CN101055273B - Animal brucella antibody colloidal gold test paper film detection reagent kit - Google Patents
Animal brucella antibody colloidal gold test paper film detection reagent kit Download PDFInfo
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- CN101055273B CN101055273B CN 200710055740 CN200710055740A CN101055273B CN 101055273 B CN101055273 B CN 101055273B CN 200710055740 CN200710055740 CN 200710055740 CN 200710055740 A CN200710055740 A CN 200710055740A CN 101055273 B CN101055273 B CN 101055273B
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Abstract
The invention discloses an animal brucella antibody colloidal gold test paper film detection kit, which bases on immunology basic principle of antigen and antibody specific combination, utilizes colloidal gold immunity chromatography technique and silver dye reinforcement technique, uses combined golden yellow staphylococcus A protein (SPA) as gold marking antibody, uses high purity brucella LPS as peridium antigen, and utilizes goat anti-bovine IgG as quality control line to prepare colloidal gold test paper tape. The invention can accurately differentiate species of animal brucella and can synchronously detect animal brucella disease of cattle, sheep and pig species with no crossing reaction and low false negative proportion, negative and positive coincidence ratio of the invention and conventional separation and culture is 100%, detection result can be obtained in 5 min, and the kit need no assistant complicated detection instrument.
Description
Technical field
The present invention discloses a kind of animal brucella antibody colloidal gold test paper film detection reagent kit, is used for the diagnosis of brucellosis, belongs to animal epidemic diagnostic techniques field.
Background technology
Brucella is a kind of Gram-negative, specificity born of the same parents' endophyte, can infect many animals and people.At present, according to pathogenic and host's difference brucella is divided into 6 kinds, i.e. Brucella melitensis, Brucella abortus, Brucella suis, sarin mouse brucella, Xinjiang Brucella ovis, dog brucella.Wherein worldwide pandemic is ox, sheep, Brucella suis, huge to animal husbandry harm, the more important thing is that they all can cause people's infection, are threatening public health.So the research of brucellosis diagnosis aspect is received the concern of countries in the world always.The serological diagnostic method of traditional diagnosis animal brucellosis comprises: quick sliding agglutination test (RSAT), standard tube agglutination test (SAT), 2 mercapto ethanol tube agglutination test (2ME), complement fixation test (CFT) (CFT) etc.They all exist following deficiency, at first, with other bacterium (as: Yersinia ruckeri O9, Escherichia coli O 157) the cross reaction phenomenon occur and are difficult to avoid; The second, traditional diagnosis method is time-consuming, require great effort and be prone to false positive; The 3rd, traditional diagnostic method can not be distinguished the cause of disease kind type of brucellosis.Therefore, set up a kind of easy, quick, special diagnostic method, have great importance for diagnosis, quarantine and the epidemiology survey of animal brucellosis.
Summary of the invention
The present invention discloses a kind of animal brucella antibody colloidal gold test paper film detection reagent kit, and this kit can detect ox, sheep, Brucella suis antibody, and is with traditional serological method ratio, easier, quick, special.Be applicable to diagnosis, quarantine and the epidemiology survey of animal brucellosis.
Solution of the present invention is the immunology principle according to antigen, antibody capable specific bond, utilize colloidal gold immunochromatographimethod technology and silver to dye the reinforcement technology, staphylococcal protein A (SPA) with restructuring is marked albumen as gold, as envelope antigen, utilize goat-anti ox IgG to prepare colloidal gold test paper film for nature controlling line with highly purified brucella LPS.This test kit comprises colloidal gold test paper film, positive and negative reagent, plastic suction pipe centrifuge tube and instructions.
The concrete method for making of kit
Extract the LPS of Brucella abortus 544A and Brucella melitensis 16M with hot phenol method and cold phenol method, behind Sephadex G-50 chromatographic column purifying, the content of LPS is 1.21～2.31mg/mL, and agar diffusion test shows that this antigen can react with corresponding antibody, and kind of interior an intersection do not occur.Utilize the LPS of purifying as the detection line envelope antigen, as gold mark albumen, be assembled into test paper film according to the ultimate principle of colloidal gold immunity chromatography with recombination staphylococcus aureus A albumen (SPA).Result of study shows, ox, sheep, pig animal brucella antibody Test paper lowest detection protein content are respectively 1 μ g/mL, 2 μ g/mL, not with Escherichia coli O 157, YE O9, salmonella, the actinomycetic positive serum generation of pleuropneumonia cross reaction, 4 ℃ of storage lives are 12 months.20 parts of brucella positive serums are with after the PBS1:1 dilution, and the test paper of assembling with this experiment detects, as a result 19 parts positive, coincidence rate is 95%.18 parts of brucella sample serum use respectively plate agglutination test (PAT) and test paper to detect, and the result is negative, positive coincidence rate is 100%.
Kit is comprised of following structure:
(1) test paper film:
1. sample pad: closely being connected in the gold mark pad of test paper, is the part that test paper detects.Main contact sample liquid utilizes the absorption of sample pad that liquid is advanced, but liquid level can not surpass the MAX line of sample pad.
2. glass fibre membrane: being positioned at the top of sample pad, is the protecpectic material of gold mark.The glass fibre absorption of Brucella antibody test paper has gold mark probe, i.e. so-called gold mark pad.Gold mark probe is to be formed by colloid gold label recombination staphylococcus staphylococcus A albumen, can specific absorption Brucella antibody.
3. nitrocellulose membrane (NC): be positioned at the top of glass fibre membrane, detection line (T) and nature controlling line (C) are arranged on the film.The T line of brucella antibody colloidal gold test paper is coated with the special LPS of brucella, and the C line is coated with goat-anti ox IgG.
4. thieving paper: be positioned at the topmost of test paper, be close to the NC film.The power of test paper adsorption sample liquid is mainly by thieving paper and sample pad.
5. plastic base plate: mainly play the support test paper, be hard plastic board.
(2) positive and negative reagent:
1. negative agents: when kit uses, as negative control.
2. positive reagent: when kit uses, as positive control.
(3) plastic suction pipe: the collection that is mainly used in the brucellosis sample.
(4) centrifuge tube: centrifugal use during mainly for separating of sample.
(5) instructions: the using method of article kit.
Below be the using method of antibody test paper film:
When [principle] arrives gold mark pad when sample, carry out specific bond with the SPA of colloid gold label, because the capillarity sample will continue to move forward along this film, during to the detection line that is fixed with antigen (T) zone, antigen, antibody generation specific binding, then sample continues the goat-anti ox IgG antibody reaction on mobile and the nature controlling line (C), because the effect of label collaurum makes T, C line district show that redness is positive reaction, thereby realizes specific immunodiagnosis.If negative reaction only has the colour developing of C line, the T line does not develop the color.
[sample process] gathers about blood 5mL, and 37 ℃ leave standstill 30min, the centrifuging and taking supernatant.Through the filter degerming of 0.25 μ m～0.45 μ m and remove the thickness composition, then do the 1:1 dilution with physiological saline, as tested serum.
[using method] got the tested serum of 1mL and added in the 2mL centrifuge tube, and test paper costal fold straight cutting is entered in the liquid, makes liquid be no more than the MAX control line of test paper film lower end, can read the result in 5 minutes.
[result's judgement] positive: the white displays district presents up and down two red lines.
Negative: the white displays district presents up and down a red control line C.
Invalid: as to occur without control line C in the 5min.
The invention has the advantages that: the antibody that can not only effectively detect animal brucella, and the kind (ox, sheep, pig kind) of energy district office pathogen infection, cross reaction does not occur with the positive serums of the close bacteriums such as Yersinia 09, colon bacillus 0157, salmonella and Actinobacillus pleuropneumoniae in a cross reaction in not occuring kind yet.Be 100% with the positive and negative coincidence rate of conventional Serology test.Detect fast, can report the result in 5 minutes.Do not need special instrument and equipment.
Description of drawings
Fig. 1 is colloid gold test paper structure assembling mode chart of the present invention.
Fig. 2 is Test paper film figure of the present invention.B: detect positive findings; C: detect negative findings.
Specific embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood to be to explaination of the present invention but not to any type of restriction of the present invention.
Embodiment 1
1, the extraction and identification of animal brucella LPS
Extracted respectively the LPS of Brucella abortus 544A and Brucella melitensis 16M with hot phenol method and cold phenol method, behind Sephadex G-50 chromatographic column purifying, purity, the immunological characteristic of the LPS that purifies have been identified.The result shows that the content of LPS is 1.21mg/mL～2.31mg/mL, and purity is 96.1%～97.2%, only reacts with corresponding antibody, and cross reaction does not occur between the LPS of different strain.
2, the preparation of collaurum and the selection of suitable particle diameter
Utilize sodium citrate reducing process and tannic acid-sodium citrate reducing process to prepare 20nm, 25nm, 30nm, 40nm colloid gold particle, observe its dispersion degree and uniformity coefficient result under the Electronic Speculum, then select the suitableeest collaurum 20nm, 25nm particle diameter to carry out mark.
Embodiment 2
1, animal brucella gold mark probe optimum mark pH's determines
Utilize collaurum gradient method and O value curve method, the optimal pH of determining colloid gold label SPA is 5.2～6.8.
2, animal brucella gold mark probe optimum mark amount determines
Utilize collaurum gradient method and O value curve method, determine that colloid gold label SPA optimum mark consumption is 20～60 μ g/mL.
3, the preparation and purification of gold mark probe
Get two test tubes, add respectively 5mL20nm, 25nm collaurum; Add 30 μ, 1～50 μ l0.2mol/L K
2CO
3, the pH value is adjusted into 5.2～6.8; The total amount that goes out needed protein to be marked according to the calculation of total in order to the collaurum of mark is 60 μ g/ml～90 μ g/ml, and SPA is added in the colloidal gold solution and should dropwise add, and the about 5min of whole process adds.Shake up 15min, room temperature is placed 10～15min; Add respectively 10%BSA to final concentration be 1～5%, prevent antibody protein and collaurum polymerization and precipitation.Adopt differential centrifugation and gel filtration that the good probe of mark is carried out purifying, observe quality and the dispersion degree of gold mark probe under the Electronic Speculum.
4, gold mark probe stability test
Affect stable factor and mainly contain electrolyte, collosol concentration, temperature, nonelectrolyte etc.Select the factors such as different stabilizing agents such as BSA, skimmed milk power, PEG20000 and pH, temperature, appropriateness, draw the top condition that keeps collaurum stability.It is BSA that the result selects the best stabilizer, and temperature is 37 ℃, and pH is 5.2～6.8.
5, the selection of the best spray of envelope antigen film concentration
T line on the NC film and C line are arranged several antigen concentration gradients to be selected optimum one group and marks concentration (T line) and coated concentration (C line) as gold.Coated condition is optimized, such as damping fluid, T and C line width, spray film speed.Damping fluid is selected Tris alkali as a result, and spray film concentration is 0.8～1.0mg/ml, and spray film speed is 0.75 μ l/cm.
6, the assembling of test paper film
Referring to shown in Figure 1, put on one's gloves, the nitrocellulose membrane (NC) of coated LPS is assembled on the plastic base plate, require its lower limb must align density bullet line on the mould and careful floating face.The thieving paper that 3.5cm is wide is assembled on the plastic base plate near the mould coboundary, and carefully floating.The gold mark glass fibre membrane of 5mm * 31cm is assembled on the plastic base plate near the scale lower limb, and carefully floating.The sample pad that 1.9cm is wide is assembled on the adhesive base near the mould lower limb, and carefully floating.Be cut into the wide test paper of 4.0mm with cutting cutter, in the assembly section test paper that cuts merged 0.5g drying agent one bag and put into packaging bag.
Test case
Fig. 2 is Test paper film figure of the present invention, through to 18 parts of brucella sample serum, detects with test paper of the present invention respectively, and the result is negative, positive coincidence rate is 100%.B: detect positive findings; C: detect negative findings.
Claims (2)
1. animal brucella antibody colloidal gold test paper film detection reagent kit, it is characterized in that: the staphylococcal protein A (SPA) with restructuring is marked albumen as gold, with highly purified brucella LPS as envelope antigen, utilize goat-anti ox IgG to prepare colloidal gold test paper film for nature controlling line, the colloidal gold test paper film detection line is respectively highly purified ox kind and brucella melitensis LPS, and LPS has species specificity.
2. the method for making of the described kit of claim 1, may further comprise the steps: the LPS that extracts Brucella abortus 544A and Brucella melitensis 16M with hot phenol method and cold phenol method, behind Sephadex G-50 chromatographic column purifying, the content of LPS is 1.21mg/mL～2.31mg/mL, purity is 96.1～97.2%, utilize the LPS of purifying as the detection line envelope antigen, mark albumen (T line) with recombination staphylococcus aureus A albumen (SPA) as gold, goat-anti ox IgG antibody is Quality Control antibody (C line), dresses up the test paper film according to the colloidal gold immunochromatographimethod technology groups.
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| CN 200710055740 CN101055273B (en) | 2007-06-08 | 2007-06-08 | Animal brucella antibody colloidal gold test paper film detection reagent kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101441215B (en) * | 2008-12-31 | 2013-06-26 | 吉林大学 | Brucella antibody immune colloidal gold detection test paper strip and preparing method thereof |
| CN101995465A (en) * | 2009-08-21 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof |
| CN101871939B (en) * | 2010-07-09 | 2014-09-10 | 北京易斯威特生物医学科技有限公司 | Test kit and application method thereof |
| CN103123358A (en) * | 2012-07-23 | 2013-05-29 | 石河子大学 | Human and animal brucella antibody immunochromatography test paper and preparation method thereof |
| CN106018800B (en) * | 2016-05-25 | 2018-07-24 | 南方医科大学 | A kind of detection device of Infected with Brucella |
| CN106771208A (en) * | 2016-11-10 | 2017-05-31 | 中国兽医药品监察所 | Brucella antibody test strip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632585A (en) * | 2004-04-02 | 2005-06-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof |
| WO2006068647A1 (en) * | 2004-12-20 | 2006-06-29 | United States Army Medical Research And Materiel Command | Internal positive control for probe-based nucleic acid molecule assays and methods of making and using thereof |
| CN1834652A (en) * | 2006-04-18 | 2006-09-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting Brucella and its prepn. method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1632585A (en) * | 2004-04-02 | 2005-06-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof |
| WO2006068647A1 (en) * | 2004-12-20 | 2006-06-29 | United States Army Medical Research And Materiel Command | Internal positive control for probe-based nucleic acid molecule assays and methods of making and using thereof |
| CN1834652A (en) * | 2006-04-18 | 2006-09-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting Brucella and its prepn. method |
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