CN201397334Y - Test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly - Google Patents
Test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly Download PDFInfo
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- CN201397334Y CN201397334Y CN2009201506759U CN200920150675U CN201397334Y CN 201397334 Y CN201397334 Y CN 201397334Y CN 2009201506759 U CN2009201506759 U CN 2009201506759U CN 200920150675 U CN200920150675 U CN 200920150675U CN 201397334 Y CN201397334 Y CN 201397334Y
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- antibody
- syndrome virus
- respiratory syndrome
- glass fibre
- pig breeding
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Abstract
A test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly comprises a bottom lining, a sample pad, a glass fibre membrane coated by gold-labeled antibody, a containing membrane and an absorbent paper; the bottom of the containing membrane is pasted on the upper surface of the bottom lining; the upper surfaces of two ends of the containingmembrane are respectively pasted with the glass fibre membrane and the absorbent paper; the upper surface of the other end of the glass fibre membrane is pasted with the sample pad; a detection lineand a control line are formed on the upper surface of the containing membrane; the detection line contains the antibody of breeding and respiratory tract syndrome virus of pig, and the control line contains dynamics antibody. The test paper strip has the advantages of high singularity, high sensitivity, high detection speed, low cost and easy operation; needs no other instrument or equipment used;and can be applied in the detection of breeding and respiratory tract syndrome virus by peasant household, grassroot organization and personal.
Description
Technical field
The utility model belongs to Preventive Veterinary Medicine check field, specifically relates to the colloidal gold test test strips of a kind of fast detecting pig breeding and respiratory syndrome virus.
Background technology
(Porcine reproductive and respiratory syndromevirus PRRSV) is Arteriviridae Arterivirus member to porcine reproductive and respiratory syndrome virus.It is a kind of important communicable disease cause of disease of pig, mainly causes gestational period sow premature labor, miscarriage, produces stillborn foetus, weak tire, mummy tire, and pneumonia, growth retardation and mortality ratio generally take place newborn piglet increases.This virus is found in the U.S. the earliest, subsequently Europe, Asia, a plurality of countries and regions, Oceania have all been found should virus.1996, reported first such as Guo Baoqing were separated to PRRSV from aborted fetus, thereby confirmed that also there is this virus in China.Since 2006, swept across Chinese most of province, brought catastrophic economic loss to pig industry by PRRSV mutant strain caused " highly pathogenic PRRS ".
For the ease of porcine reproductive and respiratory syndrome (PRRS) being made diagnosis accurately, set up kinds of experiments chamber diagnostic method, as: ELISA, RT-PCR, immunofluorescence, in situ hybridization, virus separation etc., but these diagnostic methods need just can be finished by the auxiliary of some specific apparatus, and less stable, troublesome poeration, efficient be low, need cryopreservation, accumulating inconvenience, and this has just limited these methods being extensive use of in grass-roots unit.
Summary of the invention
The purpose of this utility model provides the colloidal gold test test strips of a kind of fast detecting pig breeding and respiratory syndrome virus, need just can finish detection by the auxiliary of some specific apparatus with what solve that prior art exists, the problem that less stable, troublesome poeration, efficient be low, need cryopreservation, accumulating inconvenience.It adopts colloidal gold chromatography preparation of two bands colour developings, and showing the band color is red lines, can accurately detect the content of pig breeding and respiratory syndrome virus in the sample.
The technical solution of the utility model is: the glass fibre membrane, coated film and the thieving paper that comprise end liner, sample pad, coating colloid gold particle labelled antibody, the bottom surface of coated film is sticking to be posted on above the end liner, glue obedient glass fibre membrane and thieving paper, sticking obedient sample pad on the other end of this glass fibre membrane above the coated film two ends respectively at this; Be provided with detection line and control line above the coated film at this, the processed good pig breeding of this detection line bag and the antibody of respiratory syndrome virus, the processed good anti-mouse antibody of this control line bag.
The utility model adopts double antibody sandwich method, can be used to detect blood sample or cotton swab etc. and handles sample.Compare with existing detection kit,, make it can produce specific band, thereby satisfy the demand of terrain peasant household, basic unit's detection because detection line and control line have been carried out corresponding processing.
The utlity model has advantages such as high degree of specificity, sensitivity, detection speed, and need not use instrument and equipment, with low cost, easy and simple to handle, can be widely used in terrain peasant household, basic unit and individual for the detection of pig breeding with respiratory syndrome virus.This test strips can directly be used, and also can be assemblied in the plastic device to use with the check-out console form.
Description of drawings
Fig. 1 is a side structure synoptic diagram of the present utility model;
Fig. 2 is the vertical view of Fig. 1;
Fig. 3 is the positive synoptic diagram of the utility model testing result (detection line 6 on coated film 4 surfaces and control line 7 all develop the color);
Fig. 4 is the negative synoptic diagram of the utility model testing result (the control line 7 colour developing detection lines 6 on coated film 4 surfaces do not develop the color);
Fig. 5 is the invalid synoptic diagram (detection line 6 and the control line 7 on coated film 4 surfaces all do not develop the color) of test strips.
Embodiment
As depicted in figs. 1 and 2, the utility model is included on the end liner 1 in turn mutually, and overlap joint ground glues obedient sample pad 2, apply glass fibre membrane 3, coated film 4 and the thieving paper 5 of the thick colloid gold particle labelled antibody of about 20nm and form, coated film 4 has detection line 6 and control line 7, detection line 6 bags are by the antibody of pig breeding with respiratory syndrome virus, and control line 7 bags are by anti-mouse antibody; Colour developing by described detection line 6 and control line 7 comes qualitative.
Described pig breeding is highly purified anti-monoclonal antibody and/or polyclonal antibody with the antibody of respiratory syndrome virus; The monoclonal antibody of anti-pig breeding of described colloid gold particle mark and respiratory syndrome virus.
Preparation method of the present utility model is:
1, the preparation of coated film 4: on nitrocellulose filter, spray respectively the breeding of anti-pig with
The antibody of respiratory syndrome virus and anti-mouse IgG antibody, and be two lines and be arranged above and below, detection line 6 and control line 7 formed respectively.
Detection line 6: the debugging Membrane jetter, spouting liquid is 25 microlitres/35 centimetre, is cushioned the monoclonal antibody that liquid dilutes anti-pig breeding and respiratory syndrome virus with bag, and concentration is 70ug/ul, rules with the Membrane jetter spraying, and room temperature was dried 20 minutes.
Control line 7: the debugging Membrane jetter, spouting liquid is 25 microlitres/35 centimetre, is cushioned liquid with bag and dilutes anti-mouse Ig6 polyclonal antibody, concentration is 2mg/ml, the machine line, line-to-line should be careful even every 5mm, and room temperature was dried 20 minutes.37 ℃ were sealed 60 minutes, took out rearmounted 37 ℃ of oven dry and handled two hours, and envelope is standby.
2, the preparation of golden labeling antibody (colloid gold particle labelled antibody): transfer collaurum pH value to 7.5 with 0.1M sal tartari, the monoclonal antibody that adds anti-pig breeding and respiratory syndrome virus by 15 micrograms antibody/milliliter collaurum, mixing left standstill 30 minutes, centrifugal 30 minutes of 12000rpm, abandon supernatant, precipitation mark cleansing solution washed twice, it removes supernatant for the last time, will precipitate with the golden labeling antibody of 1/10th initial collaurum volumes to preserve the liquid dissolving, put 4 ℃ standby, 7 days effect phases;
3, the golden labeling antibody that will prepare is coated on the glass fibre membrane (or polyester film), makes the glass fibre membrane 3 of coated with gold labeling antibody.
The glass fibre membrane 3 of described coated with gold labeling antibody can be marked antigen with gold with commonsense method and be coated on the glass fibre membrane, also can obtain by following method: the collaurum that mark is good is layered on the glass fibre membrane equably, 10 square centimeters of every ml soln shops, freeze drying, envelope, put 4 ℃ standby.
4, on end liner 1, paste glass fibre membrane 3, coated film 4 and the thieving paper 5 of sample pad 2, coated with gold mark antigen in turn mutually overlap joint, obtain test paper plate, cut into the test strips of different in width as requested.
Prescription and preparation method that described bag is cushioned liquid and confining liquid are the general technology in this area.
The concrete preparation method of the utility model related material in above-mentioned preparation method further describes as follows:
A. antigen preparation: the monoclonal antibody that adopts highly purified anti-pig breeding and respiratory syndrome virus is as envelope antigen.
B. the preparation of antigenic membrane:
Bag is cushioned the preparation of liquid: 0.05M pH9.6 carbonate buffer solution for bag by solution, the 0.22u membrane filtration, put 4 ℃ standby, 7 days effect phases.
The configuration of confining liquid: the preparation 0.01M pH7.0 phosphate buffer (PBS), 0.22u membrane filtration mistake, put 4 ℃ standby, 7 days effect phases.Preparation sealing working fluid: 2%BSA, 2% skimmed milk, 0.01M pH7.0PBS, 0.22u membrane filtration mistake, put 4 ℃ standby, 3 days effect phases.
Detection line preparation: debugging Membrane jetter, spouting liquid are 25 microlitres/35 centimetre, are cushioned the monoclonal antibody that liquid dilutes anti-pig breeding and respiratory syndrome virus with bag, and concentration is 70ug/ml, and machine is rule, and room temperature was dried 20 minutes.
Control line preparation: debugging Membrane jetter, spouting liquid are 25 microlitres/35 centimetre, are cushioned liquid with bag and dilute anti-mouse IgG polyclonal antibody, and concentration is 2mg/ml, the machine line, and line-to-line should be careful even every 5mm, and room temperature was dried 20 minutes.37 ℃ were sealed 60 minutes, took out rearmounted 37 ℃ of oven dry and handled two hours, and envelope is standby.
C. the manufacturing rules of labeling antibody:
The preparation of gold chloride: with distilled water dissolved chlorine auric acid, be made into 1% solution, put 4 ℃ standby, 3 days effect phases.1000ml 1% chlorauric acid solution prescription: 10g gold chloride; The 1000ml distilled water.
The configuration of citrate three sodium: dissolve citrate three sodium with distilled water, be made into 1% solution, put 4 ℃ standby, 3 days effect phases.
0.1M the preparation of sal tartari: with distilled water preparation, 0.22u membrane filtration mistake, put 4 ℃ standby, 7 days effect phases.1000ml 0.1M solution of potassium carbonate prescription: 13.8g sal tartari; The 1000ml distilled water.
The preparation of 3%PEG-20000: with distilled water preparation, 0.22u membrane filtration mistake, put 4 ℃ standby, 7 days effect phases.1000ml 3%PEG solution formula: 30g PEG-20000; The 1000ml distilled water.
The preparation of mark cleansing solution: 2%BSA, 0.01M pH7.0PBS solution, 0.22u membrane filtration mistake, put 4 ℃ standby, two weeks of effect phase.The preparation of 1000ml mark cleansing solution: 20g BSA; 1000ml 0.01M pH7.0PBS solution.
The preparation that the gold labeling antibody is preserved liquid: 1%BSA, 0.5% skimmed milk, 5/0,000 NaN3,0.1%Tween-20,0.01M pH 7.0PBS solution, 0.22u membrane filtration mistake, put 4 ℃ standby, 15 days effect phases, the configuration that 1000ml gold labeling antibody is preserved liquid: 10g BSA; The 5g skimmed milk; 0.5g NaN3; 1ml Tween-20; 1000ml 0.01M pH7.0PBS solution.
Firing of collaurum: 1% gold chloride is diluted to 0.01% with distilled water, put electric furnace and boil, add 4 milliliter of 1% citrate three sodium, continue to boil by per 100 milliliter of 0.01% gold chloride, be shiny red up to liquid and promptly stop heating, supply dehydration after being cooled to room temperature.Outward appearance should be pure, and is bright, do not have precipitation and floating thing.
The preparation of gold labeling antibody: transfer collaurum pH value to 7.6 with 0.1M sal tartari, the monoclonal antibody that adds anti-pig breeding and respiratory syndrome virus by 10 micrograms antibody/milliliter collaurum, mixing left standstill 30 minutes, centrifugal 30 minutes of 12000rpm, abandon supernatant, precipitation mark cleansing solution washed twice, last supernatant discarded will precipitate with the golden labeling antibody of 1/10th initial collaurum volumes and preserve the liquid dissolving, put 4 ℃ standby, one week of effect phase.
D. the drying of gold bar antibody:
The collaurum that mark is good is layered on the glass fibre membrane equably, 10 square centimeters of every ml soln shops, drying, envelope, put 4 ℃ standby.
E. the glass fibre membrane width of Jin Biaokangtibao quilt is definite:
The collaurum glass fibre membrane is cut into 4 millimeters, 5 millimeters, 6 millimeters specifications, forms test-strips with coated film, every wide 4 millimeters.Selection can detect indoor Quality Control sample, and the gold mark glass fibre membrane width of no nonspecific reaction is decided to be the use width.
F. cutting of golden labeling antibody glass fibre membrane:
Join the width that experiment is determined according to dripping, golden labeling antibody glass fibre membrane is cut, put between drying shed standby.
G. cutting of thieving paper:
With trimmer thieving paper is cut into 35 centimeter length, 4.2 centimetres wide, places between drying shed standby.
H. cutting of glass fibre membrane: this is the glass fibre membrane that does not have labelled antibody, and it plays the effect of guiding liquids flow passage.
The glass fibre cross cutting is grown up 35 centimetres, and wide 2 centimetres is rectangular, places between drying shed standby.
I. the stickup of big plate:
Freeze-drying gold labeling antibody, the glass fibre membrane 3 that coated film 4, thieving paper 5, has been coated in glass fibre membrane sticks on the plastic bottom board on request, forms big plate.Composing room's temperature is controlled at 25 ℃, humidity 20%-30%.
J. slitting:
With cutting cutter big plate is cut into single head part, 3 millimeters of every part of width, sampling observation at random, sensitivity can detect indoor Quality Control, and band colour developing degree reaches one "+" number, specific band nothing but.
K. encapsulation and group box:
1 part of test strips that has cut and one drying prescription of being responsible for a task until it is completed is encapsulated in the aluminium foil polybag, puts into kit, with 4-20 ℃ keep in Dark Place.
The colloidal gold test test strips of a kind of fast detecting pig breeding described in the utility model and respiratory syndrome virus adopts the highly purified anti-pig breeding and the monoclonal antibody of respiratory syndrome virus to carry out special pig breeding and respiratory syndrome virus checking as envelope antigen.Its principle is the monoclonal antibody that contains equally distributed colloid gold label pig breeding and respiratory syndrome virus in the test strips, the antibody of anti-pig breeding and respiratory syndrome virus and anti-mouse IgG antibody are individually fixed on the nitryl film and are two lines and is arranged above and below, the negative up contrast of anti-mouse IgG antibody, the antibody of the breeding of anti-pig and respiratory syndrome virus below be detection line.Described detection line and control line be by the control size of colloid gold particle and what, determines having or not of pig breeding and respiratory syndrome virus thereby obtain the detection line of the different depths of color and control line.When containing the antigen of pig breeding and respiratory syndrome virus in by sample, pig breeding and respiratory syndrome virus earlier and the antibody of the breeding of the pig of golden mark and respiratory syndrome virus carry out specific adsorption, formation Ag-Ab beta composite.Because the capillarity and the chromatography effect of miillpore filter, the reaction compound moves forward along the nitryl film, when waiting to walk to the antibody line of anti-α pig breeding and respiratory syndrome virus, form Ab α-Ag-Ab β-Au compound and be enriched in bag by on the line, red precipitate line of final formation is to be the positive reaction of pig breeding and respiratory syndrome virus.And irrelevant golden mark mouse IgG continues to go upward to anti-mouse IgG antibody place on the test strips, and the antigen antibody complex that forms red-label with it is negative contrast.
When being used to detect, observing the colour developing and the shape of detection line 6 and control line 7 and judge positive still negative.As Fig. 3, when detection line 6 and control line 7 all developed the color, the result was positive; As shown in Figure 4, when control line 7 colour developing and detection line 6 when not developing the color, the result is negative.Fig. 5 is the invalid synoptic diagram (detection line 6 and the control line 7 on coated film 4 surfaces all do not develop the color) of test strips.
Claims (2)
1, the colloidal gold test test strips of a kind of fast detecting pig breeding and respiratory syndrome virus, it is characterized in that: the glass fibre membrane (3), coated film (4) and the thieving paper (5) that comprise end liner (1), sample pad (2), coating colloid gold particle labelled antibody, the bottom surface of coated film (4) is sticking to be posted on above the end liner (1), glue obedient glass fibre membrane (3) and thieving paper (5), sticking obedient sample pad (2) on the other end of this glass fibre membrane (3) above the two ends respectively at this coated film (4); On this coated film (4), be provided with detection line (6) and control line (7), the processed good pig breeding of this detection line (6) bag and the antibody of respiratory syndrome virus, the processed good anti-mouse antibody of this control line (7) bag.
2, the colloidal gold test test strips of fast detecting pig breeding according to claim 1 and respiratory syndrome virus is characterized in that: described pig breeding is highly purified anti-monoclonal antibody and/or polyclonal antibody with the antibody of respiratory syndrome virus; Described colloid gold particle labelled antibody is the monoclonal antibody of anti-pig breeding and respiratory syndrome virus; The antibody of anti-pig breeding and respiratory syndrome virus and anti-mouse IgG antibody are individually fixed on the nitryl film and are two lines and is arranged above and below, and constitute described detection line (6) and control line (7).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009201506759U CN201397334Y (en) | 2009-05-18 | 2009-05-18 | Test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009201506759U CN201397334Y (en) | 2009-05-18 | 2009-05-18 | Test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly |
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| Publication Number | Publication Date |
|---|---|
| CN201397334Y true CN201397334Y (en) | 2010-02-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009201506759U Expired - Fee Related CN201397334Y (en) | 2009-05-18 | 2009-05-18 | Test paper strip for Colloidal gold test for detecting breeding and respiratory tract syndrome virus of pig rapidly |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808927A (en) * | 2014-01-27 | 2014-05-21 | 武汉中博生物股份有限公司 | Enzyme linked immunosorbent assay kit for detecting porcine reproductive and respiratory syndrome virus |
-
2009
- 2009-05-18 CN CN2009201506759U patent/CN201397334Y/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808927A (en) * | 2014-01-27 | 2014-05-21 | 武汉中博生物股份有限公司 | Enzyme linked immunosorbent assay kit for detecting porcine reproductive and respiratory syndrome virus |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100203 Termination date: 20140518 |