CN101074956B - 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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- CN101074956B CN101074956B CN2007101190681A CN200710119068A CN101074956B CN 101074956 B CN101074956 B CN 101074956B CN 2007101190681 A CN2007101190681 A CN 2007101190681A CN 200710119068 A CN200710119068 A CN 200710119068A CN 101074956 B CN101074956 B CN 101074956B
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Abstract
An immune chromatographic test paper used for detecting antigen of legionella pneumophilia consists of sample pad, gold label pad of antibody label colloidal gold probe containing serum antigen resisting legionella pneumophilia, nitric acid fiber membrane and water absorption pad. It is featured as enveloping detection line containing antibody and quality control line containing anti-antibody on nitric acid fiber membrane and setting detection line to be separated from quality control line.
Description
Technical field
The present invention relates to a kind of legionella pneumophilia detection of antigens test paper and preparation method thereof, particularly relate to a kind of immune chromatography test paper that detects legionella pneumophilia antigen 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 provides a kind of immune chromatography test paper that detects legionella pneumophilia antigen
(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 antigen, comprise sample pad, closely be connected in the gold mark pad of the antibody labeling colloidal gold probe that contains anti-legionella pneumophilia serum antigen 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 the legionella pneumophilia antigen-specific antibodies, described nature controlling line contain can with the antiantibody of described legionella pneumophilia antigen-specific antibodies specific bond.
Described legionella pneumophilia antigen-specific antibodies is for being the antibody that antigen-immunized animal obtains with legionella pneumophilia antigen, described legionella pneumophilia antigen can be legionella pneumophilia serum 1 type antigen, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types or 10 type antigens, be preferably legionella pneumophilia serum 1 type antigen, the selection of described immune animal is diversified, as immune animal commonly used such as rabbit, monkey, chicken or mouse; The antiantibody of described legionella pneumophilia antigen-specific antibodies is preferably anti-rabbit antiantibody, especially is preferably goat anti-rabbit igg; The concentration of the legionella pneumophilia antigen-specific antibodies that described detection line contains can be 5-7mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antiantibody of legionella pneumophilia antigen-specific antibodies 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 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.
Second purpose of the present invention provides a kind of method for preparing the immune chromatography test paper of above-mentioned detection legionella pneumophilia antigen.
The method of the immune chromatography test paper of the above-mentioned detection legionella pneumophilia of preparation provided by the present invention antigen may further comprise the steps:
1) antiantibody of preparation legionella pneumophilia antigen-specific antibodies and legionella pneumophilia antigen-specific antibodies; Legionella pneumophilia antigen-specific antibodies solution is sprayed onto forms detection line on the cellulose membrane, another zone that the antiantibody solution of legionella pneumophilia antigen-specific antibodies is sprayed onto described tunica fibrosa 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 legionella pneumophilia antigen-specific antibodies 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 antigen.
In the preparation method of the immune chromatography test paper of above-mentioned detection legionella pneumophilia antigen, the antigen-specific antibodies of legionella pneumophilia described in the step 1) is for being the antibody that antigen-immunized animal obtains with legionella pneumophilia antigen, described legionella pneumophilia antigen can be legionella pneumophilia serum 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types or 10 type antigens, be preferably legionella pneumophilia serum 1 type antigen, the selection of described immune animal is diversified, as immune animal commonly used such as rabbit, monkey, chicken or mouse, be preferably rabbit; The antiantibody of described legionella pneumophilia antigen-specific antibodies is preferably anti-rabbit antiantibody, especially is preferably goat anti-rabbit igg; The concentration of the legionella pneumophilia antigen-specific antibodies that described detection line contains can be 5-7mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antiantibody of legionella pneumophilia antigen-specific antibodies specific bond.
The preparation method of the antigen-specific antibodies of legionella pneumophilia described in the step 1) can be: adopt subcutaneous multi-point injection mode to carry out immunity, every injection legionella pneumophilia antigen 2mg/mL, the freund adjuvant 0.1mL/ that annotates simultaneously only, immune 4-6 time (being preferably 5 times) altogether, each 10 days at interval, the last immunity was carried out the agarose double diffusion test to immune serum after 10 days, detect serum titer, reach more than the 1:32 and can take a blood sample, antagonistic Serum carries out purifying, obtains the legionella pneumophilia antigen-specific antibodies.
Baking temperature in the 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 legionella pneumophilia serum antigen specific antibody of 48 μ g/mL that every 100mL colloidal gold solution adds final concentration, stirred 25-35 minute, add 5mL10%BSA, stirred 20-30 minute, add 1mL10%PEG20000 again, stirred 20-30 minute, earlier 20,000-23, under the 500rpm centrifugal 25-35 minute, the sucking-off supernatant was again with supernatant under 10000-12000rpm 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 8-10mL0.05-0.1M, obtains the colloidal gold probe solution of legionella pneumophilia antigen-specific antibodies mark.
In the preparation method of the immune colloid gold probe solution of above-mentioned legionella pneumophilia antigen-specific antibodies 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 legionella pneumophilia antigen-specific antibodies mark combines better with glass fibre membrane or polyester film, can be to step 2) in the immune colloid gold probe solution of legionella pneumophilia antigen-specific antibodies 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 antigen 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 antigen be: use legionella pneumophilia antigen-specific antibodies bag by cellulose membrane, in order to catch legionella pneumophilia or the legionella pneumophilia antigen in the sample, then with mark the immune colloid gold probe of antiantibody of legionella pneumophilia antigen-specific antibodies detect.Immune chromatography test paper of the present invention has the following advantages: 1) susceptibility and specificity height: the laboratory result of appraisal show, immune chromatography test paper of the present invention can be used for detecting legionella pneumophilia serum 1-10 type antigen specimen, and with other biology cross reaction does 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 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 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 antigen
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 antigen
One, the preparation of legionella pneumophilia serum 1 type antigen-specific antibodies
1, the preparation of legionella pneumophilia serum 1 type antigen
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) is seeded on the BCYE flat board, places 37 ℃, 5%CO
2Cultivate in the incubator, observe the colonial morphology on the flat board, the typical single bacterium colony of picking, the BCYE inclined-plane is put in switching, places 37 ℃, 5%CO
2Cultivated 3-5 days in the incubator.The inclined-plane of cultivating is washed lawn with aseptic PBS (0.01M pH7.2), and centrifugal 25 minutes of 8000rpm abandons supernatant, collects bacterial sediment; Every gram weight in wet base thalline is suspended in the 10mL0.1M NaOH solution, stirring at room 1 hour; Dense acetic acid is transferred pH value of solution value to 3.0, and centrifugal 20 minutes of 8000rpm abandons precipitation, and it is 6.5-7.0 that supernatant is transferred to the pH value with 5N NaOH, and gained solution obtains legionella pneumophilia serum 1 type antigen with PBS (0.01M pH7.0) dialysis 48 hours, and freeze-drying is preserved.
2, the preparation of legionella pneumophilia serum 1 type antigen-specific antibodies
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 behind the legionella pneumophilia antigen mixing and emulsifying of Fu Shi Freund's complete adjuvant and step 1 acquisition is carried out immunity, immunizing dose is 2mg/mL/, respectively at the 20th day behind the initial immunity, the 30th day, supplementary immunization aqua 1 pin on the 40th, boost is identical with initial immunity with approach, back 10 days of last immunity is carried out the agarose double diffusion test to immune serum, detect serum titer, the result is 1:64 (1:32 is above to get final product) blood sampling, adopt conventional saturated ammonium sulfate salting out method antagonistic Serum to carry out purifying, promptly saltout 2 times with 33% saturated ammonium sulfate, collect antibody behind the dialysis desalting, obtain the legionella pneumophilia antigen-specific antibodies.Adopt the uv-spectrophotometric instrument to measure the concentration of the legionella pneumophilia antigen-specific antibodies albumen of above-mentioned preparation, method is: albumen is suitably diluted, measure the A value respectively under 280nm and 260nm wavelength, calculate protein concentration according to following formula:
Protein content (mg/mL)=(1.45A
280nm-0.74A
260nm) extension rate.
Result's concentration of the legionella pneumophilia antigen-specific antibodies albumen of purifying gained according to the method described above is 180mg/mL.
3, 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
4(available from Sigma company, 1g/ bottle packing) is mixed with 0.01% aqueous solution, gets 100mL and is heated to boiling, stirs 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
3Solution (or 0.1M HCl solution) is regulated colloidal gold solution pH 8.5-9.5, prepares 6 clean tube, adds the 1mL colloidal gold solution respectively.Purified legionella pneumophilia serum 1 type antigen-specific antibodies dilution is 1mg/mL, in 5 test tubes, add 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 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 40 μ l, i.e. 40 μ g/mL, and selecting the coupling antibody concentration is 48 μ g/mL.
3) preparation of gold mark pad: preparation contains the legionella pneumophilia serum 1 type antigen-specific antibodies immune colloid gold probe solution 50mL that concentration is 48 μ g/mL as stated above, stir 30 minutes (25-35 minute all can), add 10%BSA5mL, stir 25 minutes (20-30 minute all can), add 1mL 10%PEG20000, stir 25 minutes (20-30 minute all can), 20,000～23, centrifugal 25 minutes of 500rpm (20-30 minute all can), abandon supernatant, will precipitate with after the sodium tetraborate washing 1 time and preserve liquid collecting precipitation 5mL with sodium tetraborate.Get legionella pneumophilia serum 1 type antigen-specific antibodies immune colloid gold probe solution 5mL and add 0.5g sucrose, fully dissolving evenly is added on the glass fibre membrane,-35 ℃ (20 ℃～-50 ℃ all can) place 11 hours (10-12 hour all can), and freeze dryer is drained, and obtains gold mark pad.
4, the preparation of legionella pneumophilia antigen quick detection test paper
(a is the Facad structure figure of the immune chromatography test paper of detection legionella pneumophilia antigen among the figure as shown in Figure 1, b is the vertical section structure figure of the immune chromatography test paper of detection legionella pneumophilia antigen among the figure), detect the immune chromatography test paper of legionella pneumophilia antigen and form by adsorptive pads 4, nitrocellulose filter (NC film) 3, gold mark pad 2 and glass fibre membrane sample pad 1 four parts; Nitrocellulose membrane 3 is provided with detection line 5 and nature controlling line 6, and 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) and dilution legionella pneumophilia serum 1 type antigen-specific antibodies, concentration is 6mg/mL, is used to wrap tested survey line.With 0.01MpH7.2PBS (NaH
2PO
42H
2O 0.39g, Na
2HPO
41.07g, NaCl 8.5g, deionized water 1000mL) and the dilution goat anti-rabbit igg, concentration is 4mg/mL, is used for bag by nature controlling line.Be sprayed on respectively on the thick nitrocellulose filter of 2.5mm with the XYZ3000 of BIODOT company Membrane jetter, form detection line 5 and 6,37 ℃ of dry 2.5-3.5h of nature controlling line of being separated from each other.
2) preparation of legionella pneumophilia antigen 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 antigen by required size cutting, add drying agent after sealing preserve.
5, detect the preparation of the immune chromatography reagent kit of legionella pneumophilia antigen
Use for convenience, with the following plastic back plate that closely connects again of the immune chromatography test paper of the legionella pneumophilia antigen 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.
6, the using method of legionella pneumophilia antigen 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 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 combines to the anti-rabbit antiantibody of nature controlling line 6 and solid phase, and shows red Quality Control lines.Otherwise, the then reactionless lines of negative sample, and only show the Quality Control lines.
Embodiment 2, legionella pneumophilia detection of antigens reach the cross matching with other Related Bacteria
One, different serotypes legionella pneumophilia detection of antigens
The preparation method of legionella pneumophilia serum 1 type antigen is equally applicable to the preparation of other serotype antigen of legionella pneumophilia in embodiment 1 step 1, and using legionella pneumophilia serum 1,2,3,4,5,6,7,9, the 10 type bacterial strains that provided by Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre to be diluted to concentration respectively by the antigen of the method preparation of embodiment 1 is that the sample detection liquid of 100 μ g/mL, 10 μ g/mL, 1 μ g/mL, 100ng/mL, 10ng/mL is standby.
Get the kit that the present invention detects the immune chromatography test paper of legionella pneumophilia antigen 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 antigen 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 antigen 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.
Bag with embodiment 1 preparation is detected above-mentioned legionella pneumophilia serum 1,2,3,4,5,6,7,9,10 types detection liquid by the immune chromatography test paper of legionella pneumophilia serum 1 type antigen-specific antibodies and goat anti-rabbit igg, 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 antigen of each serotype.
Two, the cross matching of other Related Bacteria
The concentration that is provided by Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre is that 100 μ g/mL plague bacillus, 410050 strain F1 antigens are standby as sample detection liquid.
Bag with embodiment 1 preparation is detected above-mentioned plague bacillus 410050 strain F1 antigen samples detection liquid by the immune chromatography test paper of legionella pneumophilia serum 1 type antigen-specific antibodies and goat anti-rabbit igg, the result is negative, prove that immune chromatography test paper of the present invention can be used for the fast detecting of the legionella pneumophilia antigen of each serotype, specificity height.
Embodiment 3, laboratory examination
1, specimen preparation
Bacterium 10 strains are used in experiment altogether, wherein 9 strain legionella pneumophilias (strain number is: 510101-5101010, available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre), 1 strain plague bacillus (strain number is: 410050, available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research centre).Above-mentioned bacterium is seeded to suitable culture base separately respectively, and under different conditions, cultivate the back and extract antigen, it is 100 μ g/mL, 10 μ g/mL, 1 μ g/mL, 100ng/mL and 10ng/mL that legionella pneumophilia serum 1,2,3,4,5,6,7,9,10 type antigens are diluted to concentration respectively, F 1 antibody of plague bacterium concentration is 100 μ g/mL, and liquid is standby as detecting.
2, experimental technique
Bag with embodiment 1 preparation is detected by the legionella pneumophilia antigen quick detection reagent of the immune chromatography test paper of legionella pneumophilia serum 1 type antigen-specific antibodies and goat anti-rabbit igg to step 1 preparation, get above-mentioned legionella pneumophilia antigen quick detection reagent with the different strains preparation, 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 only to locate to occur 1 red precipitate line in Quality Control observation window " C " (nature controlling line), does not promptly have legionella pneumophilia antigen 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 antigen 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 legionella pneumophilia antigen quick detection reagent are as shown in table 1, as can be seen from Table 1, the immune chromatography test paper of detection legionella pneumophilia antigen of the present invention can detect the legionella pneumophilia serum 1 type antigen of 10ng/mL, 10 μ g/mL legionella pneumophilia serum, 2,3,4,5,6,7,9,10 type antigens, and not with 100ug/mL F 1 antibody of plague bacterium generation cross reaction.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 antigen of each serotype, and has higher sensitivity and specificity.
The examination of table 1 legionella pneumophilia antigen quick detection reagent (colloidal gold method) laboratory
Claims (6)
1. immune chromatography test paper that detects legionella pneumophilia antigen, comprise sample pad, closely be connected in the gold mark pad of the immune colloid gold probe that contains legionella pneumophilia antigen-specific antibodies mark 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 the legionella pneumophilia antigen-specific antibodies, described nature controlling line contain can with the antiantibody of described legionella pneumophilia antigen-specific antibodies specific bond;
The preparation method of the immune chromatography test paper of described detection legionella pneumophilia antigen may further comprise the steps:
1) antiantibody of preparation legionella pneumophilia antigen-specific antibodies and legionella pneumophilia antigen-specific antibodies; Legionella pneumophilia antigen-specific antibodies solution is sprayed onto forms detection line on the cellulose membrane, another zone that the antiantibody solution of legionella pneumophilia antigen-specific antibodies is sprayed onto described tunica fibrosa 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 legionella pneumophilia antigen-specific antibodies 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 antigen;
Legionella pneumophilia antigen-specific antibodies in the described step 1) is for being the antibody that antigen-immunized animal obtains with legionella pneumophilia antigen; Described legionella pneumophilia antigen is legionella pneumophilia serum 1 type antigen, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types or 10 type antigens; Described immune animal is rabbit, monkey, chicken or mouse; The concentration of the legionella pneumophilia antigen-specific antibodies that described detection line contains is 5-7mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antiantibody of legionella pneumophilia antigen-specific antibodies specific bond;
The preparation method of legionella pneumophilia antigen-specific antibodies is in the described step 1): subcutaneous multi-point injection behind Fu Shi Freund's complete adjuvant and the legionella pneumophilia antigen mixing and emulsifying is carried out immunity, immunizing dose is 2mg/mL/, immune 4-6 time altogether, each 10 days at interval, the last immunity was carried out the agarose double diffusion test to immune serum after 10 days, detected serum titer, reached above back blood sampling in 1: 32, antagonistic Serum carries out purifying, obtains the legionella pneumophilia antigen-specific antibodies;
Described 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% 2 hydration trisodium citrate aqueous solution that accurately adds 1.6mL down, continue heated and boiled, stablize into the grape wine redness up to liquid color, obtain colloidal gold solution, cooling back water returns to original volume;
B. the pH value of the colloidal gold solution that steps A is obtained transfers to 8.5-9.5, it is the legionella pneumophilia serum antigen specific antibody of 48 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, the sucking-off supernatant was again with supernatant under 10000-12000rpm 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 the colloidal gold probe solution of legionella pneumophilia antigen-specific antibodies mark.
2. the immune chromatography test paper of detection legionella pneumophilia antigen according to claim 1, it is characterized in that: the antiantibody of described legionella pneumophilia antigen-specific antibodies is anti-rabbit antiantibody.
3. the immune chromatography test paper of detection legionella pneumophilia antigen according to claim 1 is characterized in that: the back side of the immune chromatography test paper of described detection legionella pneumophilia antigen closely connects a backboard.
4. one kind prepares the described method that detects the immune chromatography test paper of legionella pneumophilia antigen of claim 1, may further comprise the steps:
1) antiantibody of preparation legionella pneumophilia antigen-specific antibodies and legionella pneumophilia antigen-specific antibodies; Legionella pneumophilia antigen-specific antibodies solution is sprayed onto forms detection line on the cellulose membrane, another zone that the antiantibody solution of legionella pneumophilia antigen-specific antibodies is sprayed onto described tunica fibrosa 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 legionella pneumophilia antigen-specific antibodies 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 antigen;
Legionella pneumophilia antigen-specific antibodies in the described step 1) is for being the antibody that antigen-immunized animal obtains with legionella pneumophilia antigen; Described legionella pneumophilia antigen is legionella pneumophilia serum 1 type antigen, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types or 10 type antigens; Described immune animal is rabbit, monkey, chicken or mouse; The concentration of the legionella pneumophilia antigen-specific antibodies that described detection line contains is 5-7mg/mL, and what described nature controlling line contained can be 3-5mg/mL with the concentration of the antiantibody of legionella pneumophilia antigen-specific antibodies specific bond;
The preparation method of legionella pneumophilia antigen-specific antibodies is in the described step 1): subcutaneous multi-point injection behind Fu Shi Freund's complete adjuvant and the legionella pneumophilia antigen mixing and emulsifying is carried out immunity, immunizing dose is 2mg/mL/, immune 4-6 time altogether, each 10 days at interval, the last immunity was carried out the agarose double diffusion test to immune serum after 10 days, detected serum titer, reached above back blood sampling in 1: 32, antagonistic Serum carries out purifying, obtains the legionella pneumophilia antigen-specific antibodies;
Described 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% 2 hydration trisodium citrate aqueous solution that accurately adds 1.6mL down, continue heated and boiled, stablize into the grape wine redness up to liquid color, obtain colloidal gold solution, cooling back water returns to original volume;
B. the pH value of the colloidal gold solution that steps A is obtained transfers to 8.5-9.5, it is the legionella pneumophilia serum antigen specific antibody of 48 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, the sucking-off supernatant was again with supernatant under 10000-12000rpm 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 the colloidal gold probe solution of legionella pneumophilia antigen-specific antibodies mark.
5. the preparation method of the immune chromatography test paper of detection legionella pneumophilia antigen according to claim 4 is characterized in that: use K among the described step B
2CO
3Solution or HCl solution adjust pH; 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.
6. according to the preparation methods of the immune chromatography test paper of claim 4 or 5 described detection legionella pneumophilia antigens, 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 antigen that obtains in described step 3); Described adsorptive pads is a thieving paper; Described sample pad is a glass fibre membrane.
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| CN102175863B (en) * | 2011-02-23 | 2013-11-27 | 谭森 | Early warning method for Legionella |
| CN106290836A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of leginella antigen gold-immunochromatographyreagent reagent for assay box |
| 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 |
| CN106290835A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of leginella antigen emulsion technique detection kit |
| CN106290834A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of leginella antigen near-infrared fluorescent detection kit and application thereof |
| CN110540972B (en) * | 2018-12-20 | 2021-04-13 | 湖北云璐生物工程有限公司 | Human legionella pneumophila surface protein monoclonal antibody and antigen capture ELISA kit |
| 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 |
| CN110907638B (en) * | 2020-02-14 | 2020-09-22 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
Citations (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 |
| CN1313771A (en) * | 1998-08-25 | 2001-09-19 | 比南股份有限公司 | Method for detection of i(legionella) bacteria employing purified antigen-specific antibodies |
-
2007
- 2007-06-19 CN CN2007101190681A patent/CN101074956B/en not_active Expired - Fee Related
Patent Citations (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 |
| CN1313771A (en) * | 1998-08-25 | 2001-09-19 | 比南股份有限公司 | Method for detection of i(legionella) bacteria employing purified antigen-specific antibodies |
Non-Patent Citations (5)
| Title |
|---|
| J. Dominguez et al..Evaluation of a rapid immunochromatographic assay for thedetection of legionella antigen in urine samples.Eur J Clin Microbiol Infect Dis18.1999,18896-898. * |
| L. Molinos.Detection of Antigens in urine.Arch Bronconeumol.42 3.2006,42(3),101-103. |
| L. Molinos.Detection of Antigens in urine.Arch Bronconeumol.42 3.2006,42(3),101-103. * |
| 汪黎 等.军团菌的免疫检测技术研究进展.微生物学免疫学进展34 2.2006,34(2),76-79. |
| 汪黎 等.军团菌的免疫检测技术研究进展.微生物学免疫学进展34 2.2006,34(2),76-79. * |
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