CN101074955A - Immune chromatography test paper for inspecting legionella pneumophilia antibody and its production - Google Patents
Immune chromatography test paper for inspecting legionella pneumophilia antibody and its production Download PDFInfo
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Abstract
An immune chromatographic test paper used for detecting antibody of legionella pneumophilia consists of sample pad, gold label pad containing A label colloidal gold probe of staphylococcus aureus protein, nitric acid fiber membrane and water absorption pad. It is featured as enveloping detection line containing antigen and quality control line containing antibody on nitric acid fiber membrane and setting said detection line to be separated from said quality control line.
Description
Technical field
The present invention relates to detection test paper of a kind of legionella pneumophilia antibody and preparation method thereof, particularly relate to a kind of immune chromatography test paper that detects legionella pneumophilia antibody and preparation method thereof.
Background technology
Legionella (Legionella) is discovery in 1976.US veteran population was gathered in Philadelphia at that time, during outbreak of epidemic a kind of agnogenic serious pneumonia, participant 149 people infect, 34 people's death.From the dead's lung tissue, separate and obtain a kind of new bacterium, called after Legionella.At present, the Legionnella of having found has 43 kinds, 65 serotypes, wherein with the human diseases relation the closest be legionella pneumophilia (Legionella pneumophila, LP), studies show that 80%～85% Legionnaires Pneumonia is caused by LP, has now found that the LP of 15 serotypes.
(Legionnarires ' disease LD) is a kind of acute bacterial respiratory infectious disease that is caused by infection with legionella to legionaires' disease, serves as main performance, easily outbreak of epidemic with Legionnaires Pneumonia and Pontiac fever.In recent years, the report of many legionaires' disease epidemic situations is arranged abroad: 2000～2002 years, only legionaires' disease was just reported 189 times altogether in Europe, and 10322 people infect.Also there is the legionaires' disease generation of epidemic situation on a small scale in China: in January, 2000, legionaires' disease has taken place in BASIC campsite, Beijing, and the incidence of disease is 89%; In July, 2000, the Pontiac fever type legionaires' disease that causes because of the air-conditioning system condensate water takes place in certain office building employee, and the incidence of disease is 61.9%.
Most of legionaires' disease patients have the consistent clinical manifestation of SARS (Severe Acute Respiratory Syndrome) that causes with other pathogen, and as cough, heating and myalgia, chest X ray shows lung's infiltration phenomenon etc., so often cause mistaken diagnosis, fail to pinpoint a disease in diagnosis, untreated legionaires' disease mortality reaches 25%.Because legionella pneumophilia is the main pathogens that causes Legionnaires Pneumonia, so its fast detecting is seemed particularly important.The method of legionella pneumophilia Detection of antigen is a lot, for example: ELISA, PCR, Immunosensors Technology And etc.But in practice, the use of these methods exposes some common inferior positions, and for example: experiment must be carried out in special laboratory; Need power supply and special instrument; Operating personnel must pass through special training; Experimental system is not easy standardization, for making credible result, need carry out repeatedly trial test and set up multinomial contrast; The test operation complex steps needs more than 2 hours at least.
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.
Summary of the invention
The object of the invention provide a kind of immune chromatography test paper that detects legionella pneumophilia antibody (Immuno-Chromatographic Assay, ICA).
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of immune chromatography test paper (ICA test paper) that detects legionella pneumophilia antibody, comprise sample pad, closely be connected in the gold mark pad that contains staphylococcus aureus protein A (SPA) mark colloidal gold probe of described sample pad one end, with the close-connected nitrocellulose membrane of the other end (NC film) of described gold mark pad with closely be connected in the adsorptive pads of the described nitrocellulose membrane other end; Described nitrocellulose membrane is coated with detection line and the nature controlling line that is separated from each other, and described detection line contains legionella pneumophilia antigen, described nature controlling line contain can with the antibody of described staphylococcus aureus protein A specific bond.
Described legionella pneumophilia antigen can be selected from one or more hybrid antigens in legionella pneumophilia serum 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types and the 10 type antigens, is preferably the hybrid antigen of legionella pneumophilia serum 1,4,5 and 6 types; Described staphylococcus aureus protein A specific antibody is for being the antibody that antigen-immunized animal obtains with the staphylococcus aureus protein A, and the selection of described immune animal is diversified, as immune animal commonly used such as sheep, rabbit, monkey, chicken or mouse; The concentration that described detection line contains the legionella pneumophilia antigen of 1,4,5,6 serotypes is 1-3mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antibody of staphylococcus aureus protein A specific bond.
Described adsorptive pads and sample pad prepare by absorbent material.
Can directly be dipped in the sample pad of the immune chromatography test paper of above-mentioned detection legionella pneumophilia antibody in the sample during test sample; Convenient for using, the back side of the immune chromatography test paper of described detection legionella pneumophilia antibody also closely connects a backboard, the selection of back veneer material is diversified, as plastics, resin or polyvinyl chloride panel (PVC plate) etc., the immune chromatography test paper that again this is had a backboard is packed in the kit, 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 legionella pneumophilia antibody.
The method of the immune chromatography test paper of the above-mentioned detection legionella pneumophilia antibody of preparation provided by the present invention may further comprise the steps:
1) preparation legionella pneumophilia antigen, the legionella pneumophilia antigenic solution is sprayed onto forms detection line on the cellulose membrane, another zone that can be sprayed onto described tunica fibrosa with the antibody-solutions of staphylococcus aureus protein A specific bond forms nature controlling line, then adsorptive pads is sticked on the end away from described calibration tape of described cellulose membrane;
2) the immune colloid gold probe solution of preparation staphylococcus aureus protein A mark, glass fibre membrane or polyester film are immersed this immune colloid gold probe solution, obtain gold mark pad, it is sticked on an end of the close described detection line of the cellulose membrane that step 1) obtains;
3) will be in step 2) in the gold mark pad that obtains go up away from an end of described detection line and paste sample pad, obtain detecting the immune chromatography test paper of legionella pneumophilia antibody.
In the preparation method of the immune chromatography test paper of above-mentioned detection legionella pneumophilia antibody, the antigen of legionella pneumophilia described in the step 1) can be selected from one or more hybrid antigens in legionella pneumophilia serum 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types and the 10 type antigens, is preferably the hybrid antigen of legionella pneumophilia serum 1,4,5 and 6 types; Described staphylococcus aureus protein A specific antibody is for being the antibody that antigen-immunized animal obtains with the staphylococcus aureus protein A, and the selection of described immune animal is diversified, as immune animal commonly used such as sheep, rabbit, monkey, chicken or mouse; The concentration that described detection line contains the legionella pneumophilia antigen of 1,4,5,6 serotypes is 1-3mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antibody of staphylococcus aureus protein A specific bond.
Legionella pneumophilia antigen in the described step 1) can prepare as follows: a. is seeded in legionella pneumophilia on the BCYE flat board, at 37 ℃, 5%CO
2Cultivated in the incubator 3-5 days, the typical single bacterium colony that picking grows on flat board, with its transferred species to the BCYE inclined-plane, at 37 ℃, 5%CO
2Cultivated in the incubator 3-5 days, the inclined-plane cultivated is washed lawn with the PBS of aseptic 0.01M, pH 7.2, centrifugal, abandon supernatant, the collection bacterial sediment; B. every gram weight in wet base thalline is suspended among the 10mL 0.1M NaOH stirring at room 1 hour; C. transfer pH value of solution to 3.0 with dense acetic acid, centrifugal, abandon precipitation, supernatant is transferred to pH 6.5-7.0 with 5N NaOH; D. gained solution was dialysed 48 hours with the PBS of 0.01M, pH 7.0, obtained legionella pneumophilia antigen, and freeze-drying is preserved.
Baking temperature in the described step 1) can be 30-45 ℃, is preferably 37 ℃, can be 2.5-3.5 hour drying time.
Step 2) preparation method of the immune colloid gold probe solution of legionella pneumophilia antigen-specific antibodies mark can may further comprise the steps in:
A. 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 continues heated and boiled (being preferably 10-12 minute), stablizes into the grape wine redness up to liquid color, obtains colloidal gold solution, and cooling back water returns to original volume;
B. the pH value of the colloidal gold solution that step 1) is obtained transfers to 8.5-9.5, it is the staphylococcus aureus protein A of 12 μ g/mL that every 100mL colloidal gold solution adds final concentration, stirred 25-35 minute, add 5mL 10%BSA, stirred 20-30 minute, add 1mL 10%PEG20000 again, stirred 20-30 minute, earlier 20,000-23, under the 500rpm centrifugal 25-35 minute, abandon supernatant, sodium tetraborate solution washing with 0.05-0.1M precipitates, and it is stored in the sodium tetraborate solution of 8-10mL 0.05-0.1M, obtains having a liking for the colloidal gold probe solution of staphylococcus aureus protein A mark.
In the preparation method of the immune colloid gold probe solution of above-mentioned staphylococcus aureus protein A mark, available K
2CO
3Solution or HCl solution adjust pH are 8.5-9.5, the K of described adjusting pH value
2CO
3The concentration of solution can be 0.15-0.25M, is preferably 0.2M; The concentration of described adjusting pH value HCl solution can be 0.08-0.12mol/L, is preferably 0.1mol/L.For remedying the moisture because of the heating evaporation loss, available water returns to original volume with colloidal gold solution.
For the immune colloid gold probe that makes the staphylococcus aureus protein A mark combines better with glass fibre membrane or polyester film, can be to step 2) in the immune colloid gold probe solution of staphylococcus aureus protein A mark in add 0.05-0.1g/mL sucrose; Gold mark pad is easier to be pasted with cellulose membrane in order to make, can with gold mark pad-20 ℃ to-50 ℃ freezing 10-12 hour, and, paste with cellulose membrane again after its freezing draining.
Described adsorptive pads and sample pad prepare by absorbent material.
Can directly be dipped in the sample pad of the immune chromatography test paper of above-mentioned detection legionella pneumophilia antigen in the sample during test sample; Convenient for using, the back side of the immune chromatography test paper of described detection legionella pneumophilia antigen also closely connects a backboard, the selection of back veneer material is diversified, as plastics, resin or polyvinyl chloride panel (PVC plate) etc., the immune chromatography test paper that again this is had a backboard is packed in the kit, 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.
In actual applications, described cellulose membrane can be nitrocellulose filter (NC film) or cellulose acetate membrane, and width is controlled at 2.5-3.0mm and is advisable; Described adsorptive pads can be thieving paper, and width is 20-40mm, and thickness is 0.1-0.2mm; The width of described gold mark pad is 5-10mm; Described sample pad is a glass fibre membrane, and width is 20-40mm.
The invention provides a kind of immune chromatography test paper that detects legionella pneumophilia antibody and preparation method thereof.This test paper has utilized immune colloidal gold technique and double-antibody sandwich detection method, with its ultimate principle that detects legionella pneumophilia or legionella pneumophilia antibody be: with the antigen coated cellulose membrane of legionella pneumophilia, in order to catch legionella pneumophilia or the legionella pneumophilia antibody in the sample, then with mark the immune colloid gold probe of staphylococcus aureus protein A detect.Immune chromatography test paper of the present invention has the following advantages: 1) susceptibility and specificity height: the laboratory result of appraisal show that immune chromatography test paper of the present invention can be used for detecting the legionella pneumophilia serum specimen, and with other biology cross reaction do not take place; 2) detection method is simple, quick: 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, after legionella pneumophilia in the sample or legionella pneumophilia antibody are analysed through about 5 minutes ply of paper, macroscopic precipitation line can occur, thereby strive for the time, be well suited for the on-the-spot and basic unit's use of accident for the treatment of legionella pneumophilia disease; 3) preparation method is simple, and is with low cost, is easy to carry out suitability for industrialized production.The present invention will play a significant role in the diagnosis of the detection of legionella pneumophilia or legionella pneumophilia antibody and relevant disease thereof and treatment, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the front and the vertical section structure synoptic diagram of the immune chromatography test paper of detection legionella pneumophilia antibody
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
Material:
Nitrocellulose membrane (NC film), sample pad and absorbent filter are available from Millipore company.
Plastic back plate is available from Beijing Yan Hua company.
The preparation of the immune chromatography test paper of embodiment 1, detection legionella pneumophilia antibody
One, the preparation of legionella pneumophilia antigen
With legionella pneumophilia serum 1 type bacterial strain (available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences's strain library, preserving number 510101) (prescription is the CM655 basal medium to be seeded in the BCYE flat board, the SR110A growth additive, the SR118E selective additives) on, at 37 ℃, 5%CO
2Incubator is cultivated, and observes the dull and stereotyped colonial morphology of going up, the typical single bacterium colony of picking, and transferred species is put the BCYE inclined-plane, at 37 ℃, 5%CO
2Cultivated 3-5 days in the incubator; The inclined-plane of cultivating is washed lawn with aseptic PBS (0.01M pH 7.2), and centrifugal 25 minutes of 8000rpm abandons supernatant, collects bacterial sediment; Every gram weight in wet base thalline is suspended among the 10mL 0.1M NaOH, stirring at room 1 hour; Transfer pH value of solution to 3.0 with dense acetic acid, centrifugal 20 minutes of 8000rpm abandons precipitation, and supernatant is transferred to 6.5-7.0 with 5N NaOH; Gained solution obtains legionella pneumophilia serum 1 type antigen with PBS (0.01M pH 7.0) dialysis 48 hours, and freeze-drying is preserved.
Prepare legionella pneumophilia serum 4,5,6 type antigens (all available from the entire PLA of Military Medical Science Institute Micro biological Tests research centre strain library, preserving number is respectively 510104,510105,510106 to legionella pneumophilia serum 4,5,6 type bacterial strains) with above-mentioned same procedure.
Two, preparation immune colloid gold probe
1, preparation colloidal gold solution
Adopt the citrate reducing process to prepare colloid gold particle, concrete grammar is: HAuCl4 (available from Sigma company, 1g/ bottle packing) is mixed with 0.01% aqueous solution, gets 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.The cooling back returns to original volume with distilled water, makes the colloid gold particle that particle diameter is 25nm.
2, determine collaurum coupling antibody saturation concentration
Use 0.2M K
2CO
3(or 0.1M HCl) regulates colloidal gold solution pH 8.5-9.5, prepares 4 clean tube, adds the 1mL colloidal gold solution respectively.Staphylococcus aureus protein A (purchasing the company in Amersham PharmaciaBiotech) dilution is 1mg/mL, in 3 test tubes, add 5 μ l, 10 μ l, 15 μ 1 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 10 μ l, i.e. 10 μ g/mL, and selecting the coupling antibody concentration is 12 μ g/mL.
3, the preparation of gold mark pad
Preparation contains the staphylococcus aureus protein A immune colloid gold probe solution 50mL that concentration is 12 μ g/mL as stated above, stir 30 minutes (25-35 minute all can), add 1mL 10%PEG20000, stir 25 minutes (20-30 minute all can), 20,000～23, the centrifugal 25-30 of 500rpm minute, abandon supernatant, will precipitate with after the sodium tetraborate washing 1 time and preserve liquid collecting precipitation 5mL with sodium tetraborate.Get gold mark probe 5mL and add 0.5g sucrose, fully evenly be added on the glass fibre membrane after the dissolving ,-35 ℃ (20 ℃～-50 ℃ all can) place 11 hours (10-12 hour all can), and freeze dryer is drained, and obtains gold mark pad.
Three, the preparation of legionella pneumophilia antibody quick detection test paper
As shown in Figure 1, the present invention's immune chromatography test paper of detecting legionella pneumophilia antibody 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 of this test paper may further comprise the steps:
1) the bag quilt of NC film 3
PB damping fluid (NaH with 0.01M, pH7.2
2PO
42H
2O 0.39g, Na
2HPO
41.07g, deionized water 1000mL) dilution legionella pneumophilia serum 1,4,5,6 type antigens, diluted four kinds of antigenic solutions are mixed, and extremely the content of legionella pneumophilia serum 1,4,5,6 type antigens is respectively 2mg, 1mg, 3mg, 2.5mg in every ml soln, is used to wrap tested survey line.With 0.01M pH 7.2 PBS (NaH
2PO
42H
2O 0.39g, Na
2HPO
41.07g, NaCl8.5g, deionized water 1000mL) and (the IgG immune sheep of personnel selection obtains the dilution goat anti-human igg, concrete preparation method sees " immunochemistry progress ", Li Chengwen work, China Science Tech Publishing House, 1993) to concentration be 4mg/mL, be used for the bag by nature controlling line.Be sprayed on respectively on the thick nitrocellulose filter of 2.5mm (available from Millipore company) with the XYZ3000 of BIODOT company Membrane jetter, form the detection line 5 be separated from each other and 6,37 ℃ of dry 3h of nature controlling line (2.5-3.5h all can).
2) preparation of legionella pneumophilia antibody quick detection test paper
Adsorptive pads 4 usefulness double faced adhesive tapes are sticked on an end of the nitrocellulose membrane 3 of bag quilt; The NC film 3 usefulness double faced adhesive tapes of bag quilt are sticked on an end of the gold mark pad 2 of preparation in the step 2; On gold mark pad 2, use double faced adhesive tape sticking glass tunica fibrosa sample pad 1; At last they are sticked on the plastic back plate with double faced adhesive tape again,, are the immune chromatography test paper that detects legionella pneumophilia antibody by required size cutting, add drying agent after sealing preserve.
Four, detect the preparation of the immune chromatography reagent kit of legionella pneumophilia antibody
Use for convenience, with the following plastic back plate that closely connects again of the immune chromatography test paper of the legionella pneumophilia antibody fast detecting 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.
Five, the using method of legionella pneumophilia antibody quick detection test paper and principle
During mensuration test strips sample pad 1 is immersed in the liquid sample, sample pad 1 is that imbitition moves to the upper end, the immune Au composite solution that the gold mark pad of flowing through made on the dry plate in 2 o'clock, and drive it and ooze to nitrocellulose membrane 3 and move.If specific antibody 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 solid phase antigen, shows red reaction lines on film.Superfluous immune Au composite continues to move ahead, and combines with the solid phase goat anti-human igg to nature controlling line 6, 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, legionella pneumophilia antibody reaches the cross matching with other Related Bacteria blood serum sample
One, the detection of different serotypes legionella pneumophilia antibody
Legionella pneumophilia serum 1,2,3,4,5,6,7,9, the 10 type strain antigens immunizing rabbits that provide available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre are provided, and (immunizing dose is 2mg/mL/, immune time is 4-6 time, each immunity 10 days at interval) the antiserum sample that obtains is standby as sample detection liquid after diluting by 1: 40 ratio with physiological saline.
Get the kit that the present invention detects the immune chromatography test paper of legionella pneumophilia antibody that is equipped with of embodiment 1 preparation, respectively at adding 3 of above-mentioned sample detection liquid (about 150ul) in the point sample mouth, begin observations after 2 minutes, observation in 15 minutes stops.
Result's report: only locate to occur 1 red precipitate line explanation legionella pneumophilia serodiagnosis feminine gender, promptly do not have Legionella pneumophila infection in Quality Control observation window " C " (nature controlling line); Locate to occur 2 red precipitate line explanation legionella pneumophilia serodiagnosis positives in detecting observation window " T " (detection line) and Quality Control observation window " C " (nature controlling line), Legionella pneumophila infection is promptly arranged; Locate not occur the red precipitate line as Quality Control observation window " C " (nature controlling line), then explanation detects the test paper inefficacy, and no matter detect observation window " T " (detection line) this moment is located precipitation line whether to occur, and testing result is false.
Bag with embodiment 1 preparation is detected above-mentioned legionella pneumophilia serum 1,2,3,7,9,10 type antibody test liquid by legionella pneumophilia serum 1,4,5,6 type antigens and goat anti-human igg's immune chromatography test paper, testing result is all positive, proves that immune chromatography test paper of the present invention can be used for the fast detecting of the legionella pneumophilia antibody of each serotype.
Two, the cross matching of other Related Bacteria blood serum sample
Use the antiserum sample of colon bacillus (preserving number 270014) available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre, salmonella (preserving number 460046), shigella dysenteriae (preserving number 51258), Pseudomonas aeruginosa (preserving number 10101), plague bacillus (preserving number 410050), brucella (preserving number 55227), staphylococcus aureus (preserving number 26067) standby as sample detection liquid, detecting liquid with the antiserum of legionella pneumophilia 1-10 type is contrast.
Result report: with 1: 40 above positive diagnostic criteria of serum titer, only locate to occur 1 red precipitate line (contrast) in Quality Control observation window " C " (nature controlling line), for the legionella pneumophilia no cross reaction; Locate to occur 2 red precipitate lines (sample and contrast) in detecting observation window " T " (detection line) and Quality Control observation window " C " (nature controlling line), for cross reaction being arranged with legionella pneumophilia; Locate not occur the red precipitate line as Quality Control observation window " C " (nature controlling line), then explanation detects the test paper inefficacy, and no matter detect observation window " T " (detection line) this moment is located precipitation line whether to occur, and testing result is false.
Bag with embodiment 1 preparation is detected above-mentioned antiserum sample by legionella pneumophilia serum 1,4,5,6 type antigens and goat anti-human igg's immune chromatography test paper, the result contrasts positive, other test sample is negative, prove that immune chromatography test paper of the present invention can be used for the fast detecting of the legionella pneumophilia antibody of each serotype, the specificity height.
1, specimen preparation
Will be available from the antiserum sample of the legionella pneumophilia 1-10 type in Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre, colon bacillus, salmonella, shigella dysenteriae, Pseudomonas aeruginosa, plague bacillus, brucella, staphylococcus aureus with physiological saline by standby after 1: 40 dilution proportion as sample detection liquid.
2, experimental technique
Get the kit that the present invention detects the immune chromatography test paper of legionella pneumophilia antibody that is equipped with of embodiment 1 preparation, respectively at adding 3 of above-mentioned sample detection liquid (about 150ul) in the point sample mouth, begin observations after 2 minutes, observation in 15 minutes stops.
Result's report: it is negative only to locate to occur 1 red precipitate line in Quality Control observation window " C " (nature controlling line), does not promptly have legionella pneumophilia antibody and detects; To locate to occur 2 red precipitate lines positive in detecting observation window " T " (detection line) and Quality Control observation window " C " (nature controlling line), promptly has legionella pneumophilia antibody to detect; Locate not occur the red precipitate line as Quality Control observation window " C " (nature controlling line), then explanation detects the test paper inefficacy, and no matter detect observation window " T " (detection line) this moment is located precipitation line whether to occur, and testing result is false.
3, experimental result
The laboratory result of appraisal of the immune chromatography test paper of detection legionella pneumophilia antibody of the present invention are as shown in table 1, as can be seen from Table 1, the immune chromatography test paper of detection legionella pneumophilia antibody of the present invention can detect legionella pneumophilia serum 1-10 type antibody, and with colon bacillus, salmonella, shigella dysenteriae, Pseudomonas aeruginosa, plague bacillus, brucella, staphylococcus aureus cross reaction does not take place.Above-mentioned testing result proves that immune chromatography test paper of the present invention can be used for the fast detecting of the legionella pneumophilia antibody of each serotype, and has higher sensitivity and specificity.
Table 1 the present invention detects the laboratory result of appraisal of the immune chromatography test paper (colloidal gold method) of legionella pneumophilia antibody
| Bacterium | Strain number | Serotype | Diluted sample degree 1: 40 |
| Legionella pneumophilia | 510101 | 1 | + |
| Legionella pneumophilia | 510102 | 2 | + |
| Legionella pneumophilia | 501103 | 3 | + |
| Legionella pneumophilia | 510104 | 4 | + |
| Legionella pneumophilia | 510105 | 5 | + |
| Legionella pneumophilia | 510106 | 6 | + |
| Legionella pneumophilia | 510107 | 7 | + |
| Legionella pneumophilia | 510108 | 8 | + |
| Legionella pneumophilia | 510109 | 9 | + |
| Legionella pneumophilia | 510110 | 10 | + |
| Colon bacillus | 270014 | - | |
| Salmonella | 460046 | - | |
| Shigella dysenteriae | 51258 | - | |
| Pseudomonas aeruginosa | 10101 | - | |
| Plague bacillus | 410050 | - | |
| Brucella | 55227 | - | |
| Staphylococcus aureus | 26067 | - |
Claims (10)
1, a kind of immune chromatography test paper that detects legionella pneumophilia antibody, comprise sample pad, closely be connected in the gold mark pad that contains staphylococcus aureus protein A mark colloidal gold probe of described sample pad one end, with the close-connected nitrocellulose membrane of the other end of described gold mark pad with closely be connected in the adsorptive pads of the described nitrocellulose membrane other end; Described nitrocellulose membrane is coated with detection line and the nature controlling line that is separated from each other, and described detection line contains legionella pneumophilia antigen, described nature controlling line contain can with the antibody of described staphylococcus aureus protein A specific bond.
2, the immune chromatography test paper of detection legionella pneumophilia antibody according to claim 1 is characterized in that: described legionella pneumophilia antigen is selected from one or more hybrid antigens in legionella pneumophilia serum 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types and the 10 type antigens; Described staphylococcus aureus protein A specific antibody is for being the antibody that antigen-immunized animal obtains with the staphylococcus aureus protein A, and described immune animal is rabbit, monkey, chicken or mouse; The concentration that described detection line contains the legionella pneumophilia antigen of 1,4,5,6 serotypes is 1-3mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antibody of staphylococcus aureus protein A specific bond.
3, the immune chromatography test paper of detection legionella pneumophilia antibody according to claim 2 is characterized in that: described legionella pneumophilia antigen is the hybrid antigen of legionella pneumophilia serum 1,4,5 and 6 types.
4, the immune chromatography test paper of detection legionella pneumophilia antibody according to claim 1 is characterized in that: the back side of the immune chromatography test paper of described detection legionella pneumophilia antibody closely connects a backboard.
5, a kind of method for preparing the immune chromatography test paper of the described detection legionella pneumophilia antibody of claim 1 may further comprise the steps:
1) preparation legionella pneumophilia antigen, the legionella pneumophilia antigenic solution is sprayed onto forms detection line on the cellulose membrane, another zone that can be sprayed onto described tunica fibrosa with the antibody-solutions of staphylococcus aureus protein A specific bond forms nature controlling line, then adsorptive pads is sticked on the end away from described calibration tape of described cellulose membrane;
2) the immune colloid gold probe solution of preparation staphylococcus aureus protein A mark, glass fibre membrane or polyester film are immersed this immune colloid gold probe solution, obtain gold mark pad, it is sticked on an end of the close described detection line of the cellulose membrane that step 1) obtains;
3) will be in step 2) in the gold mark pad that obtains go up away from an end of described detection line and paste sample pad, obtain detecting the immune chromatography test paper of legionella pneumophilia antibody.
6, preparation method according to claim 5 is characterized in that: the legionella pneumophilia antigen in the described step 1) is the hybrid antigen of legionella pneumophilia serum 1,4,5 and 6 types; Described staphylococcus aureus protein A specific antibody is for being the antibody that antigen-immunized animal obtains with the staphylococcus aureus protein A, and described immune animal is sheep, rabbit, monkey, chicken or mouse; The concentration that described detection line contains the legionella pneumophilia antigen of 1,4,5,6 serotypes is 1-3mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antibody of staphylococcus aureus protein A specific bond.
7, preparation method according to claim 5 is characterized in that: the preparation method of the legionella pneumophilia antigen in the described step 1) is: a. is seeded in legionella pneumophilia on the BCYE flat board, at 37 ℃, 5%CO
2Cultivated in the incubator 3-5 days, the typical single bacterium colony that picking grows on flat board, with its transferred species to the BCYE inclined-plane, at 37 ℃, 5%CO
2Cultivated in the incubator 3-5 days, the inclined-plane cultivated is washed lawn with the PBS of aseptic 0.01M, pH 7.2, centrifugal, abandon supernatant, the collection bacterial sediment; B. every gram weight in wet base thalline is suspended among the 10mL 0.1M NaOH stirring at room 1 hour; C. transfer pH value of solution to 3.0 with dense acetic acid, centrifugal, abandon precipitation, supernatant is transferred to pH6.5-7.0 with 5N NaOH; D. gained solution was dialysed 48 hours with the PBS of 0.01M, pH 7.0, obtained legionella pneumophilia antigen.
8, preparation method according to claim 5 is characterized in that: the baking temperature in the described step 1) is 30-45 ℃, and be 2.5-3.5 hour drying time; Step 2) preparation method of the immune colloid gold probe solution of legionella pneumophilia antigen-specific antibodies mark may further comprise the steps in:
A. 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 continues heated and boiled, stablizes into the grape wine redness up to liquid color, obtains colloidal gold solution, and cooling back water returns to original volume;
B. the pH value of the colloidal gold solution that step 1) is obtained transfers to 8.5-9.5, it is the staphylococcus aureus protein A of 12 μ g/mL that every 100mL colloidal gold solution adds final concentration, stirred 25-35 minute, add 5mL 10%BSA, stirred 20-30 minute, add 1mL 10%PEG20000 again, stirred 20-30 minute, earlier 20,000-23, under the 500rpm centrifugal 25-35 minute, abandon supernatant, sodium tetraborate solution washing with 0.05-0.1M precipitates, and it is stored in the sodium tetraborate solution of 8-10mL 0.05-0.1M, obtains having a liking for the colloidal gold probe solution of staphylococcus aureus protein A mark.
9, preparation method according to claim 8 is characterized in that: in the preparation method of the immune colloid gold probe solution of described staphylococcus aureus protein A mark, use K
2CO
3Solution or HCl solution adjust pH are 8.5-9.5, the K of described adjusting pH value
2CO
3The concentration of solution is 0.15-0.25M; The concentration of described adjusting pH value HCl solution is 0.08-0.12mol/L.
10, according to the preparation method of the immune chromatography test paper of each described detection legionella pneumophilia antibody of claim 5-9, it is characterized in that: to described step 2) in the immune colloid gold probe solution of legionella pneumophilia antigen-specific antibodies mark in add 0.05-0.1g/mL sucrose; With gold mark pad-20 ℃ to-50 ℃ freezing 10-12 hour, and, paste with cellulose membrane again after its freezing draining; A backboard is closely pasted at the back side of the immune chromatography test paper of the detection legionella pneumophilia antibody that obtains in described step 3); Described cellulose membrane is nitrocellulose filter or cellulose acetate membrane; Described adsorptive pads is a thieving paper; Described sample pad is a glass fibre membrane.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101692089A (en) * | 2009-09-29 | 2010-04-07 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunochromatographic test paper for detecting mycobacterium bovis antibodies and preparation method thereof |
| CN106290266A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Legionella Ab near-infrared fluorescent detection kit and application thereof |
| CN110540967A (en) * | 2018-12-20 | 2019-12-06 | 湖北诺美华抗体药物技术有限公司 | Human legionella pneumophila surface protein monoclonal antibody and application |
| CN114002437A (en) * | 2021-11-01 | 2022-02-01 | 西安文理学院 | Human serum albumin polyclonal antibody colloidal gold test strip and preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5741662A (en) * | 1995-12-18 | 1998-04-21 | Quidel Corporation | Direct stain specific binding assays for microorganisms |
| US9134303B1 (en) * | 1998-08-25 | 2015-09-15 | Alere Scarborough, Inc. | ICT immunoassay for Legionella pneumophila serogroup 1 antigen employing affinity purified antibodies thereto |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101692089A (en) * | 2009-09-29 | 2010-04-07 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunochromatographic test paper for detecting mycobacterium bovis antibodies and preparation method thereof |
| CN106290266A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Legionella Ab near-infrared fluorescent detection kit and application thereof |
| CN110540967A (en) * | 2018-12-20 | 2019-12-06 | 湖北诺美华抗体药物技术有限公司 | Human legionella pneumophila surface protein monoclonal antibody and application |
| CN114002437A (en) * | 2021-11-01 | 2022-02-01 | 西安文理学院 | Human serum albumin polyclonal antibody colloidal gold test strip and preparation method and application thereof |
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