CN1834650A - Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof - Google Patents
Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof Download PDFInfo
- Publication number
- CN1834650A CN1834650A CN 200610072299 CN200610072299A CN1834650A CN 1834650 A CN1834650 A CN 1834650A CN 200610072299 CN200610072299 CN 200610072299 CN 200610072299 A CN200610072299 A CN 200610072299A CN 1834650 A CN1834650 A CN 1834650A
- Authority
- CN
- China
- Prior art keywords
- glanders
- antibody
- bulkholderia cepasea
- test paper
- chromatography test
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention discloses immune chromatography test paper and its preparation method that used to detect glanders bokehuoerde germ. The paper includes sample mat 1, golden mark mat that nearly connected to one end of the mat 1 and contains glanders bokehuoerde germ antibody mark colloidal gold probe 2, nitrate cellulose film 3 that connected to another end of the gold mat and water absorbing mat 4 connected to another end of the NC film, the NC film is coated by separated detection line 5 and quality control line 6, the detecting line is glanders bokehuoerde germ antibody, the quality control line is rabbit antibody. This invention is used to the antibody detecting of clinical specimen, pollution and environmental glanders bokehuoerde germ, also can be used to identification of pure culture glanders bokehuoerde germ.
Description
Technical field
The present invention relates to a kind of test paper that detects the glanders bulkholderia cepasea, particularly a kind of immune chromatography test paper that detects the glanders bulkholderia cepasea also relates to its preparation method.
Background technology
Glanders is a kind of lethal and the propagated all very strong bacteriosis that is caused by glanders bulkholderia cepasea (Burkholderia mallie).The glanders bacterium is called after glanders Fei Shi bacillus (Pfeifferellamallei once, 1918), glanders malignant disease bacillus (Malleonydes mallei, 1933), actinomyces farcinicus (Adtinoei mallei, 1933), glanders Lv Shi bacillus (Loefferella mallei, 1935), glanders acinetobacter calcoaceticus (Acinetobacter mallei, 1964) and pseudomonas mallei (Pseudomonas, mallei1966), called after glanders bulkholderia cepasea in 1993.
Because the glanders bacterium obtains easily, pathogenic strong, the fatal rate height, as not treatment in time, case fatality rate is up to 100%.Be acknowledged as classical biological warfare agent, be put into " international Biological Weapons Convention " and verify inventory.During the World War I, German spy is premeditated in the battlefield, east to have infected large quantities of Muscovite horses and ox with the glanders bacterium, and making after the war, the morbidity number of Russian's glanders increases; During the World War II, the biological weapons that the Japanese may use in the research of the Harbin of China deliberate horse, the common people and the prisoner of war are infectd glanders; The U.S. and USSR (Union of Soviet Socialist Republics) are studied the glanders bacterium always as potential biological weapons after World War II.After " 9.11 ", American National anti-terrorism prediction scheme is classified the glanders bacterium as may be used for the strong pathogenic microorganism that bio-terrorism attacks.Therefore, the fast detecting of glanders bacterium is important content (the Bossi P of anti-bio-terrorism, Guihot A, Bricaire F.Emerging or re-emerging infections that can be used for bioterrorismPresse Med.2005 Jan 29; 34 (2Pt 2): 149-155).
The classical way that the glanders bacterium detects: the complicated clinical manifestation mutability of glanders, be difficult for diagnosis, must make a decision by breadboard test findings, comprise bacteriology checking and serological test.
Clinical collection patient nasal secretion, phlegm, skin ulcer secretion or abscess puncture thing, the direct smear microscopy is inoculated 4% glycerin bouillon agar simultaneously, according to separation and Culture, biochemical reaction, the aggegation of slide serum and the check of zoogenetic infection test routine.
The glanders bacterium is the gram-Negative bacillus of the blunt circle in two ends, and 3～5 μ m are long, and 0.5～1 μ m is wide, is dispersed in distribution, atrichia, no gemma, no pod membrane.The glanders bacterium is an aerobic bacteria, well-grown on 4% glycerin agar.Glanders bacterium and glander-like disease bacterium and Pseudomonas aeruginosa can be differentiated according to biochemical reaction and dynamic test.
Polymerase chain reaction (PCR) is a most frequently used technology in the glanders bacterium method for quick, at first according to glanders bacterium 23S rRNA gene design primer: CVMP231:5 ' AAA CCG ACA CAG GTG G 3 '; M232:5 ' CAC CGA AAC TAG CA 3 ', evaluation (the Adolf Bauernfeind that is used for the glanders bacterium, CarstenRoller Molecular procedure for rapid detection of Burkholderia malleiand Burkholderia pseudomallei.J.Clin.Microbiol, 1998,36:2737-2741); Use glanders bacterium 23S rRNA gene design primer, also can obtain clear and definite glanders dientification of bacteria result (Gee JE, Sacchi CT, Glass MB, De BK.Use of 16S rRNA gene sequencing for rapididentification and differentiation of Burkholderia pseudomallei and B.mallei.J Clin Microbiol.2003,41 (10): 4647-4654); In recent years real-time quantitative PCR (real-time PCR) technology is used for the detection of clinical samples glanders bacterium, can detect 1～10cfu/ml glanders bacterium (Tomaso H, Scholz HC, Al Dahouk S, Eickhoff M Development of a5 '-nuclease real-time PCR assay targeting fliP for the rapididentification of Burkholderia mallei in clinical samples.Clin Chem.2006,52 (2): 307-310; Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ.Developmentand evaluation of a real-time PCR assay targeting the type III secretionsystem of Burkholderia pseudomallei J Clin Microbiol.200644 (1): 85-90).
Immune colloidal gold technique is the Fast Detection Technique that develops rapidly in recent years, this technology and other method are relatively, advantage is: sample disposal is simple in the testing process, do not need specialized equipment and staff training, non-specialized-technical personnel can operate to specifications, and observations rapidly, be well suited for the on-the-spot and basic unit's use of accident.
Still do not have at present and see the report that immune chromatography test paper detects the glanders bulkholderia cepasea.
Summary of the invention
The purpose of this invention is to provide a kind of immune chromatography test paper that detects the glanders bulkholderia cepasea (Immuno-Chromatographic Assay, ICA).
The immune chromatography test paper of detection glanders bulkholderia cepasea provided by the present invention, comprise sample pad, closely be connected in the gold mark pad that contains glanders bulkholderia cepasea specific antibody mark colloidal gold probe of described sample pad, with described gold mark pad close-connected nitrocellulose membrane (NC film) with closely be connected in the adsorptive pads of the described nitrocellulose membrane other end; Described nitrocellulose membrane is coated with a detection line and nature controlling line that is separated from each other, and described detection line is a glanders bulkholderia cepasea specific antibody, described nature controlling line be can with the antiantibody of described glanders bulkholderia cepasea antibody specific bond.
Described glanders bulkholderia cepasea specific antibody is preferably rabbit antibody, and described nature controlling line is preferably anti-rabbit antiantibody.
Can directly be dipped in the sample pad that detects the immune chromatography test paper of glanders bulkholderia cepasea in the sample during test sample; Convenient in order to use, the following backboard that also closely is connected with of described adsorptive pads, the material of backboard can be diversified, as plastics, polyvinyl chloride panel (PVC plate) etc.The test paper that again this is had a backboard is packed in the kit, and this kit is provided with the point sample mouth corresponding to the position of sample pad, is provided with observation window corresponding to the position of detection line and nature controlling line.
Second purpose of the present invention provides a kind of method for preparing the immune chromatography test paper of above-mentioned detection glanders bulkholderia cepasea.
The method of the immune chromatography test paper of the above-mentioned detection glanders of preparation provided by the present invention bulkholderia cepasea may further comprise the steps:
1) preparation glanders bulkholderia cepasea antibody is sprayed onto glanders bulkholderia cepasea antibody-solutions on the tunica fibrosa, and bag is obtained detection line by a zone of NC film; The antiantibody solution of anti-rabbit igg is sprayed onto on the tunica fibrosa, and bag is obtained nature controlling line by another zone of NC film; 37 ℃ dry 2.5-5.5 hour, then it is sticked on the suction paper washer on.
2) preparation contains glanders bulkholderia cepasea antibody labeling immune colloid gold probe solution; Get 5ml, add 0.5-0.65g sucrose and fully dissolve, glass fibre membrane or polyester film are immersed this immune colloid gold probe solution, obtain gold mark pad, place after 10-11 hour for-20 ℃～-50 ℃, freeze dryer is drained, and it is sticked on an end of the close described detection line of the tunica fibrosa that step 1) obtains.
3) will be in step 2) in paste sample pad again above the gold mark pad that obtains, obtain detecting the immune chromatography test paper of glanders bulkholderia cepasea.
Convenient in order to use, the following also adhesive back of described adsorptive pads.
Glanders bulkholderia cepasea antibody labeling colloidal gold probe of the present invention can be prepared by following method:
1) with HAuCl
4Be mixed with 0.01% aqueous solution, get 100ml and be heated to and boil, stir the 1% trisodium citrate (Na that accurately adds 1.6ml down
3C
6H
5O
72H
2O) aqueous solution is stablized into the grape wine redness up to liquid color, obtains colloidal gold solution, and cooling back water returns to original volume;
2) use K
2CO
3Or the HCl adjust pH is 9.2-9.5, adds glanders bulkholderia cepasea antibody by 30 μ g/ml.Stirred 20-25 minute, and added 10%BSA 5ml then, stirred 20-25 minute, add 1ml 10%PEG20000, stirred 20-25 minute, the centrifugal 10-15 of 2800-2900rpm minute, the sucking-off supernatant the centrifugal 25-35 of 10000-12000rpm minute, was abandoned supernatant; Precipitation is once preserved the liquid collection with sodium tetraborate in the back with the sodium tetraborate washing, obtains collaurum mark probe.
The K of described adjusting pH value
2CO
3Concentration can be 0.15-0.25M, be preferably 0.2M; The concentration of the HCl of described adjusting pH value can be 0.05-0.2M, is preferably 0.1M.
The ultimate principle that immune colloidal gold technique detects glanders bacterium antigen is: use glanders bacterium specific antibody bag by cellulose nitrate (NC) film, in order to catch glanders bacterium or the glanders bacterium antigen in the sample, the immune colloid gold probe in detecting of specific antibody of having used mark then.Glanders bacterium in the sample or glanders bacterium antigen macroscopic precipitation line promptly occurs after analysing through about 5 minutes ply of paper.
Glanders bulkholderia cepasea polysaccharide antigen is the specific antigen of glanders bacterium.Therefore, contain the whole cell antigen-immunized animal that enriches polysaccharide antigen, can obtain glanders bulkholderia cepasea specific antibody with the preparation of glanders bulkholderia cepasea toxic strain.Immune chromatography test paper of the present invention adopts double antibody sandwich method, to resist glanders bulkholderia cepasea specific antibody to be coated on the nitrocellulose filter, be used for catching the glanders bulkholderia cepasea antigen of sample, the immune colloid gold probe with the specific antibody mark detects then.
Glanders bacterium antigen quick detection reagent (colloidal gold method) the laboratory result of appraisal of the present invention's development show, can detect glanders bacterium 10 in the sample
6Cfu/ml, and do not intersect with Related Bacteria.
Advantage of the present invention is that sample disposal is simple in the testing process, does not need specialized equipment and staff training, and non-specialized-technical personnel can operate to specifications, and observations rapidly, is well suited for the on-the-spot and basic unit's use of accident.
Description of drawings
Fig. 1 is the structural representation of glanders bulkholderia cepasea immune chromatography test paper.Immune chromatography test paper is made up of adsorptive pads 4, nitrocellulose membrane 3, gold mark pad 2 and glass fibre membrane sample pad 1 four parts; Be coated with detection line 5 and nature controlling line 6 on the nitrocellulose membrane 3.
Embodiment
Main material: gold chloride (HAuCl
4) (available from Sigma company, 1g/ bottle packing); Nitrocellulose membrane (NC film), sample pad and absorbent filter (available from Millipore company).
Used bacterial classification provides by Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences's strain library among the embodiment: glanders bulkholderia cepasea (preserving number: 350018), draw and supervise institute in department of agriculture and forestry; Glander-like disease 4 strains (Vietnam's strain, Guangdong strain, Guangxi strain, each 1 strain of Hainan strain), Pseudomonas aeruginosa 3 strains.
Experimental technique among the following embodiment if no special instructions, is conventional method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The preparation of the immune chromatography test paper of embodiment 1, detection glanders bulkholderia cepasea
1, the preparation of glanders bulkholderia cepasea specific antibody
1) preparation of glanders bulkholderia cepasea antigen
Glanders bacterium (preserving number 350018) inoculation contains on the 4% glycerine plain agar flat board, and 37 ℃ of incubators are cultivated, and observes the dull and stereotyped colonial morphology of going up, the typical single bacterium colony of picking, and transferred species 4% glycerine plain agar inclined-plane was cultivated 24-28 hour in 37 ℃ of incubators.Wash the thickness lawn in saline bottle with 5% formaldehyde salt solution, place 37 ℃ and spend the night.Sterility test was observed 7-8 days, prove no viable bacteria after, with bacterium liquid 10,000g-12, the centrifugal 10-15 of 000g minute, collecting precipitation was mixed with the turbid concentration of 2 multiple proportions (about 10 with stroke-physiological saline solution
9Cfu bacterium/ml), be glanders bulkholderia cepasea antigen.
2) preparation of glanders bulkholderia cepasea antibody
Select body weight 2kg Healthy female white big ear rabbit (available from Academy of Military Medicine, PLA's animal center) for use, subcutaneous multi-point injection immunity Fu Shi Freund's complete adjuvant glanders bacterium somatic antigen 2 * 10
8Cfu bacterium/1ml/ only, respectively at the 20th day behind the initial immunity, the 30th day, the 40th supplementary immunization aqua 1 pin, boost and approach be with identical for the first time, last immunity examination in back 10 days blood, slide agglutination test detects serum titer and reaches 1: 1280 above blood sampling.
Adopt conventional saturated ammonium sulfate salting out method that the glanders bulkholderia cepasea antibody in the blood is carried out purifying, identify that through the agar double diffusion test antibody that obtains has specificity and compatibility preferably.
2, preparation immune colloid gold probe
1) preparation colloidal gold solution: adopt the citrate reducing process to prepare colloid gold particle, concrete grammar is: with HAuCl
4Be mixed with 0.01% aqueous solution, get 100mL and be heated to boiling, stir the 1% trisodium citrate (Na that accurately adds 1.6mL down
3C
6H
5O
72H
2O) aqueous solution, liquid color are stablized into the grape wine redness, promptly obtain colloidal gold solution.
2) determine collaurum coupling antibody saturation concentration: use 0.2M K
2CO
3Regulate colloidal gold solution pH9.2, prepare 5 clean tube, add the 1ml colloidal gold solution respectively.With purified glanders bulkholderia cepasea antibody dilution is 1mg/ml, in 4 test tubes, add 20 μ l, 25 μ l, 30 μ l, 35 μ l respectively, another is contrast, under room temperature, placed 5 minutes behind the mixing, add the 10%NaCl aqueous solution, mixing leaves standstill after 10-20 minute and observes liquid color.Contained minimum antibody amount was the optimum concentration of stablizing the required antibody of 1ml collaurum when the colloidal gold solution color was constant, increased by 20% antibody amount based on this, was collaurum coupling antibody saturation concentration.The result shows: keeping the constant antibody amount of colloidal gold solution color is 25 μ l, i.e. 25 μ g/ml, and selecting the coupling antibody concentration is 30 μ g/ml.
3) preparation of gold mark pad 2: preparation contains the immune colloid gold probe solution 50ml that concentration is the glanders bulkholderia cepasea antibody of 30 μ g/mL as stated above.Stirred 20-25 minute, and added 10%BSA 5ml then, stirred 20-25 minute, add 1ml 10%PEG20000, stirred 20-25 minute, the centrifugal 10-15 of 2800-2900rpm minute, the sucking-off supernatant the centrifugal 25-35 of 10000-12000rpm minute, was abandoned supernatant; Precipitation is once preserved liquid collecting precipitation 5ml with sodium tetraborate in the back with the sodium tetraborate washing.Get gold mark probe 5ml and add 0.5-0.65g sucrose, fully dissolving evenly is added on the glass fibre membrane, places 10-11 hour hour for-20 ℃～-50 ℃, and freeze dryer is drained, and obtains gold mark pad 2.
3, detect the preparation of the immune chromatography test paper of glanders bulkholderia cepasea
As shown in Figure 1, the immune chromatography test paper that detects the glanders bulkholderia cepasea is made up of adsorptive pads 4, nitrocellulose membrane 3, gold mark pad 2 and glass fibre membrane sample pad 1 four parts; Be coated with detection line 5 and nature controlling line 6 on the nitrocellulose membrane 3.The preparation method may further comprise the steps:
1) the bag quilt of NC film 3
The bag quilt of glanders bulkholderia cepasea antibody and goat anti-rabbit igg: with 0.01M pH7.2PB dilution glanders bulkholderia cepasea antibody to final concentration is 2mg/ml, is used to wrap tested survey line.PBS dilution goat anti-rabbit igg to final concentration with 0.01M pH 7.2 is 4mg/ml, is used for bag by nature controlling line.Be sprayed on respectively that 300mm is long, on the wide nitrocellulose filter of 25mm, form detection line 5 and 6,37 ℃ of dry 2.5-5.5h of nature controlling line of being separated from each other with the XYZ3000 of BIODOT company Membrane jetter.
2) preparation of the immune chromatography test paper of detection glanders bulkholderia cepasea
Suction paper washer 4 usefulness double faced adhesive tapes are pasted on an above-mentioned end that is coated with the NC film 3 of glanders bulkholderia cepasea antibody and goat anti-rabbit igg near nature controlling line; The gold mark pad 2 usefulness double faced adhesive tapes that will prepare in step 2 are pasted on the end of NC film 3 near detection line; On gold mark pad 2, use double faced adhesive tape sticking glass tunica fibrosa sample pad 1 again; At last they are sticked on the plastic back plate with double faced adhesive tape,, are the immune chromatography test paper that detects the glanders bulkholderia cepasea by required size cutting, add drying agent after sealing preserve.
4, detect the preparation of the immune chromatography reagent kit of glanders bulkholderia cepasea
Use for convenience, with the following plastic back plate that closely connects again of the immune chromatography test paper of the detection glanders bulkholderia cepasea of step 3 preparation, the test paper that again this is had a backboard is packed in the kit, add drying agent after sealing preserve.This kit is provided with the point sample mouth corresponding to the position of sample pad, is provided with observation window corresponding to the position that contrasts band and calibration tape.
5, detect the using method and the principle of the immune chromatography test paper of glanders bulkholderia cepasea
During mensuration test strips sample pad 1 is immersed in the liquid sample, sample pad 1 is that imbitition moves to the upper end, and the gold mark pad of flowing through made the immune Au composite on the dry plate redissolve at 2 o'clock, and drives it and ooze to nitrocellulose membrane 3 and move.If specific antigen to be measured is arranged in the sample, its can with the antibodies of immune Au composite, this antigen antibody complex flow to detection line 5 and is promptly obtained by insolubilized antibody, shows red reaction lines on film.Superfluous immune Au composite continues to move ahead, and closes to nature controlling line 6 and solid phase two resistive connections, and shows red Quality Control lines.Otherwise, the then reactionless lines of negative sample, and only show the Quality Control lines.
The detection of embodiment 2, glanders bacterium reaches the cross matching with other Related Bacteria
1, the detection of glanders bacterium
1) by Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre provide, 350018 strains of glanders bacterium, 350019 strains of glanders bacterium, concentration is 1 * 10
6Cfu/ml, bacteria suspension is standby as sample detection liquid.
2) bag through embodiment 1 preparation is positive findings by the detection of the immune chromatography test paper of glanders bacterium specific antibody and goat anti-rabbit igg.
2, the cross matching of other Related Bacteria
1) by Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre provide, 350102 strains of glander-like disease bacterium, Pseudomonas aeruginosa 430023 strains, concentration is 1 * 10
8Cfu/ml, bacteria suspension is standby as sample detection liquid.
2) bag through embodiment 1 preparation is negative findings by the detection of the immune chromatography test paper of glanders bacterium specific antibody and goat anti-rabbit igg.
Embodiment 3, laboratory examination
1, specimen preparation
Different bacterium is inoculated suitable culture base separately respectively, and under different conditions, cultivate, wash with stroke-physiological saline solution after growing lawn, turbidimetry preparation glanders bacterium bacteria suspension 1 * 10
6Cfu/ml, 1 * 10
7Cfu/ml and 1 * 10
8Cfu/ml, other bacterium bacteria suspension 1 * 10
8Cfu/ml, liquid is standby as detecting.
2, experimental technique
Get glanders bacterium antigen quick detection reagent, respectively at adding 3 of above-mentioned sample detection liquid (about 150 μ l) on the sample pad, begin observations after 2 minutes, observation in 15 minutes stops.Result's report: it is negative that 1 precipitation line appears in the nature controlling line place, promptly do not have glanders bacterium antigen and detect; It is positive that 2 precipitation lines appear in detection line and nature controlling line place, promptly has glanders bacterium antigen to detect.
3, experimental result
Glanders bacterium antigen quick detection reagent (colloidal gold method) the laboratory result of appraisal see Table 1.
The examination of table 1 glanders bacterium antigen quick detection reagent (colloidal gold method) laboratory
| Bacteria name | Strain number | Bacterium amount (cfu/ml) | ||
| 10 6 | 10 7 | 10 8 | ||
| The glanders bacterium | 350011 | + | + | + |
| 350017 | + | + | + | |
| 350018 | + | + | + | |
| 350019 | + | + | + | |
| The glander-like disease bacterium | 350101 (Vietnam's strains) | - | ||
| 350103 (Hainan strains) | - | |||
| 350129 (Guangdong strains) | - | |||
| 350139 (Guangxi strains) | - | |||
| Pseudomonas aeruginosa | 430022 | - | ||
| 430023 | - | |||
| 430024 | - | |||
As can be seen from Table 1, glanders bacterium antigen quick detection reagent (colloidal gold method) can detect 1 * 10
6Cfu/ml glanders bacterium is homophyletic not, not with 1 * 10
8Cfu/ml glander-like disease burkholderia different regions separated strain and Pseudomonas aeruginosa generation cross reaction.
Claims (6)
1, a kind of immune chromatography test paper that detects the glanders bulkholderia cepasea, comprise sample pad (1), closely be connected in the gold mark pad (2) that contains glanders bulkholderia cepasea antibody labeling colloidal gold probe of described sample pad one end, with the close-connected nitrocellulose membrane of the other end (3) of described gold mark pad with closely be connected in the adsorptive pads (4) of the described nitrocellulose membrane other end; Described nitrocellulose membrane is coated with detection line (5) and the nature controlling line (6) that is separated from each other, and described detection line is a glanders bulkholderia cepasea antibody, described nature controlling line be can with the antiantibody of described glanders bulkholderia cepasea antibody specific bond.
2, the immune chromatography test paper of detection glanders bulkholderia cepasea according to claim 1 is characterized in that: nature controlling line is preferably anti-rabbit antiantibody.
3, the immune chromatography test paper of detection glanders bulkholderia cepasea according to claim 1 is characterized in that: described sample pad, adsorptive pads, gold mark pad are made by absorbent material; The following backboard that also closely is connected with of described adsorptive pads.
4, a kind of method for preparing the immune chromatography test paper that detects the glanders bulkholderia cepasea may further comprise the steps:
1) preparation glanders bulkholderia cepasea antibody is sprayed onto glanders bulkholderia cepasea antibody-solutions on the tunica fibrosa, and bag is obtained detection line by a zone of NC film; Anti-rabbit antiantibody solution is sprayed onto on the tunica fibrosa, and bag is obtained nature controlling line by another zone of NC film; 37 ℃ dry 2.5-5.5 hour, then it is sticked on the suction paper washer on.
2) preparation collaurum mark probe adopts the citrate reducing process to prepare colloid gold particle, and concrete grammar is: with HAuCl
4Be mixed with 0.01% aqueous solution, get 100mL and be heated to boiling, stir the 1% trisodium citrate (Na that accurately adds 1.6mL down
3C
6H
5O
72H
2O) aqueous solution, liquid color are stablized into the grape wine redness, promptly obtain colloidal gold solution.Use 0.2M K
2CO
3Regulate colloidal gold solution pH9.2-9.5, add glanders bulkholderia cepasea antibody by 30 μ g/ml, stirred 20-25 minute, and added 10%BSA 5ml then, stirred 20-25 minute, add 1ml 10%PEG20000, stirred 20-25 minute, the centrifugal 10-15 of 2800-2900rpm minute, the sucking-off supernatant, the centrifugal 25-35 of 10000-12000rpm minute, abandon supernatant; Precipitation is once preserved liquid collecting precipitation 5ml with sodium tetraborate in the back with the sodium tetraborate washing.Described percentage composition is the quality percentage composition.
3) preparation contains glanders bacteria antibody labelled immune colloidal gold probe solution; Get 5ml, add 0.5-0.65g sucrose and fully dissolve, glass fibre membrane or polyester film are immersed this immune colloid gold probe solution, obtain gold mark pad, place after 10-11 hour for-20 ℃～-50 ℃, freeze dryer is drained, and it is sticked on an end of the close described detection line of the tunica fibrosa that step 1) obtains.
4) will be in step 2) in paste sample pad again above the gold mark pad that obtains, obtain detecting the immune chromatography test paper of glanders bulkholderia cepasea.
5, method according to claim 4 is characterized in that: the concentration of described glanders bulkholderia cepasea antibody is 2mg/ml; Anti-rabbit igg concentration is 4mg/ml; The following backboard that also is pasted with of described adsorptive pads; The K of described adjusting pH value
2CO
3Concentration be 0.2M.。
6, the kit that contains the immune chromatography test paper of arbitrary described detection glanders bulkholderia cepasea among the claim 1-3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610072299 CN1834650A (en) | 2006-04-18 | 2006-04-18 | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610072299 CN1834650A (en) | 2006-04-18 | 2006-04-18 | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1834650A true CN1834650A (en) | 2006-09-20 |
Family
ID=37002475
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610072299 Pending CN1834650A (en) | 2006-04-18 | 2006-04-18 | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1834650A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363866B (en) * | 2008-05-26 | 2012-09-19 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications |
| CN113046452A (en) * | 2021-03-23 | 2021-06-29 | 中国人民解放军陆军军医大学 | Composition for detecting Boeck hollandia farci and application thereof |
| CN113755619A (en) * | 2021-10-19 | 2021-12-07 | 中国医学科学院北京协和医院 | Digital PCR detection kit for Burkholderia |
-
2006
- 2006-04-18 CN CN 200610072299 patent/CN1834650A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363866B (en) * | 2008-05-26 | 2012-09-19 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications |
| CN113046452A (en) * | 2021-03-23 | 2021-06-29 | 中国人民解放军陆军军医大学 | Composition for detecting Boeck hollandia farci and application thereof |
| CN113046452B (en) * | 2021-03-23 | 2022-02-25 | 中国人民解放军陆军军医大学 | Composition for detecting Boeck hollandia farci and application thereof |
| CN113755619A (en) * | 2021-10-19 | 2021-12-07 | 中国医学科学院北京协和医院 | Digital PCR detection kit for Burkholderia |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2020233148A1 (en) | Inert carrier salmonella sp. and potential application thereof | |
| CN1214816C (en) | Process and materials for the repid detection of i(streptococcus pneumoniae) employing purified antigen-specific antibodies | |
| CN110308274A (en) | A kind of kit and preparation method thereof detecting listeria monocytogenes | |
| WO2020233147A1 (en) | Inert carrier escherichia coli and potential use thereof | |
| CN101074956A (en) | Immune chromatography test paper for inspecting legionella pneumophilia antibody and its production | |
| CN1834652A (en) | Immunity chromatography test paper for detecting Brucella and its prepn. method | |
| CN105717310A (en) | Immuno-fluorescent staining method for detecting mycobacterium tuberculosis in leukocytes and kit | |
| CN103499685B (en) | People's urinary system cause of disease bacterium infects the preparation method of the immunofluorescence diagnostic reagent of clinical diagnosis | |
| CN1880961A (en) | Immunochromatographic assay test paper for detecting staphylococcal enterotoxin B and preparation method thereof | |
| CN1834650A (en) | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof | |
| CN1888076A (en) | Fast identification method for ox tubercle bacillus and its drug sensitive test kit | |
| CN105974113A (en) | Sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7 | |
| CN1159580C (en) | Reagent for colloidal gold chromatographic analysis of SARS coronavirus antigen | |
| CN101000347B (en) | Immunological chromatographic test paper for testing francisella tularensis and its preparation method | |
| CN102033128B (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN105891412B (en) | A kind of citrobacter freundii test strip and preparation method thereof | |
| CN101074955A (en) | Immune chromatography test paper for inspecting legionella pneumophilia antibody and its production | |
| CN1279358C (en) | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof | |
| CN1834651A (en) | Immunity chromatography test paper for detecting farcinia Boeck Hold's bacteria infection and prepn. method thereof | |
| CN1265200C (en) | Immune chromatographic test paper for detecting Yersinia pestis infection and preparing process thereof | |
| CN105277694B (en) | Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications | |
| CN101676724A (en) | Immunity-chropadography test paper for detecting Vibrio Cholerae and preparation method thereof | |
| CN105769212A (en) | Condom | |
| CN1632586A (en) | Immune chromatographic test paper for detecting tularemia infection and preparing process thereof | |
| CN1831536A (en) | Immunization chromatograph test paper for detecting plague yersinia and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |