayed on nitrocellulose filter, forms detection line; Polyclonal antibody is diluted to 2mg/ml, and the speed with Membrane jetter with 1 μ l/cm is sprayed on nitrocellulose filter, formation control line; After film is dried, in 25-37 ℃ of confining liquid, soak 60 minutes, fully dry, standby after taking out;
(4) the colored particle labelled antibody of preparation in step (2) is coated on glass fibre membrane, forms colored particle storage pad;
(5) on base plate, the stickup sample liquid-adsorption layer of overlap joint, colored particle are stored pad, nitrocellulose filter, water sucting plate mutually in turn.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810147353.9A CN101650366B (en) | 2008-08-11 | 2008-08-11 | Quick test paper for detecting enterovirus and method for preparing same |
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| CN102323413A (en) * | 2010-06-04 | 2012-01-18 | 北京庄笛浩禾生物医学科技有限公司 | Colloidal gold rapid detection test paper for enterovirus 71 (EV71) |
| 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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| SG11202100009YA (en) * | 2018-07-23 | 2021-02-25 | Agency Science Tech & Res | A method of detecting hfmd |
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