CN209280731U - A kind of quickly detection viral infection of measles kit - Google Patents
A kind of quickly detection viral infection of measles kit Download PDFInfo
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- CN209280731U CN209280731U CN201821816525.2U CN201821816525U CN209280731U CN 209280731 U CN209280731 U CN 209280731U CN 201821816525 U CN201821816525 U CN 201821816525U CN 209280731 U CN209280731 U CN 209280731U
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Abstract
The utility model provides a kind of quickly detection viral infection of measles kit, comprising: test strip, sample treatment solution, sample treatment solution storing containers；The test strip includes that sample pad, colloidal-carbon bonding pad, chromatographic film, water absorption pad are successively pasted on hydrophobic supporting pad and constitute, and the MAbH1 of colloid carbon markings is coated on colloidal-carbon bonding pad；The chromatographic film includes T line and C line；The T line is coated with MAbH2；The C line is coated with goat anti-human igg's monoclonal antibody；The utility model can go out the symptoms such as fever and dermexanthesis as caused by measles virus by primary sample, rapid screening, provide strong foundation for accurate treatment in next step；The kit sensibility and specificity of the utility model is higher, fast and convenient intuitive, is suitable for promoting the use of.
Description
Technical field
The utility model relates to disease detection fields, more particularly to a kind of quickly detection viral infection of measles kit.
Background technique
Morbilli is one of most common Acute respiratory infectious disease of children, and infectiousness is very strong, densely populated and not general
The easy happening and prevelence in area of kind vaccine, generation in about 2~3 years are once very popular.Clinically may occur in which fever, inflammation of upper respiratory tract,
The symptoms such as eye conjunctivitis, and occur having in red maculopapule and buccal mucosa spots,Filatov's and rash to step back with skin to leave pigment heavy
With bran sample furfur be main feature.
Measles virus is spherical or silk shape, and diameter about 120nm~250nm, core is single strand RNA, non-segmented negative, gene
Group overall length about 16kb, genome have N, P, M, F, H, L6 genes, are separately encoded 6 structure and function albumen: nucleoprotein
(nucleoprotein, NP), phosphorylated protein (phosphoprotein, P), M albumen (membraneprotein, M), fusion
The RNA polymerase of albumen (fusionprotein, F), hemagglutinin (hemagglutinin, H) and dependenc RNA
(largepolymerase, L), nucleocapsid is helically symmetrical, has coating outside, there are two types of furcellas, i.e. hemagglutinin on surface
(hemagglutinin, H) and hemolysin (haemolyxin, HL), their ingredient are all glycoprotein, but distinct, and H is only
Monkey red blood cells can be aggregated, moreover it is possible to adsorbed with host cell receptor, HL there is haemolysis and make cell that fusion occur to form multicore big and small
The effect of born of the same parents, H and HL have antigenicity, and the corresponding antibodies of generation have protective effect.Clinical labororatory's detection method has, and takes trouble
Blood, throat wash or the throat swab of person's morbidity early stage is inoculated in human embryo kidney (HEK), monkey kidney or human amniotic cell after antibiotic treatment
Culture detects IgM antibody with indirect fluorescent antibody technique or ELISA, is checked in patient's catarrhal period Pharyngeal aspirate with fluorescent labeled antibody
Mucomembranous cell whether there is or not measles virus antigens, also can detect intracellular viral nucleic acid with hybridization.But this
A little methods are not only bothersome, time-consuming, laborious, and need higher professional technique and experience.Colloid gold test paper is developed in recent years
Item is easy to operate, quick, is widely applied, but costly, is not easy to mark, and color identifying is unobvious.Colloidal carbon
Particle technique overcomes disadvantages described above.World Health Organization's innovation diagnostics foundation confirmed colloidal-carbon as immunochromatographic method
The advantage of signal label, it is seen that light chart scanner Scanning Detction band colloidal-carbon gray value, by analyzing software quantification testing concentration
Sample can be quantified.The a variety of antigens of detection are widely used in using colloidal-carbon as signal label both at home and abroad.This application is used
Colloidal-carbon applies to the detection of measles virus antigens as signal label, and the quick and quantitative Diagnosis of measles virus may be implemented.
The present invention is used as antigen, measles haemagglutinin protein with measles haemagglutinin protein (hemagglutinin, H)
(hemagglutinin, H) can stimulate body to generate effective immune response, be the two receptoroid film confactors of measles virus
The major site of PROTEIN C D46 and signal transduction lymphocyte activator molecule SLAM effect, while can also be with the envelope membrane surface of virus
Fusion protein interaction.The mutation of research surface H gene directly results in the haemagglutination of measles virus, cell-cytotoxic reaction
With virus and all various variations such as acceptor interaction, and the measles haemagglutinin protein in blood of human body
(hemagglutinin, H) is longer and more and more with the time of infection, therefore measles haemagglutinin protein
The correspondence monoclonal antibody of (hemagglutinin, H) is expected to the candidate molecules as measles virus quick diagnosis.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of this utility model is that using colloidal-carbon immunochromatographic method,
A kind of quickly detection viral infection of measles kit is provided, for establishing a kind of quickly detection measles haemagglutinin protein
(hemagglutinin, H) and the immune diagnostic method for being able to achieve semi-quantitative analysis.To overcome current fast diagnosis reagent
The defect of box, using can be by the colloidal-carbon of gray scale scanning realization half-quantitative detection as marker, utilization in the utility model
Double antibody sandwich method, using the monoclonal antibody of measles haemagglutinin protein as labelled protein and coating chromatographic film capture albumen,
Establish a kind of quick detection viral infection of measles kit based on double antibody sandwich method.
In order to achieve the above objects and other related objects, the utility model provides a kind of quickly detection viral infection of measles examination
Agent box includes: test strip, sample treatment solution, sample treatment solution storing containers；It include anti-morbilli in the test strip
The monoclonal antibody 1(MAbH1 of hemagglutinin (hemagglutinin, H)) and anti-measles haemagglutinin protein monoclonal antibody
2(MAbH2), respectively with the antibody of different epitopes；The test strip includes sample pad, colloidal-carbon bonding pad, chromatography
Film, water absorption pad are successively pasted on hydrophobic supporting pad and constitute；The MAbH1 of colloid carbon markings is coated on the colloidal-carbon bonding pad
And goat anti-human igg；The chromatographic film is equipped with T line and C line, and the monoclonal antibody MAbH2 of anti-H is coated on the T line, described
C line on be coated with goat anti-human igg；The sample pad connects chromatographic film by colloidal-carbon bonding pad, and the chromatographic film connection is inhaled
Water cushion；Described MAbH1, MAbH2 are manually to prepare antibody.
Preferably, coated MAbH2 concentration is the mg/ml of 1.5mg/ml~2.0 on the T line, and discharge rate is 0.10 μ l/mm
~0.25 μ l/mm.
Preferably, coated goat anti-human igg's concentration is the mg/ml of 1.0 mg/ml~2.0, discharge rate 5ul/ on the C line
Cm~10ul/cm.
Preferably, the MAbH1 of the colloid carbon markings is prepared by following methods: the colloidal-carbon for the use of concentration being 0.1%
Solution, adjusting its pH value is 9, and the antibody of 1000ug is added in 10ml colloidal carbon solution, is uniformly mixed, mixes repeatedly at room temperature
Even reaction 3 hours, 10%BSA, which is added, makes final concentration to 1%, 10% polyethylene glycol is added to final concentration of 0.2%, it is small that room temperature mixes 3
When, 4 DEG C of 13000g are centrifuged 30min, discard supernatant liquid, precipitate the 0.01M pH7.6 phosphoric acid containing 0.4%BSA, 0.2%PEG
Salt buffer suspends, and repeated centrifugation is twice.Precipitating is suspended with the dilution of the colloidal-carbon labelled antibody of original solution volume 1/10, is added
Enter 0.01% thimerosal anti-corrosion to obtain.
Preferably, the goat anti-human igg of the colloid carbon markings is prepared by following methods: the glue for the use of concentration being 0.1%
Body carbon solution, adjusting its pH value is 9, and the goat anti-human igg of 1000ug is added in 10ml colloidal carbon solution, is uniformly mixed, room
Temperature mixes reaction 3 hours repeatedly, and 10%BSA, which is added, makes final concentration to 1%, 10% polyethylene glycol is added to final concentration of 0.2%, room temperature
It mixes 3 hours, 4 DEG C of 13000g are centrifuged 30min, discard supernatant liquid, 0.01M of the precipitating containing 0.4%BSA, 0.2%PEG
PH7.6 phosphate buffer suspends, and repeated centrifugation is twice.Precipitating is with the dilution of the colloidal-carbon labelled antibody of original solution volume 1/10
Liquid suspends, and 0.01% thimerosal anti-corrosion is added and obtains.
Preferably, the colloidal carbon solution is prepared by following methods: the borate buffer solution of 0.01M pH9.0, addition are received
Rice carbon is configured to 0.1% suspension, and 0.1% Triton100 is added, and 300W ultrasonic treatment, work 6s, stops 4s, and 80 recycle,
200rpm is centrifuged 10min, and supernatant is taken to obtain colloidal carbon solution.
Preferably, the dilution of the colloidal-carbon labelled antibody is prepared by following methods: 0.01 M pH7.6 precipitate phosphoric acid
1%BSA, 0.2%PEG, 3% sucrose, 0.5% Triton100,0.5%PVP is added in salt buffer.
Preferably, coated MAbH1 concentration is 10ug/ml~20ug/ml on the colloidal-carbon bonding pad.
Preferably, on the colloidal-carbon bonding pad coated colloid carbon markings goat anti-human igg's concentration be 10ug/ml~
20ug/ml。
Preferably, the colloidal-carbon labelled antibody MAbH1, the T line coating MAbH2 is manually to prepare antibody.
Preferably, the test strip further includes cover board and bottom plate, there is sample-adding window and detection window on the cover board,
The cover board is located on the bottom plate, so that inner containment chamber is formed between the cover board and the bottom plate, the sample pad,
Colloidal-carbon bonding pad, chromatographic film, water absorption pad, hydrophobic supporting pad are located in the inner containment chamber, and the sample-adding window is located at institute
It states in sample pad, the detection window is located in the chromatographic film.
Preferably, a kind of quickly detection viral infection of measles kit further includes sample treatment solution storing containers, institute
It states in sample treatment solution storing containers equipped with sample treatment solution.
Preferably, the sample treatment solution is prepared by the following steps to obtain: weighing 0.15M NH4Cl, the 1.00g of 8.00g
10mM KHCO3,0.37g 1mM NaEDTA, be dissolved in the tri-distilled water of 980ml, with 1M HCl or 1M NaOH adjust pH value
To between 6.0~6.4, add tri-distilled water constant volume to 1000ml, it is spare.
As described above, one kind of the utility model quickly detects viral infection of measles kit, in use, by sample to be tested
10ul is added drop-wise to the sample-adding window of test strip, and 10ul sample treatment solution is added dropwise, the morbilli that sample discharges after treatment
Hemagglutinin (hemagglutinin, H) first forms compound with the MAbH1 of the colloid carbon markings on colloidal-carbon bonding pad, by
It is acted in chromatography, reaction compound is moved forward along chromatographic film, is captured and assembled by MAbH2, the amount of aggregation is more, then sample
Positive degree is stronger, and testing result is viral infection of measles at this time；And working as in sample does not have measles haemagglutinin protein or its content
When lower than 1ng/ml, there is no or have minimal amount of compound to assemble in T line, testing result is feminine gender at this time.Meanwhile C line is coated with
IgG react to form certain band, then show that testing result is normal, if there is not band on C line, illustrate that sample to be tested exists
It is not chromatographed normally in test strips.
Detailed description of the invention
Fig. 1 is shown as the main schematic diagram that a kind of quickly detection viral infection of measles kit of the utility model removes cover board.
Fig. 2 is shown as a kind of main schematic diagram of quickly detection viral infection of measles kit of the utility model.
Fig. 3 is shown as measles when a kind of specific implementation of quickly detection viral infection of measles kit of the utility model
The schematic top plan view of poison infection positive findings.
Fig. 4 is shown as measles when a kind of specific implementation of quickly detection viral infection of measles kit of the utility model
The schematic top plan view of malicious negative findings.
Result when Fig. 5 is shown as a kind of specific implementation of quickly detection viral infection of measles kit of the utility model without
The schematic top plan view of effect.
Description of symbols: 1, sample pad；2, colloidal-carbon bonding pad；3, chromatographic film；4, water absorption pad；5, hydrophobic supporting pad；
6, T line；7, C line；8, it is loaded window；9, detection window；10, cover board.
Specific embodiment
Embodiment 1
A kind of quickly detection viral infection of measles kit, by sample pad 1, colloidal-carbon bonding pad 2, chromatographic film 3, water absorption pad
4 are successively pasted on hydrophobic supporting pad 5 and constitute, between material must close contact, cannot be there are gap, joint is to be overlapped
1.0~1.5mm is advisable, and the hydrophobic supporting pad 5 cuts smooth, thick 0.2mm, long 7.0cm, wide 3.0mm, in the chromatographic film 3
It is provided with T line at a distance 2cm of upper connection bonding pad 2, and is provided with C line 7 at the 1cm of distance T line, the bonding pad
It is coated with MAbH1, the concentration 15ug/ml of colloid carbon markings on 2, the goat-anti of colloid carbon markings is coated on the bonding pad 2
Human IgG concentration is 15ug/ml, and MAbH2, concentration 2mg/ml are coated on the T line 6, and discharge rate is 0.25 μ l/mm, the C line
Coated goat anti-human igg's concentration is 2mg/ml on 7, and discharge rate is 3 μ l/mm.
Embodiment 2
The embodiment the preparation method comprises the following steps:
A. purifying is prepared according to a conventional method obtains measles haemagglutinin protein；
B. the monoclonal antibody of more plants of mouse, purifying acquisition anti-H is immunized using conventional method, ratio is reacted with corresponding antigens
Compared with, filter out preferable two class of effect respectively, one plant as label monoclonal antibody MAbH1, one plant as coating monoclonal antibody MAbH2；
C. colloid carbon markings goat anti-human igg: the colloidal carbon solution for the use of concentration being 0.1%, adjusting its pH value is 9, will
The goat anti-human igg of 100ug is added in 1ml colloidal carbon solution, is uniformly mixed, and mixes reaction 3 hours repeatedly at room temperature, is added
10%BSA makes final concentration to 1%, 10% polyethylene glycol is added to final concentration of 0.2%, room temperature mixes 3 hours, 4 DEG C of 13000g centrifugations
30min discards supernatant liquid, and precipitating is suspended with the 0.01M pH7.6 phosphate buffer containing 0.4%BSA, 0.2%PEG, repeats
Twice, precipitating is suspended with the dilution of the colloidal-carbon labelled antibody of original solution volume 1/10 for centrifugation, and it is anti-that 0.01% thimerosal is added
It is rotten；Colloid carbon markings MAbH1: the colloidal carbon solution for the use of concentration being 0.1%, adjusting its pH value is 9, and the MAbH1 of 1000ug is added
Enter into 10ml colloidal carbon solution, be uniformly mixed, at room temperature the even reaction of back mixing 3 hours, 10%BSA, which is added, makes final concentration to 1%, adds
Enter 10% polyethylene glycol to final concentration of 0.2%, room temperature mixes 3 hours, and 4 DEG C of 13000g are centrifuged 30min, discard supernatant liquid, precipitates
It is suspended with the 0.01M pH7.6 phosphate buffer containing 0.4%BSA, 0.2%PEG, repeated centrifugation is twice.Precipitating is with original solution
The dilution of the colloidal-carbon labelled antibody of volume 1/10 suspends, and 0.01% thimerosal anti-corrosion is added and obtains；
D. colloidal-carbon bonding pad is handled: after impregnating 10min with the 0.01M pH7.6 phosphate buffer containing 0.2%PVA
It is placed in 37 DEG C to dry, then is placed on 37 DEG C with colloidal-carbon labelled antibody immersion bonding pad 10min and dries, kept dry；
E. prepared by T line: taking MAbH2 concentration is 1.5mg/ml, and discharge rate is 0.25 μ l/mm, is coated in chromatographic film；
F. prepared by C line: taking goat anti-human igg's concentration is 2mg/ml, and discharge rate is 3 μ l/mm, is coated in chromatographic film；
G. it chromatographs film process: the chromatographic film for being coated with T line and C line being placed in 50 DEG C and is baked for 24 hours, kept dry；
H. sample pad 1, colloidal-carbon bonding pad 2, chromatographic film 3, water absorption pad 4 are successively pasted on hydrophobic supporting pad 5, material
Between must close contact, cannot be there are gap, joint is overlapped 1.0mm, is cut into single with cutting machine；
Test strips using embodiment 1 are easy to operate, have quick, easy, intuitive and do not need specific apparatus, sensitive
Property and specificity be respectively 96.00% and 95.00%, more existing detection means compared to not only quickly and also accuracy it is higher.
Claims (5)
1. a kind of quickly detection viral infection of measles kit, which is characterized in that a kind of quickly detection viral infection of measles
Kit includes: test strip, sample treatment solution, sample treatment solution storing containers；It include anti-fiber crops in the test strip
The monoclonal antibody 1 of rash hemagglutinin and the monoclonal antibody 2 of anti-measles haemagglutinin protein, the list of anti-measles haemagglutinin protein
Clonal antibody 1 is abbreviated as MAbH1, and the monoclonal antibody 2 of anti-measles haemagglutinin protein is abbreviated as MAbH2, respectively has difference
The antibody of epitope；The test strip include sample pad, colloidal-carbon bonding pad, chromatographic film, water absorption pad be successively pasted onto it is hydrophobic
It is constituted on supporting pad；The MAbH1 and goat anti-human igg of colloid carbon markings are coated on the colloidal-carbon bonding pad；The chromatographic film
It is equipped with T line and C line, the monoclonal antibody MAbH2 of anti-H is coated on the T line, is coated with goat-anti people on the C line
IgG；The sample pad connects chromatographic film by colloidal-carbon bonding pad, and the chromatographic film connects water absorption pad；Described MAbH1, MAbH2
It is manually to prepare antibody.
2. a kind of quickly detection viral infection of measles kit according to claim 1, it is characterised in that: on the T line
Coated MAbH2 concentration is 1.5mg/ml~2.0mg/ml, and discharge rate is 0.10 μ of μ l/mm~0.25 l/mm.
3. a kind of quickly detection viral infection of measles kit according to claim 1, it is characterised in that: on the C line
Coated goat anti-human igg's concentration is 1.0mg/ml~2.0mg/ml, and discharge rate is 5ul/cm~10ul/cm.
4. a kind of quickly detection viral infection of measles kit according to claim 1, it is characterised in that: the colloidal-carbon
The MAbH1 concentration of coated colloid carbon markings is 10ug/ml~20ug/ml on bonding pad；It is coated on the colloidal-carbon bonding pad
Colloid carbon markings goat anti-human igg's concentration be 10ug/ml~20ug/ml.
5. a kind of quickly detection viral infection of measles kit according to claim 1, it is characterised in that: the detection examination
Paper slip further includes cover board and bottom plate, has sample-adding window and detection window, the cover board to be located on the bottom plate on the cover board, from
And inner containment chamber is formed between the cover board and the bottom plate, the sample pad, colloidal-carbon bonding pad, chromatographic film, water suction
Pad, hydrophobic supporting pad are located in the inner containment chamber, and the sample-adding window is located in the sample pad, the detection window position
In in the chromatographic film.
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| CN201821816525.2U CN209280731U (en) | 2018-11-07 | 2018-11-07 | A kind of quickly detection viral infection of measles kit |
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| CN201821816525.2U CN209280731U (en) | 2018-11-07 | 2018-11-07 | A kind of quickly detection viral infection of measles kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114859040A (en) * | 2022-05-24 | 2022-08-05 | 广州万孚生物技术股份有限公司 | Respiratory tract pathogen antigen detection reagent strip with sampling indication and preparation method thereof |
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2018
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114859040A (en) * | 2022-05-24 | 2022-08-05 | 广州万孚生物技术股份有限公司 | Respiratory tract pathogen antigen detection reagent strip with sampling indication and preparation method thereof |
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