CN202256349U - GICA (gold immunochromatography assay) system for detecting equine encephalitis virus antibody - Google Patents
GICA (gold immunochromatography assay) system for detecting equine encephalitis virus antibody Download PDFInfo
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Abstract
The utility model discloses a GICA (gold immunochromatography assay) system for detecting an equine encephalitis virus antibody. The GICA system comprises a gold immunity analyzer used for quantitative detection, a gold immunity test strip A used for detecting an eastern equine encephalitis virus antibody, a gold immunity test strip B used for detecting a western equine encephalitis virus antibody and a gold immunity test strip C used for detecting a Venezuela equine encephalitis virus antibody. According to the utility model, the GICA technology principle and the indirect-method immunochromatography assay technology principle are combined, thus the sensibility and specificity of a detection method are ensured. The system disclosed by the utility model has the advantages of simplicity in operation, short detection time, no need of professionals, suitability for rapid on-site sieving analysis, capability of achieving the quantitative detection purpose, and the like.
Description
Technical field
The utility model relates to a kind of gold-marking immunity tomographic system that can detect eastern equine encephalitis virus antibody, western equine encephalitis virus antibody, Venezuelan equine encephalitis virus antibody simultaneously.
Background technology
Eastern equine encephalitis virus (Eastern Equine Encephalitis Virus; EEEV) belong to A group arboviruse Togaviridae alphavirus; Contain the sub-thread positive chain RNA, this virus mainly is to propagate through the mosquito matchmaker, and can be human through respiratory tract infection through the gasoloid that contains virus; The meningoencephalitis that it causes is a kind of communicable disease of acute, viral, the natural epidemic disease of infecting both domestic animals and human source property.Infection sequela rate is high, and infectiousness is strong, and fatal rate is high, lacks effective prevention method, and sequelae is left in the back of recovering more, and is big to crowd's harmfulness, so classified as one of anti-main diseases kind of making bio-terrorism by international community, enjoys countries in the world to pay close attention to.
China still is in blank to researchs such as the detection of eastern equine encephalitis virus biological warfare agent and medical protections; In more lacking, development plan studies for a long period of time; Still there are not supporting diagnostic reagent and effective immunization therapy and preventive means to the attack of terrorism of eastern equine encephalitis virus; So it is very urgent to invent quick, easy, as to be fit to the on-the-spot detection Eastern equine encephalitis virus that detects method, the image of China in international community and detection status are had very important significance.
Western equine encephalitis (Western Equine Encephalitis is abbreviated as WEE) (being designated hereinafter simply as western horse) is a kind of acute viral encephalitis that can make multiple animal lethal such as human and horse that is caused by western horse disease poison.West horse disease poison can infect through respiratory infectious, but mainly is by killing propagation, and the antigenicity of western horse disease poison is by membrane glycoprotein E1 and equine encephalitis virus decision.E1 contains in one and antigen site, infects behind the western horse disease poison to produce corresponding antibodies, can learn whether have the infection should virus so detect western horse disease poison E1C antibody.It is malicious to isolate western horse disease the anopheles that nineteen ninety He Haihuai etc. gather from Usu, Xinjiang county and the ixodes persulcatus in Bole county.Lv Xinjun etc. find that in to China's human serum investigation western horse disease poison antibody positive rate is 2.71%.These situation have all confirmed the existence of western horse disease poison in China, have explained that western horse disease poison might become one of new cause of disease of China's infectious diseases.
Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus; Be abbreviated as VEEV) (be designated hereinafter simply as committee horse disease poison) be to belong to Togaviridae, alphavirus, genome is the potent virus of the caused Venezuelan equine encephalitis of sub-thread positive chain RNA.This virus mainly by the mosquito propagation of sucking blood, can cause that horses, the mankind or other animals are ill, and research shows that gasoloid also is a kind of more important route of transmission.This virus has the higher incidence of disease and fatal rate, also can leave sequelae after recovering, and is classified as the bio-terrorism agent by U.S. disease prevention and control center.Though China does not also have the report of finding Venezuelan equine encephalitis virus within the border; But along with international tourism and trade are associated frequent day by day; Carrying the danger that risk factor that this virus imports China into and potential bio-terrorism attack increases day by day; So China should enhance your vigilance domestic especially entry and exit port, strengthen detection to committee's horse disease poison, prevent this virus breaking out in China with popular.
Gold-marking immunity chromatography (GICA) is a novel vitro diagnostic techniques that on monoclonal antibody technique, golden mark technology and new material technology basis, has grown up since the nineties in 20th century.Development in recent years is rapid, has particularly obtained widespread use in the medical test at biomedical sector.This technology mainly is that specific antigen or antibody are fixed on the NC film with ribbon; Gold mark labelled reagent is adsorbed on the pad; Be added on the sample pad of test strips one end when testing sample after, move forward, react to each other behind the gold mark labelled reagent on the dissolving pad through siphon principle; When moving to fixing antigen or antibody regional again; Specificity takes place again with it and combines and be trapped in the compound of determinand and gold marked reagent, accumulates in to detect and is with, the result that developed the color intuitively of the gold mark label through range estimation.The label of wandering about as a refugee is then crossed and is detected band, reaches the purpose of separating automatically with binding label.Since fast and convenient, accurate, high degree of specificity and hypersensitivity had, and visual result is reliable; And reagent and amount of samples are few; Each sample only needs 1-2 μ l, need not instrument and equipment, has simplified loaded down with trivial details routine operation process; Also reduced simultaneously the error that causes because of operation, the gold-marking immunity chromatographic technique is a kind of composite immune chromatographic technique with fastest developing speed at present.The gold sample need not add chromogenic reagent as redness, to the human non-toxic evil.At present should technology in the clinical medical inspection widespread use, be considered in the diagnosis of microorganism and infectious disease and parasitic disease one of the most promising new technology.
The utility model content
To the problem that existing situation exists, the purpose of the utility model is to provide the gold-marking immunity tomographic system that a kind of fast qualitative, specificity and susceptibility are high, can detect eastern equine encephalitis virus antibody, western equine encephalitis virus antibody, Venezuelan equine encephalitis virus antibody simultaneously.
For realizing above-mentioned purpose; A kind of gold-marking immunity tomographic system that detects equine encephalitis virus antibody of the utility model; Comprise gold-marking immunity analyser, the gold-marking immunity test strips A that is used to detect eastern equine encephalitis virus antibody, the gold-marking immunity test strips B that is used to detect western equine encephalitis virus antibody that is used for detection by quantitative, the gold-marking immunity test strips C that is used to detect Venezuelan equine encephalitis virus antibody; Three test strips include the reaction stilt, closely are connected in the nitrocellulose membrane of reaction stilt upper surface middle part; The upper surface of nitrocellulose membrane initiating terminal overlap with adsorptive pads be connected, upper surface that nitrocellulose membrane is terminal overlaps with golden labeling antibody diaphragm and is connected the overlapping sample pad that is connected with of the upper surface portion of golden labeling antibody diaphragm;
Wherein, Golden labeling antibody diaphragm among the gold-marking immunity test strips A comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization eastern equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-eastern equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization eastern equine encephalitis virus near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips B comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization western equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization western equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips C comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization Venezuelan equine encephalitis virus E antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization Venezuelan equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane.
Further, said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C are made into integration side by side.
Further, said reaction holder is the PVC plate, and adsorptive pads is a filter paper for oil.
Further, said golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
Further, said sample pad is glass fibre membrane, polyester film or filter paper fibre.
Further; Said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C wrap up separately whole outside and are provided with shell; Be provided with sample well corresponding to said sample pad place on this shell, be provided with view window corresponding to said Quality Control band, test strip place on the shell.
A kind of gold-marking immunity tomographic system that detects equine encephalitis virus antibody of the utility model in conjunction with the know-why of golden mark mark and indirect method, dual-antigen sandwich method immunochromatography, adopts spreading agent and sealer to carry out pre-service the sample pad; Confirmed optimum reaction condition; The susceptibility and the specificity of detection method have been guaranteed, have had simple to operately that detection time is short; Need not the professional, be fit to advantages such as fast on-the-spot detection.And, by means of relevant device, can accomplish qualitative and detection by quantitative, thereby improve its promotion and application greatly.
Description of drawings
Fig. 1 is the front schematic view of the utility model immuno-chromatographic test paper strip inner structure;
Fig. 2 is the side structure synoptic diagram of the utility model immuno-chromatographic test paper strip inner structure;
Fig. 3 is the positive test symbol synoptic diagram of the utility model immuno-chromatographic test paper strip;
Fig. 4 is the negative result synoptic diagram of the utility model immuno-chromatographic test paper strip;
Fig. 5 is the invalid detection synoptic diagram as a result of the utility model immuno-chromatographic test paper strip;
The 3 kind gold-marking immunity test strips structural representations of Fig. 6 for being wholely set side by side in the utility model.
Embodiment
Below, with reference to accompanying drawing, the present invention is more comprehensively explained, exemplary embodiment of the present invention has been shown in the accompanying drawing.Yet the present invention can be presented as multiple multi-form, and should not be construed as the exemplary embodiment that is confined to narrate here.But, these embodiment are provided, thereby make the present invention, and scope of the present invention is fully conveyed to those of ordinary skill in the art comprehensively with complete.
In order to be easy to explanation, here can use such as " on ", D score " left side " space relative terms such as " right sides ", be used for element shown in the key diagram or characteristic relation with respect to another element or characteristic.It should be understood that except the orientation shown in the figure spatial terminology is intended to comprise the different azimuth of device in using or operating.For example, if the device among the figure is squeezed, be stated as the element that is positioned at other elements or characteristic D score will be positioned at other elements or characteristic " on ".Therefore, the exemplary term D score can comprise upper and lower orientation both.Device can otherwise be located (revolve turn 90 degrees or be positioned at other orientation), and the relative explanation in used here space can correspondingly be explained.
Among Fig. 1, Fig. 2,1: adsorptive pads; 2: nitrocellulose membrane, T: encapsulate reorganization equine encephalitis virus Protein Detection band; C: the Quality Control band that encapsulates goat anti-rabbit igg or goat-anti reorganization equine encephalitis virus protein I gG; 3: contain gold mark mark SPA (staphylococcal protein A, Staphylococal Protein A, SPA) or the golden labeling antibody diaphragm of reorganization equine encephalitis albumen; 4: sample pad; 5: the reaction holder.
Fig. 3, Fig. 4, Fig. 5 are the testing result synoptic diagram of the utility model; T appears, two lines of C are positive; It is negative line of C to occur; Then test strips is invalid lines not occur.The whole outer wrap of test strips is provided with shell 6, is provided with sample well 7 corresponding to the sample pad place on the shell 6, is provided with view window 8 corresponding to Quality Control band, test strip place on the shell.
Fig. 6 is the gold-marking immunity test strips structural representation that can detect eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus antibody simultaneously that is wholely set side by side in the utility model; It comprises gold-marking immunity test strips A, gold-marking immunity test strips B or the gold-marking immunity test strips C that is wholely set, and 3 kinds of gold-marking immunity chromatograph test strips is placed respectively can reach testing goal simultaneously.
Be used in the utility model detect eastern equine encephalitis virus antibody gold-marking immunity test strips A, be used to detect western equine encephalitis virus antibody gold-marking immunity test strips B, be used to detect the gold-marking immunity test strips C of Venezuelan equine encephalitis virus antibody; The basic structure of these three test strips is identical, and its key distinction is golden labeling antibody diaphragm:
Golden labeling antibody diaphragm among the gold-marking immunity test strips A comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization eastern equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-eastern equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization eastern equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips B comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization western equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization western equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips C comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization Venezuelan equine encephalitis virus E antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization Venezuelan equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane.
A kind of gold-marking immunity chromatograph test strip that detects equine encephalitis virus antibody of the utility model; Qualitative detection eastern equine encephalitis virus antibody sensitivity 60 ng/mL; Detect the sensitivity of western equine encephalitis virus antibody and reach 120 ng/mL, detect the sensitivity of Venezuelan equine encephalitis virus antibody and reach 1/20000 times serum dilution.
A kind of gold-marking immunity chromatograph test strip that detects equine encephalitis virus antibody of the utility model; Comprising with sample to be measured and sample diluting liquid mixing; Again sample mix liquid is added test paper sample well place, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result; Said immuno-chromatographic test paper strip comprises:
(1) reaction holder;
(2) adsorptive pads;
(3) nitrocellulose membrane, this film are coated with reorganization equine encephalitis virus albumen and Quality Control detection of antibodies band and Quality Control band;
(4) golden labeling antibody diaphragm, wherein contain gold mark mark SPA (staphylococcal protein A, Staphylococal Protein A, SPA) or reorganization equine encephalitis virus albumen;
(5) sample pad;
Wherein react holder and select the PVC plate for use; Adsorptive pads is selected filter paper for oil for use; Be provided with Quality Control band that the anti-reorganization of goat anti-rabbit igg or rabbit equine encephalitis virus IgG encapsulates, be provided with the test strip that reorganization equine encephalitis virus albumen encapsulates near its initiating terminal place on the nitrocellulose membrane near its end.The material of gold labeling antibody diaphragm is selected from polyester film, spun glass or filter paper fibre, contains the SPA or the reorganization equine encephalitis virus albumen of gold mark mark on it; The sample pad material is selected from polyester film, spun glass or filter paper fibre.
Golden labeling antibody diaphragm in the related test paper of the utility model is processed by the SPA of gold mark or reorganization equine encephalitis virus albumen are uniformly coated on polyester film, glass fibre membrane or the filter paper fibre film.
A kind of structure that detects the gold-marking immunity chromatograph test strip of equine encephalitis virus antibody of the utility model is: reaction holder 5 is positioned at bottom; Nitrocellulose membrane 2 is positioned at the middle part on the reaction holder 5; The T place of nitrocellulose membrane 2 is test strips that reorganization equine encephalitis virus albumen encapsulates, and the C place is the Quality Control band that goat anti-rabbit igg or the anti-reorganization of rabbit equine encephalitis virus protein I gG encapsulate; Gold labeling antibody diaphragm 3 is positioned at a side on nitrocellulose membrane 2 tops and overlaps with it, and this film contains the reorganization equine encephalitis virus antibody of gold mark mark; Adsorptive pads 1 is positioned at the opposite side for golden labeling antibody diaphragm 3 on nitrocellulose membrane 2 tops and overlaps with nitrocellulose membrane 2; Sample pad 4 is positioned on the nitrocellulose membrane 2 side opposite with adsorptive pads 1 and overlaps with golden labeling antibody diaphragm 3.
In the above-mentioned test paper, adsorptive pads 1 one sides are initiating terminal, and golden labeling antibody diaphragm 3 one sides are terminal, and test strip is positioned near terminal, and the Quality Control band approaches initiating terminal.
In the above-mentioned test paper, the concentration of reorganization equine encephalitis virus albumen is 0.1-5mg/ml, and the concentration of Quality Control goat anti-rabbit igg is 0.2-5mg/ml.
In the above-mentioned test paper, the concentration of goat anti-rabbit igg is preferably 0.5-2.5 mg/ml.
In the above-mentioned test paper, SPA mark 1ml gold target amount is 2-6 μ g.
The utility model detects the preparation method of the gold-marking immunity chromatograph test strip of equine encephalitis virus antibody, and this method comprises:
(1) uses and on nitrocellulose membrane, spray equine encephalitis virus albumen at a distance from stream metal spraying pen machine with certain spray film speed and resist the equine encephalitis virus protein I gG that recombinates to be the Quality Control band as test strip and goat anti-rabbit igg or rabbit;
(2) a kind of SPA of gold mark mark or golden labeling antibody diaphragm of reorganization equine encephalitis virus albumen of containing of preparation; The SPA or the reorganization equine encephalitis virus albumen of gold mark are uniformly coated on the glass fibre membrane; And after oven dry or the freeze drying, process golden labeling antibody diaphragm.
The described preparation method of the utility model, in the step of wherein said spraying coated film, spray film speed is 10-100mm/s.
The described preparation method of the utility model, wherein said reorganization equine encephalitis virus albumen, its addition is 0.1-5mg/ml, the dilution of this albumen can be PB solution.
The described preparation method of the utility model, wherein the concentration of the anti-reorganization of goat anti-rabbit igg or rabbit equine encephalitis virus protein I gG is 0.1-5 mg/ml in the Quality Control band.
The described preparation method of the utility model, wherein SPA or the reorganization equine encephalitis virus albumen with the gold mark is uniformly coated on the glass fibre membrane, and wherein pH is 6.0-6.4.
The utility model relates to said test paper in the application that detects equine encephalitis virus antibody; Comprising with sample to be measured and sample diluting liquid mixing; Again sample mix liquid is added test paper sample well 7 places, the liquid in the sample relies on syphonic effect up, but 10-15 minute sentence read result.
The utility model relates to the immune chromatography test paper of the detection equine encephalitis virus antibody of method for preparing.
Use the immune chromatograph testing strip consumptive material in the utility model, consumptive material for example is the pad of spun glass, nitrocellulose filter (for example NC film, SHF 1350225), and sample pad and adsorptive pads, filter paper can be available from Millipore companies.
The gold-marking immunity chromatographic test paper of the above-mentioned detection equine encephalitis virus antibody that the utility model relates to, it also comprises golden labeling antibody diaphragm.
The gold-marking immunity chromatographic test paper of the above-mentioned detection equine encephalitis virus antibody that the utility model relates to wherein reacts holder and selects the PVC plate for use.
The gold-marking immunity chromatographic test paper of the above-mentioned detection equine encephalitis virus antibody that the utility model relates to, wherein adsorptive pads is selected filter paper for use.
The gold-marking immunity chromatographic test paper of the above-mentioned detection equine encephalitis virus antibody that the utility model relates to, wherein golden labeling antibody diaphragm is selected polyester film, spun glass or filter paper fibre for use.
The utility model can reach the purpose of detection by quantitative, and its experimental apparatus that adopts for example is the gold-marking immunity analyser, can derive from Shanghai Optics and Precision Mechanics institute, Chinese Academy of Sciences, China Inst. of Quarantine Inspection Sciences's joint research and development.Detection by quantitative result and judgement: detect sample to be checked with the gold-marking immunity chromatograph test strip, reacted test strips through the scanning of gold-marking immunity analyser, writes down corresponding reflective light intensity successively, representes with optical density detected value T, Quality Control value C and T/C.
(1) confirms decision content (Cut-off)
Utilize the quantitative gold-marking immunity analyser of DJM-3 to detect 20 negative values, confirm decision content (Cut-off) with the ratio calculation of T/C, Cut-off=average (Average)+3 * standard deviation (Stdeva).Positive more than or equal to Cut-off.
(2) matched curve is set up
With the several variable concentrations of object standard items to be checked (comprising feminine gender) is horizontal ordinate, detects the T/C ratio that uses the colloidal-gold strip analyser to record after each concentration with the gold test strip bar and is ordinate, sets up matched curve, and judges detection by quantitative sensitivity.
(3) detection by quantitative
With the T/C ratio that sample to be checked records through gold mark analyser, the substitution matched curve can calculate the concentration of sample, thereby obtains the concentration of sample, reaches detection by quantitative.
One, the preparation of gold-marking immunity chromatograph test strip
Material and method
1, antigen and antibody
The anti-eastern equine encephalitis virus E1C of the rabbit antibody of reorganization eastern equine encephalitis virus E1C antigen, western equine encephalitis virus E1C antigen, Venezuelan equine encephalitis virus E2 proteantigen, purifying, the anti-western equine encephalitis virus E1C of rabbit antibody, the anti-Venezuelan equine encephalitis virus E2 of rabbit antibody are by this research department's preparation.
2, immuno-chromatographic test paper strip consumptive material
Pad (spun glass), nitrocellulose filter (NC film, SHF 1350225), sample pad and adsorptive pads, filter paper are available from Millipore company.
3, the preparation of gold-marking immunity chromatograph test strip
3.1 gold-marking binding pad
SPA or the reorganization equine encephalitis virus antigenic mark of pH value 6.0-6.4, concentration 0.2mg/ml are marked particle, 37 ℃ of dryings in the 25nm of sodium citrate method preparation gold.
3.2 nitrocellulose filter
Detect band: reorganization equine encephalitis virus albumen 1 mg/ml; Quality control band: goat anti-rabbit igg or goat-anti reorganization equine encephalitis virus protein I gG:1mg/ml, 37 ℃ of dryings.
3.3 assembling
Sample pad, pad, adsorptive pads are attached to the end liner card that has the mixture that slits successively, are cut into the bar of 0.4cm, drying, the dress shell, room temperature storage is subsequent use.
Two, the detection of sample
1, the processing of analog sample
Adopt people's blood serum sample to detect, carry out after 100 times of dilutions as sample to be checked with sample preparation liquid; In addition, detect simultaneously adding the anti-equine encephalitis virus antibody of rabbit in the human serum after the dilution.
2 sample detection
Sample and sample preparation liquid (as negative sample) 100 μ l after handling are added to the chromatography strip sample pad end for preparing, leave standstill 15min, observations.Detection band and quality control band redness all occurs and are judged to the positive, and it is negative that only redness appears in quality control band, and detection is with and quality control band does not all develop the color, and then are that test strips lost efficacy.
Three, eastern equine encephalitis virus antibody sensitivity test
1, visual inspection sensitivity is estimated
Optimum reaction condition by confirming prepares the gold-marking immunity chromatograph test strip, detects the Eastern equine encephalitis virus antibody of variable concentrations.Eastern equine encephalitis virus antibody is diluted to concentration with sample preparation liquid is followed successively by 15 ng/mL, 30 ng/mL, 60 ng/mL, 75 ng/mL, 150 ng/mL, 300 ng/mL, 600 ng/mL, 1200ng/mL, detect simultaneously.The result shows, sensitivity 60 ng/mL that directly estimate.
2, the sensitivity of detection by quantitative
Detect variable concentrations Eastern equine encephalitis virus antibody, the gold mark analyser value of reading is 0.012 through computational discrimination value (Cut-off), and average T/C ratio that concentration is lower than the eastern equine encephalitis virus antibody of 60 ng/ml is 0,0.006, is lower than 0.012, and is negative; The average T of the eastern equine encephalitis virus antibody of 60～1200ng/ml/C ratio is respectively 0.067,0.079,0.111,0.221,1.32,1.331, all is higher than 0.012, and is positive; So detection by quantitative sensitivity is 60ng/ml (table 1).
Each concentration testing result of table 1 eastern equine encephalitis virus gold-marking immunity chromatographic test paper
| East horse disease poison AC (ng/mL) | Detected value T (V) | Quality Control value C (V) | The T/C value | The |
| 0 | 0 | 2.437 | 0 | - |
| 15 | 0.001 | 2.364 | 0 | - |
| 30 | 0.014 | 2.475 | 0.006 | - |
| 60 | 0.115 | 2.328 | 0.067 | + |
| 75 | 0.184 | 2.726 | 0.079 | + |
| 150 | 0.257 | 2.311 | 0.111 | + |
| 300 | 0.523 | 2.376 | 0.221 | + |
| 600 | 4.451 | 3.370 | 1.32 | + |
| 1200 | 4.392 | 3.298 | 1.331 | + |
3, the sample practicality detects
With healthy subjects serum with 100 times of dilutions of sample preparation liquid after as sample to be checked; Detect the eastern equine encephalitis virus antibody that adds variable concentrations in the healthy subjects serum after the dilution; The result shows that healthy subjects serum is negative, and the sample sensitivity of eastern equine encephalitis virus antibody is 60 ng/ml.
Four, western equine encephalitis virus antibody test sensitivity test
1, visual inspection sensitivity is estimated
Optimum reaction condition by confirming prepares colloidal gold immuno-chromatography test paper strip, detects the western equine encephalitis virus antibody of variable concentrations.Western equine encephalitis virus antibody is diluted to concentration with sample preparation liquid is followed successively by 90 ng/mL, 120 ng/mL, 180ng/mL, 300 ng/mL, 450 ng/mL, 600 ng/mL, 900 ng/mL, 1200 ng/mL, detect simultaneously.
The result shows that directly sensitivity 120 ng/mL of range estimation colour developing is more clear.
2, the sensitivity of detection by quantitative
Detecting variable concentrations western equine encephalitis virus antibody, is 0.011 through computational discrimination value (Cut-off), and it is positive that concentration is that the value that average T/C ratio is respectively of the western equine encephalitis virus antibody of 120ng/ml all is higher than 0.011 result; When western equine encephalitis virus AC during greater than 120ng/ml, T/C average of relatives value is greater than 0.011, so detection by quantitative sensitivity is 120ng/ml.
Each concentration testing result of table 2 western equine encephalitis virus antibody gold-labeled immunization chromatographic test paper
| West horse disease poison AC (ng/mL) | The T/C value | The |
| 0 | 0 | - |
| 90 | 0.002 | - |
| 120 | 0.014 | + |
| 180 | 0.025 | + |
| 300 | 0.042 | + |
| 450 | 0.054 | + |
| 600 | 0.074 | + |
| 900 | 0.114 | + |
| 1200 | 0.139 | + |
3, typical curve
To the west of horse disease poison AC be horizontal ordinate (X), the gold mark analyser value of reading T/C ratio (Y) is ordinate, sets up collaurum examination criteria curve.When western horse disease poison AC is 120～1200 ng/ml, be good linear relationship between the concentration of antibody and the gold mark analyser value of the reading T/C ratio, equation of linear regression is Y=0.0001X+0.0025, R
2Be 0.9947.
Five, Venezuelan equine encephalitis virus antibody test sensitivity test
1, visual inspection sensitivity is estimated
Optimum reaction condition by confirming prepares colloidal gold immuno-chromatography test paper strip, detects the Venezuelan equine encephalitis virus antibody of variable concentrations.The horse disease of will entrusting poison E2 rabbit anteserum is diluted to concentration with sample preparation liquid and is followed successively by 1/700,1/1000,1/3000,1/7000,1/10000,1/20000 and 1/30000 times of dilution and detects simultaneously.
The result shows that sensitivity 1/20000 colour developing of directly range estimation is more clear.
Six, specificity test
1, eastern equine encephalitis virus antibody gold-labeled immunization chromatographic test paper
Detect Venezuelan equine encephalitis virus E2-19AA rabbit anteserum, Venezuelan equine encephalitis virus E2 rabbit anteserum, Western equine encephalitis virus E1C antibody, dengue virus Deng-E-3 rabbit anteserum, H1N1 Antibody of Influenza and west nile virus NS1 rabbit anteserum with the gold-marking immunity chromatograph test strip for preparing; Positive reaction does not all appear in testing result; Explain and these antibody, the equal no cross reaction of serum; Detect 5 parts of rabbit anteserums, 15 parts of mouse serum, 51 parts of healthy subjects serum; The result is all negative, shows that this detection method specificity is good.
2, Venezuelan equine encephalitis virus antibody gold-labeled immunization chromatographic test paper
Optimum reaction condition by confirming prepares the gold-marking immunity chromatograph test strip, detects horse beautiful eyes GP2 virus rabbit anteserum, Eastern equine encephalitis virus E1C rabbit anteserum, western equine encephalitis virus E1C rabbit anteserum, dengue virus E 2 rabbit anteserums, H1N1virus antibody, west nile virus NS1 rabbit anteserum with the sample diluting liquid colloidal gold immuno-chromatography test paper strip that Processing of Preparation is good respectively.Positive reaction does not all appear in testing result, proves and the equal no cross reaction of these antibody that this detection method specificity is good.
3, western equine encephalitis virus antibody gold-labeled immunization chromatographic test paper
Optimum reaction condition by confirming prepares the gold-marking immunity chromatograph test strip, with the anti-horse beautiful eyes of rabbit GP2 albumen, smallpox M1R albumen, bird flu NP albumen, dengue virus Deng-E-D3 albumen, Rickettsia prowazeki 120N protein antibodies, 63 parts of healthy mouse serum and 105 parts of healthy subjects serum of sample diluting liquid detection.Detect with the gold-marking immunity chromatograph test strip for preparing, and with the western equine encephalitis virus antibody sample contrast of same concentration, detect blank simultaneously.The result shows that respond well to the detection of western equine encephalitis virus antibody specificity, with sample preparation liquid 1:10 dilution back detection, the result has 6 parts to detect the positive with 63 parts of healthy mouse serum, and false positive rate is 9.52%; 105 parts of healthy subjects serum are detected, and the result has 7 parts to detect the positive, and false positive rate is 6.67%.This test strip is for all specific phenomenon appearance nothing but of these antibody.
Seven, stability test
Freshly prepd gold-marking immunity chromatograph test strip was deposited for 2 weeks in 37 ℃, at room temperature place 4 week its specificitys compare with freshly prepd test strips with susceptibility, sensitivity is obviously decline not, and specificity is good.
Claims (6)
1. gold-marking immunity tomographic system that detects equine encephalitis virus antibody; It is characterized in that; This system comprises gold-marking immunity analyser, the gold-marking immunity test strips A that is used to detect eastern equine encephalitis virus antibody, the gold-marking immunity test strips B that is used to detect western equine encephalitis virus antibody that is used for detection by quantitative, the gold-marking immunity test strips C that is used to detect Venezuelan equine encephalitis virus antibody; Three test strips include the reaction stilt, closely are connected in the nitrocellulose membrane of reaction stilt upper surface middle part; The upper surface of nitrocellulose membrane initiating terminal overlap with adsorptive pads be connected, upper surface that nitrocellulose membrane is terminal overlaps with golden labeling antibody diaphragm and is connected the overlapping sample pad that is connected with of the upper surface portion of golden labeling antibody diaphragm;
Wherein, Golden labeling antibody diaphragm among the gold-marking immunity test strips A comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization eastern equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-eastern equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization eastern equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips B comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization western equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization western equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;
Golden labeling antibody diaphragm among the gold-marking immunity test strips C comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization Venezuelan equine encephalitis virus E antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of reorganization Venezuelan equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane.
2. the system of claim 1 is characterized in that, said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C are made into integration side by side.
3. system as claimed in claim 2 is characterized in that, said reaction holder is the PVC plate, and adsorptive pads is a filter paper for oil.
4. system as claimed in claim 2 is characterized in that, said golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
5. system as claimed in claim 2 is characterized in that, said sample pad is glass fibre membrane, polyester film or filter paper fibre.
6. system as claimed in claim 2; It is characterized in that; Said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C wrap up separately whole outside and are provided with shell; Be provided with sample well corresponding to said sample pad place on this shell, be provided with view window corresponding to said Quality Control band, test strip place on the shell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011203678314U CN202256349U (en) | 2011-09-29 | 2011-09-29 | GICA (gold immunochromatography assay) system for detecting equine encephalitis virus antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011203678314U CN202256349U (en) | 2011-09-29 | 2011-09-29 | GICA (gold immunochromatography assay) system for detecting equine encephalitis virus antibody |
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| CN202256349U true CN202256349U (en) | 2012-05-30 |
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| CN2011203678314U Expired - Fee Related CN202256349U (en) | 2011-09-29 | 2011-09-29 | GICA (gold immunochromatography assay) system for detecting equine encephalitis virus antibody |
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| CN (1) | CN202256349U (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445536A (en) * | 2011-09-29 | 2012-05-09 | 中国检验检疫科学研究院 | Gold mark immune paper test strip used for detecting equine encephalitis viral antibody and preparation method and application thereof |
| RU2803155C1 (en) * | 2021-11-09 | 2023-09-07 | Северо-Западный Институт Экологической Окружающей Среды И Ресурсов Китайской Академии Наук | Test card and test kit for detection of brassica yellows virus |
-
2011
- 2011-09-29 CN CN2011203678314U patent/CN202256349U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445536A (en) * | 2011-09-29 | 2012-05-09 | 中国检验检疫科学研究院 | Gold mark immune paper test strip used for detecting equine encephalitis viral antibody and preparation method and application thereof |
| RU2803155C1 (en) * | 2021-11-09 | 2023-09-07 | Северо-Западный Институт Экологической Окружающей Среды И Ресурсов Китайской Академии Наук | Test card and test kit for detection of brassica yellows virus |
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