CN104407145B - Enterovirns type 71 latex agglutination assay kit preparations and applicatio - Google Patents
Enterovirns type 71 latex agglutination assay kit preparations and applicatio Download PDFInfo
- Publication number
- CN104407145B CN104407145B CN201410586139.9A CN201410586139A CN104407145B CN 104407145 B CN104407145 B CN 104407145B CN 201410586139 A CN201410586139 A CN 201410586139A CN 104407145 B CN104407145 B CN 104407145B
- Authority
- CN
- China
- Prior art keywords
- latex
- enterovirns type
- assay kit
- agglutination assay
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The present invention relates to a kind of enterovirns type 71 latex agglutination assay kit and Synthesis and applications thereof, comprise following material: the carboxylated latex reagent of (1) sensitinogen nuclear expression enterovirns type 71 VP1 specific antigens; (2) sensitization three strain is for the carboxylated latex reagent of the mono-clonal mixed antibody of enterovirns type 71 VP1; (3) enterovirns type 71 yin and yang attribute standard serum, inactivation of viruses solution and PBS and (4) are containing can carry out the slide platform of agglutination reaction and toothpick or the plastic rod for mixing even latex and question response serum for sensitization latex. The present invention can detect the EV71 antigen in different sources sample, the shortcoming such as when overcoming albumen sensitization generic latex, sensitising antigens amount is few, unstable, albumen after sensitization easily comes off. The detection method of the present invention can be used for the scientific research fields such as the duplication regulation and control of the serum epidemiology survey of enterovirns type 71, clinical assistant diagnosis and EV71 virus, antiviral therapy drug screening.
Description
Technical field
The invention belongs to Pathobiology assay test technical field, it is specifically related to quick two-way latex agglutination assay kit of a kind of enterovirns type 71 antigen-antibody and its preparation method and application.
Background technology
Hand foot mouth disease is global infectious disease, has become one of transmissible disease of serious threat children's health in recent years. Its taking generate heat and the fash at the position such as hand, foot, pharynx or bleb as main feature. Small number of patients can concurrent aseptic meningitis, encephalitis, acute flaccid paralysis, respiratory tract infection and myocarditis etc., indivedual severe infant disease progression is fast, and death easily occurs, and severe infant case fatality rate is at 10%-25%.
Hand foot mouth disease causes primarily of the Coxsackie virus (Cox, A group 16,4,5,7,9,10 type, B group 2,5,13 type) of enterovirus genus, Echo virus (Echo) and EV71, wherein the most common with EV71 and CoxA16 type. And EV71 infection causes severe cases comparatively common. China began to see this disease in Shanghai from 1981, and all there is report in multiple province, city and region afterwards. Hand foot mouth disease was formally classified as the third class transmissible disease and carried out Notifiable disease management by China on May 2nd, 2008.
Virus invades from pharyngeal or enteron aisle, breeds in local mucous membrane or Lymphoid tissue, and by locally discharging, now can cause local symptom. Then virus invades again regional nodes, and thus enters blood circulation and cause first time viremia. Virus is bred in a large number through places such as circulation of blood intrusion reticuloendothelium, deep layer lymphoglandula, liver, spleen, marrow and is thus entered blood circulation, causes second time viremia. Virus can enter each organ of whole body with blood flow, such as places such as central nervous system, skin mucosa, hearts, breeds further and causes pathology. After susceptible person infects EV71, blood vessel transformation reactions and tissue inflammation pathology occur. When virus involves central nervous system, relatively neurotoxic effect is more strong for tissue inflammation, and central nervous system thin vessels endothelium is vulnerable to infringement most. Cytogamy, vasculitic become, thrombosis can cause ischemic and infarct. In the local organization of spinal cord rope, brain stem, a brain, brain and cerebellum, except Neural invasion effect, also exist widely around blood vessel and parenchyma inflammation. After infant infects EV71, clinical manifestation is various, makes fast progress from stealth infection, common hand foot mouth disease to the severes such as central nervous system infection, part severe conditions of patients, even dead. Patient is mainly preschool children, especially with less than 3 years old child morbidity height. Compared with the hand foot mouth disease that other enteroviruses cause, the ratio infecting the disease generation severe infection caused by EV71 is relatively high. Meanwhile, owing to enterovirus infectivity is strong, easily causes and break out or popular. Strengthen the prevention and control that EV71 infects have been become one of target of China's viral infectious management work.
At present EV71 infectious diseases being there is no to the medicine of special efficacy, main employing is suited the medicine to the illness and supportive treatment, actively prevents complication from occurring. Strengthening the monitoring of disease and cause of disease, accurately and timely dispose epidemic situation, launching health education etc. is the key that control EV71 is popular. In the middle of EV71 vaccine is being developed, the key of prevention will be become. To crowd particularly the serum epidemiology state of the EV71 of infant colony and rule study, can be the formulation of the Immune Programming of hand foot mouth disease and data refer be provided. Therefore the exploitation of the antibody fast detecting kit of EV71 seems particularly necessary.
The exploitation of related vaccines and the development of virus antigen quick detection kit are then the keys of this disease of prevention and control. The mode of detection enterovirus is except traditional virus culture identification method at present, domestic existing biotechnology company utilizes biochip technology to develop " enterovirus authentication chip " (EVtypingchip), the type that can detect out enterovirus fast is other, the primary opportunity that unlikely delay sufferer is gone to a doctor and treated. Easily lead to complications because enterovirus authentication chip infects, and within a week, often namely change into severe, if so the Virus Type infected can be known ahead of time, the very first time of symptomatic treatment can be grasped. The biochip that enterovirus authentication chip authentication chip mainly goes out designed by a few type enterovirus authentication chip such as EV71, CoxA15, CoxA16 and CoxB3 etc. that complication is more serious, utilizes the specific gene sequence of enterovirus authentication chip to differentiate the type of enterovirus authentication chip. At China's public health system, the infant sample of brothers' mouth symptom occur, pharynx swab or anus swab carry out enterovirus detection with the Real-timePCR of probe mark after sampling.
Developed at present for enterovirus IgG, IgM antibody and detection virus-virus particle ELISA kit. China Medical Sciences Academy Medical Biology Institute discloses " detection method of EV71 C-type virus C antigen " patent (application number 200910094972.0), and this patent adopts the method for enzyme linked immunological to detect EV71 antigen. Beijing Kexing Biotech Products Co., Ltd discloses " EV71 virus neutralizing epitope antigen detecting agent box or reagent and its preparation method " patent (application number 200910131382.0), the patent provides the method that EV71 virus antigen is carried out detection by quantitative by the HD6 monoclonal antibody that a kind of employing has a spectrum activity. Below it is all adopt the method for enzyme linked immunological to detect EV71 antigen, it is applicable to clinical diagnosis, owing to detection time is longer and needs by special instrument equipment, expensive, complex operation step is time-consuming, and needs to wash trigger, the volley of rifle fire, the expensive instrument such as enzyme labelling, is not suitable for promoting the use of in common small towns medical institutions. Hunan Kang Run pharmaceutcal corporation, Ltd discloses " enterovirns type 71 colloidal gold colloidal gold detection test paper strip and its preparation method and application " patent (application number 201210151666.8), this patent adopts the method for colloid gold label to detect EV71 antibody, method simple and fast, but cost height, is unfavorable for large-scale serum epidemiology survey result.
Summary of the invention
For the above-mentioned technical problem of prior art, it is an object of the invention to provide a kind of quick two-way latex agglutination assay kit for detecting enterovirns type 71 antigen-antibody and Synthesis and applications thereof, the present invention is carrier based on carboxylated latex particle, by carbodiimide target protein or antibody coupling on latex particle, for detecting enterovirns type 71.
For achieving the above object, the present invention is achieved by the following technical solutions:
A kind of enterovirns type 71 latex agglutination assay kit, is made up of following material:
(1) the carboxylated latex reagent of sensitinogen nuclear expression enterovirns type 71 VP1 specific antigens;
(2) sensitization three strain is for the carboxylated latex reagent of the mono-clonal mixed antibody of enterovirns type 71 VP1;
(3) enterovirns type 71 yin and yang attribute standard serum, inactivation of viruses solution and PBS
(4) containing can carry out the slide platform of agglutination reaction and toothpick or the plastic rod for mixing even latex and question response serum for sensitization latex.
A preparation method for enterovirns type 71 latex agglutination assay kit, comprises the following steps:
(1) three kinds of EV71 monoclonal antibodies are prepared: the EV71VP1 protein immunization BALB/c mouse purified with prokaryotic expression, conventional hybridization oncocyte technology is adopted to prepare some strain of hybridoma, nutrient solution is collected after carrying out cell cultures, then three all higher strains of three strain specificity susceptibility are filtered out with western blotting method, concentrated also freeze-drying after carrying out chromatography purification ,-80 DEG C save backup;
(2) structure of pET-28a-VP1 and protein expression and purification: the VP1 gene clone of EV71-C4 standard strain is entered prokaryotic expression carrier pET-28a, BL21 intestinal bacteria carry out prokaryotic expression EV71 virus VP 1 albumen, Ni post carries out affinity purification, and-80 DEG C save backup;
(3) foundation of carboxylated latex aggegation method: the albumen after purifying or monoclonal antibody are coupled on latex particle by carbodiimide, follow and fix other condition, change the principle of one of them condition, grope coupling protein amount, coupling time, latex concentration, selects linked reaction terminator and encapsulant;
The working fluid concentration of described carbodiimide is 1.0-2.3%, and the reaction final concentration of carbodiimide is 60-80 �� l, and the reaction times of carbodiimide is 2.5-3 hour;
Described coupling protein amount is 20-70ug, and coupling time is 4.5-9.6 hour, and described latex concentration is 110-180ul;
It is obtained that described linked reaction terminator is that the 0.1M ethanolamine solutions of 20-60 �� l adds in the latex solution that 1-2mlpH8.0 borate buffer suspends;
(4) assembling of the quick two-way latex agglutination assay kit of enterovirns type 71 antigen-antibody: above-mentioned sensitization latex, yin and yang attribute comparison, slide glass and the glue rod prepared is assembled into detection kit.
The concrete steps by carbodiimide, the albumen after purifying or monoclonal antibody being coupled on latex particle in described step (3) are:
A () is got carboxylate latex and is put into centrifuge tube, after washing 3 times with the carbonic acid buffer of pH9.6, then wash 3 times with the phosphoric acid buffer of pH4.5;
B () adds water-soluble carbodiimide room temperature effect 3h in the phosphoric acid buffer of pH4.5;
C () washes 3 times with pH8.0 borate buffer;
D () adds VP1 proteantigen or mixing monoclonal antibody in latex suspension, after on shaking table, sense is done 6 hours, be suspended in stock solution by latex antibody test reagent.
After prepared by described enterovirns type 71 latex agglutination assay kit, the specificity of test kit, susceptibility, repeatability and preservation period etc. are measured and optimized.
The application of a kind of enterovirns type 71 latex agglutination assay kit in detection enterovirns type 71 antigen and antibody.
The principle of the present invention is: adopting the VP1 albumen of three strain EV71 monoclonal antibodies or prokaryotic expression purifying by EDC coupling, sensitization carboxylated latex, adopts antigen antibody reaction and the EV71 antigen in the principle detection sample of aggegation and antibody occur.
The useful effect of the present invention is: the quick two-way latex agglutination detection method of the enterovirns type 71 antigen-antibody of the present invention is simple to operate, quick, cheap and without the need to applying special instrument equipment, be suitable for early screening and the clinical assistant diagnosis of enterovirns type 71, especially it be adapted to common small towns medical institutions and promote the use of. Meanwhile, present method resists without the need to carrying the two of mark, directly carries out the agglutination reaction of antigen-antibody, is therefore possible not only to detection human antibody and can also detect animal source antibody, such as mouse, and the blood sample in the laboratory animal sources such as rabbit. Therefore the relevant animal experiment that can be widely used in scientific experiment.
The present invention is possible not only to the IgG of the anti-EV71 detected in humanized's serum, is also adapted to animal derived serum. Antigen detecting agent box can detect the EV71 antigen in different sources sample. Owing to have employed the technology of covalent coupling, the shortcoming such as when overcoming albumen sensitization generic latex, sensitising antigens amount is few, unstable, albumen after sensitization easily comes off. Meanwhile, adopt VP1 albumen as antigen, make the method have higher biological safety compared with totivirus. The detection method that the present invention sets up can be used for the scientific research fields such as the duplication regulation and control of the serum epidemiology survey of enterovirns type 71, clinical assistant diagnosis and EV71 virus, antiviral therapy drug screening.
Accompanying drawing explanation
Fig. 1 is the VP1 albumen polyacrylamide gel electrophoresis of preliminary purification after ultrasonic disruption;
Fig. 2 is the VP1 albumen polyacrylamide gel electrophoresis that Ni post carries out affinity purification;
Fig. 3 is that Western-blot detects VP1 albumen;
Fig. 4 is that carboxylated latex microballoon carboxylic group and carbodiimide connect with chemical bond;
Fig. 5 is the anti-EV71VP1 antibody in latex agglutination antibody assay kit detection different sources serum sample;
Fig. 6 is that EV71 virus produces cytopathy on VERO cell;
Fig. 7 is latex agglutination antigen detecting agent box detection VP1 albumen and virus culture supernatant, and wherein stoste is 2MOI.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
Embodiment 1
The preparation of EV71 monoclonal antibody
The critical materials of this product is EV71 monoclonal antibody and VP1 purifying protein, and the research and development of the two and preparation place are Wuhan Virology Institute,Chinan academy of Sciences.
Prepared by EV71 monoclonal antibody: the EV71VP1 albumen purified with prokaryotic expression, immunity BALB/c mouse respectively, conventional hybridization oncocyte technology carries out cytogamy, screen positive hybridoma cell by ELISA method and measure and tire, filter out have anti-EV71 antibody height secretion property, high affinity hybridoma as initiating cell strain prepare put into production, liquid nitrogen is deposited preservation. Take out screening the cell seed obtained as stated above from-173 DEG C of liquid nitrogen containers, melt as early as possible, cultivate at 37 DEG C and obtain primary seed solution, same CMC model obtains secondary seed solution, then seed liquor is moved to, in cell cultures cylinder, cultivating in the titanium dioxide incubator of 37 DEG C, collect nutrient solution. Above-mentioned nutrient solution obtains supernatant liquor after centrifugal, then filters through film separating system, and the supernatant liquor collected is the crude extract containing EV71 monoclonal antibody. By crude extract respectively through affinity chromatography, collect antibody, then concentrate to normality through evaporating pipe. 2 kinds of preparation comprise the antibody-solutions of different EV71 core position, respectively filling, be lyophilized into freeze-drying pipe ,-80 DEG C save backup. Wherein a kind of preparation for colloid gold label albumen during production, another kind is for being coated in nitrocellulose membrane.
Embodiment 2
The production of EV71VP1 purifying protein, namely VP1 gene clone EV71-C4 standard strain enters prokaryotic expression carrier pET-28a, BL21 intestinal bacteria carry out prokaryotic expression EV71 virus VP 1 albumen, Ni post carries out affinity purification: as shown in Figure 1-Figure 3, flat board is selected positive bacterium colony be inoculated on that flat board of card, it is 0.4mM that 37 DEG C of shaking culture add IPTG to final concentration when about equaling 0.6 to OD600, continue to cultivate 3h, 1ml bacterium fluid samples is got under aseptic condition, the centrifugal 1min of 10000r/min, get precipitation and carry out SDS-PAGE electrophoresis according to a conventional method, observe protein expression situation. get the centrifugal 10min of bacterial cultures 8000r/min of abduction delivering 3h, collecting bacterium, the buffer A of the former culture volume that adds 1/10 is resuspended, strong ultrasonication about 8 times, 10s/ time (operating on ice), until liquid turns into transparent, 4 DEG C of centrifugal 15min of 12000r/min, get 100 �� L of supernatant for subsequent use, precipitation is added equal-volume (before centrifugal) buffer A resuspended, getting the 100 �� L �� L2 �� sds gel sample loading buffer that adds 100, mixed even rear boiling water bath 5min, as SDS-PAGE electrophoresis sample. it is remaining that to put-80 DEG C for subsequent use. the immunogenicity of the albumen of the expressed purifying of Western-blot method qualification.
Embodiment 3
The foundation of the quick two-way latex agglutination assay kit of enterovirns type 71 antigen-antibody, as Figure 4-Figure 7:
1, by carbodiimide, the albumen after purifying or monoclonal antibody are coupled on latex particle. Concrete steps are: a. gets 125 �� L10% carboxylate latexs and puts into centrifuge tube; B. carbonic acid buffer (pH9.6) washes 3 times; C. phosphoric acid buffer (pH4.5) washes 3 times; D. water-soluble carbodiimide (EDC) room temperature effect 3h in phosphoric acid buffer (pH4.5) is added; E. borate buffer (pH8.0) washes 3 times; F. adding VP1 proteantigen or mixing monoclonal antibody in latex suspension, on shaking table, sense does 6 hours; G. latex antibody test reagent is suspended in stock solution.
2, follow and fix other condition, change the principle of one of them condition, grope the coupling protein amount of the best of the inventive method, best coupling time, best latex concentration, the selection etc. of linked reaction terminator and encapsulant.
The working fluid concentration of best carbodiimide is 1.0-2.3%, such as 2%, namely 5g is dissolved in 250ml distilled water, the reaction final concentration of best carbodiimide is 60-80 �� l, as 2% carbodiimide joins in the latex solution that the phosphate buffered saline buffer of 1mlpH4.5 suspends, the reaction times of best carbodiimide is 2.5-3 hour;
Coupling protein amount is 20-70ug, and coupling time is 4.5-9.6 hour, and described latex concentration is 110-180ul;
It is obtained that linked reaction terminator is that the 0.1M ethanolamine solutions of 20-60 �� l adds in the latex solution that 1-2mlpH8.0 borate buffer suspends;
Best coupling protein amount is 20-70ug, and best coupling time is 4.5-9.6 hour, and best latex concentration is 110-180ul; Best damping fluid consumption is 1ml damping fluid/150 �� l carboxylated latex;
It is obtained that best linked reaction terminator is that the 0.1M ethanolamine solutions of 20-60 �� l adds in the latex solution that 1-2mlpH8.0 borate buffer suspends.
3, the assembling of the quick two-way latex agglutination assay kit of enterovirns type 71 antigen-antibody: by the above-mentioned sensitization latex prepared, yin and yang attribute compares, and slide glass, glue rod is assembled into detection kit.
Embodiment 4
The detection method of enterovirns type 71 latex agglutination assay kit of the present invention and result.
1, detection method: draw 15 �� L latex reagents on slide glass with pipettor, draw 15 �� L serum to be checked or mix even containing viral sample and latex, and stir even with plastic rod rotation, after sense does 2-5 minute, result judgement can be carried out, according to agglutination reaction intensity by weak to can be divided into by force "-", "+", " ++ ", " +++ ", " ++++".
2, detected result: the test strip of the present invention carries out clinical examination in disease prevention and control center of Shaoxin City, comprise hand foot mouth disease outpatient service patient, hand foot mouth disease in-patient, hepatopathy center hospitalized child, the various disease hospitalized child of Infectious Disease, examine the serum sample of 600 clinical infants and 400 parts of health examination childs altogether. The result of clinical experiment checking shows the quick two-way latex agglutination assay kit of enterovirns type 71 antigen-antibody, accuracy height, specificity is good, wherein to the serum sample antibody test result of appraisal: accuracy is 89.5%, specific degree is 92.4%, sensitivity is 81.3%, and the coincidence rate of positive sample and business-like ELISA detection kit is 82.6%, and the coincidence rate of negative sample and PCR is 94.5%; The result of appraisal to Detection of antigen: the cells and supernatant comprising stoste to the 100 times dilution of 2MOIEV71 virus by the Detection of antigen latex reagent box Detection of antigen of the present invention. The recall rates such as the low pharynx swab of viral level, anus swab is lower, and real-timePCR detects, the sample that virus copy number is higher be latex agglutination test "+" or " ++ ".
After prepared by the enterovirns type 71 latex agglutination assay kit of the present invention, the specificity of test kit, susceptibility, repeatability and preservation period etc. are measured and optimized.
The detection of the applicable EV71 antibody in people and animal-origin serum of the antibody test reagent of the present invention, and easy and simple to handle, cheap, can immediately learn result, the quick auxiliary diagnosis of very convenient each medical institutions. Detection of antigen latex agglutination test kit can detect in cells and supernatant virus replication level height difference, has important application prospect for aspects such as the duplication regulation and control of research EV71 virus, antiviral therapy drug screenings.
Embodiment recited above is only the preferred embodiment of the present invention be described; not the scope of the present invention is limited; do not departing under inventive design spirit prerequisite; the various distortion that technical solution of the present invention is made by those skilled in the art and improvement, in the protection domain that the claim book that all should fall into the present invention is determined.
Claims (4)
1. the preparation method of an enterovirns type 71 latex agglutination assay kit, it is characterised in that comprise the following steps:
(1) three kinds of EV71 monoclonal antibodies are prepared: the EV71VP1 protein immunization BALB/c mouse purified with prokaryotic expression, conventional hybridization oncocyte technology is adopted to prepare some strain of hybridoma, nutrient solution is collected after carrying out cell cultures, then three all higher strains of three strain specificity susceptibility are filtered out with western blotting method, concentrated also freeze-drying after carrying out chromatography purification ,-80 DEG C save backup;
(2) structure of pET-28a-VP1 and protein expression and purification: the VP1 gene clone of EV71-C4 standard strain is entered prokaryotic expression carrier pET-28a, BL21 intestinal bacteria carry out prokaryotic expression EV71 virus VP 1 albumen, Ni post carries out affinity purification, and-80 DEG C save backup;
(3) foundation of carboxylated latex aggegation method: the albumen after purifying or monoclonal antibody are coupled on latex particle by carbodiimide, follow and fix other condition, change the principle of one of them condition, grope coupling protein amount, coupling time, latex concentration, selects linked reaction terminator and encapsulant;
The working fluid concentration of described carbodiimide is 1.0-2.3%, and the reaction final concentration of carbodiimide is 60-80 �� l, and the reaction times of carbodiimide is 2.5-3 hour;
Described coupling protein amount is 20-70ug, and coupling time is 4.5-9.6 hour, and described latex concentration is 110-180ul;
It is obtained that described linked reaction terminator is that the 0.1M ethanolamine solutions of 20-60 �� l adds in the latex solution that 1-2mlpH8.0 borate buffer suspends;
(4) assembling of the quick two-way latex agglutination assay kit of enterovirns type 71 antigen-antibody: the sensitization latex prepared, yin and yang attribute comparison, slide glass and glue rod are assembled into detection kit;
Described enterovirns type 71 latex agglutination assay kit is made up of following material:
(1) the carboxylated latex reagent of sensitinogen nuclear expression enterovirns type 71 VP1 specific antigens;
(2) sensitization three strain is for the carboxylated latex reagent of the mono-clonal mixed antibody of enterovirns type 71 VP1;
(3) enterovirns type 71 yin and yang attribute standard serum, inactivation of viruses solution and PBS
(4) containing can carry out the slide platform of agglutination reaction and toothpick or the plastic rod for mixing even latex and question response serum for sensitization latex.
2. the preparation method of enterovirns type 71 latex agglutination assay kit as claimed in claim 1, it is characterised in that: the concrete steps by carbodiimide, the albumen after purifying or monoclonal antibody being coupled on latex particle in described step (3) are:
A () is got carboxylate latex and is put into centrifuge tube, after washing 3 times with the carbonic acid buffer of pH9.6, then wash 3 times with the phosphoric acid buffer of pH4.5;
B () adds water-soluble carbodiimide room temperature effect 3h in the phosphoric acid buffer of pH4.5;
C () washes 3 times with pH8.0 borate buffer;
D () adds VP1 proteantigen or mixing monoclonal antibody in latex suspension, after on shaking table, sense is done 6 hours, be suspended in stock solution by latex antibody test reagent.
3. the preparation method of enterovirns type 71 latex agglutination assay kit as claimed in claim 1, it is characterized in that: after prepared by described enterovirns type 71 latex agglutination assay kit, the specificity of test kit, susceptibility, repeatability and preservation period have been measured and optimized.
4. the preparation method of enterovirns type 71 latex agglutination assay kit as claimed in claim 1, it is characterised in that: the application of described enterovirns type 71 latex agglutination assay kit in detection enterovirns type 71 antigen and antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410586139.9A CN104407145B (en) | 2014-10-28 | 2014-10-28 | Enterovirns type 71 latex agglutination assay kit preparations and applicatio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410586139.9A CN104407145B (en) | 2014-10-28 | 2014-10-28 | Enterovirns type 71 latex agglutination assay kit preparations and applicatio |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104407145A CN104407145A (en) | 2015-03-11 |
| CN104407145B true CN104407145B (en) | 2016-06-01 |
Family
ID=52644791
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410586139.9A Expired - Fee Related CN104407145B (en) | 2014-10-28 | 2014-10-28 | Enterovirns type 71 latex agglutination assay kit preparations and applicatio |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104407145B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849470B (en) * | 2015-04-21 | 2016-08-24 | 湖北省农业科学院畜牧兽医研究所 | A kind of enterorrhagia Bacillus coil 0157: H7 latex agglutination assay kit and application |
| CN105567643B (en) * | 2015-12-28 | 2020-01-17 | 菲鹏生物股份有限公司 | Hybridoma cell capable of secreting anti-EV 71 virus protein VP1 monoclonal antibody, monoclonal antibody and application |
| CN106680505A (en) * | 2017-01-04 | 2017-05-17 | 南京鼓楼医院 | Latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies |
| CN107356752B (en) * | 2017-07-10 | 2020-07-17 | 湖北省农业科学院畜牧兽医研究所 | Riemerella anatipestifer latex agglutination antibody detection kit, detection method and storage method thereof |
| CN108896758A (en) * | 2018-05-09 | 2018-11-27 | 中国农业科学院兰州兽医研究所 | A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101025420A (en) * | 2005-09-29 | 2007-08-29 | 华中农业大学 | Avian influenza virus latex agglutination assay kit and its use |
| CN101147065A (en) * | 2005-07-25 | 2008-03-19 | 爱科米株式会社 | Immunoassay method and immunoassay kit to be used therein |
| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN102465116A (en) * | 2010-11-11 | 2012-05-23 | 华中农业大学 | Latex agglutination detection method for porcine circovirus type 2 and application thereof |
| CN102749445A (en) * | 2012-06-29 | 2012-10-24 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for improving rabies neutralizing antibody detection sensitivity in latex agglutination test |
| CN102854317A (en) * | 2011-09-27 | 2013-01-02 | 上海博沃生物科技有限公司 | EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method |
-
2014
- 2014-10-28 CN CN201410586139.9A patent/CN104407145B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101147065A (en) * | 2005-07-25 | 2008-03-19 | 爱科米株式会社 | Immunoassay method and immunoassay kit to be used therein |
| CN101025420A (en) * | 2005-09-29 | 2007-08-29 | 华中农业大学 | Avian influenza virus latex agglutination assay kit and its use |
| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN102465116A (en) * | 2010-11-11 | 2012-05-23 | 华中农业大学 | Latex agglutination detection method for porcine circovirus type 2 and application thereof |
| CN102854317A (en) * | 2011-09-27 | 2013-01-02 | 上海博沃生物科技有限公司 | EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method |
| CN102749445A (en) * | 2012-06-29 | 2012-10-24 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for improving rabies neutralizing antibody detection sensitivity in latex agglutination test |
Non-Patent Citations (4)
| Title |
|---|
| Development of a Dot-immunoenzyme Filtration Assay for the Detection of Enterovirus 71 Antigen in Human Serum via Immunoglobulin M;A ZHANG等;《The Journal of International Medical Research》;20121231;第40卷;1122-1129 * |
| EV71结构蛋白VP1的原核表达及VP1单克隆抗体的制备;李晨阳等;《细胞与分子免疫学杂志》;20120930;第28卷(第9期);第957页第1.2.5节，摘要 * |
| Monoclonal Antibodies to VP1 Recognize a Broad Range of Enteroviruses;Lynn Yihong Miao等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20091031;第47卷(第10期);3108-3113 * |
| 牛型分枝杆菌MPB70蛋白胶乳凝集方法的建立及应用;钦博等;《中国预防兽医学报》;20070131;第29卷(第1期);第59页第1.1-1.4节，第60页第1.5-1.9节，摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104407145A (en) | 2015-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104407145B (en) | Enterovirns type 71 latex agglutination assay kit preparations and applicatio | |
| Estes et al. | Human norovirus cultivation in nontransformed stem cell-derived human intestinal enteroid cultures: success and challenges | |
| Abzug et al. | Neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin | |
| Zapor | Persistent detection and infectious potential of SARS-CoV-2 virus in clinical specimens from COVID-19 patients | |
| CN102967710B (en) | Competitive ELISA kit of PPR antibody test and preparation method thereof | |
| Bai et al. | The human virome: viral metagenomics, relations with human diseases, and therapeutic applications | |
| CN105527437B (en) | A kind of detection kit and its application | |
| CN102539782B (en) | Immune chromatography test strip for detecting cystic echinococcosis and alveolar echinococcosis | |
| CN102818898B (en) | Test strip for identifying foot-and-mouth-disease virus infected and vaccine immunized animal at one step and preparation method of test strip | |
| CN106841604A (en) | A kind of Respirovirus quick detection kit and preparation method thereof | |
| CN105203754A (en) | Method and kit for fast detection of moraxella catarrhalis based on magnetic resolution and quantum dot labelling | |
| CN105319373A (en) | Rapid human respiratory syncytial virus detection method and kit based on magnetic separating and quantum dot labeling | |
| CN104198710A (en) | Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously | |
| CN101130765B (en) | Reagent kit for detecting syncytial virus of respiratory passage | |
| CN106771182A (en) | Brucella abortus IgM subclass antibodies indirect ELISA testing kits | |
| CN103499685B (en) | People's urinary system cause of disease bacterium infects the preparation method of the immunofluorescence diagnostic reagent of clinical diagnosis | |
| Chua et al. | Saffold virus infection in children, Malaysia, 2009 | |
| CN101660001A (en) | Reagent kit for detecting rotavirus nucleic acid | |
| CN106645714B (en) | EV71 virus IgA antibody test strips and its application | |
| CN106226520B (en) | The application of antigen of mycobacterium tuberculosis albumen Rv0865 and its B cell epitope peptide | |
| CN101458256A (en) | Hepatitis C virus envelope antigen ELISA kit and detecting method | |
| CN102608330A (en) | Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof | |
| Kennett et al. | An epidemic associated with echovirus type 18 | |
| CN103235127A (en) | Marek's disease virus rapid combined-detection test strip | |
| CN105203769A (en) | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160601 Termination date: 20161028 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |