CN104198704A - Pertussis diagnostic kit and preparation method thereof - Google Patents
Pertussis diagnostic kit and preparation method thereof Download PDFInfo
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- CN104198704A CN104198704A CN201410443710.1A CN201410443710A CN104198704A CN 104198704 A CN104198704 A CN 104198704A CN 201410443710 A CN201410443710 A CN 201410443710A CN 104198704 A CN104198704 A CN 104198704A
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Abstract
The invention discloses a pertussis diagnostic kit and a preparation method thereof. The kit comprises a detection probe of a nanogold marker mice anti-human IgA antibody, pertussis bacillus capsular antigen and a dot immunogold concentrated filtration device, wherein the centralized filtration device consists of a plastic capsule, a nitrocellulose membrane added with the detection probe of the nanogold marker mice anti-human IgA antibody and a water absorption layer; water absorption pads are arranged at a groove of the capsule; the nitrocellulose membrane is arranged between the water absorption pads and a cover of the capsule; and the water absorption layer is formed by three to five water absorption pads via stacking. According to the kit disclosed by the invention, the immunogold dots generated by the antigen-antibody reaction are concentrated and amplified again through the concentrated filtration device on the basis of a nanogold amplification technology, so that the detection sensitivity is improved, the limit of detection is enlarged and effective detection can be carried out on the incubation period, the convulsion period and the recovery period.
Description
Technical field
The present invention relates to medical test technical field, particularly relate to a kind of pertussis diagnostic kit and preparation method.
Background technology
Pertussis is a kind of Acute respiratory infectious disease being caused by Bordetella pertussis, and clinical manifestation is paroxysmal spasmodic cough, and cough end is with special air-breathing roar, and the course of disease is longer, can reach several weeks even about 3 months, therefore claim pertussis.Pertussal cough has been found that and can cause subconjunctival hemorrhage, fracture of rib, the retention of urine, hernia, after cough, faint, and vertebral artery interlayer, violent cough can cause pleura to break, cause pneumothorax, especially concerning children's, endanger very seriously, the length of stage of spasmodic cough is sooner or later relevant with treatment, diagnosis as early as possible could be treated as early as possible, so pertussal quick diagnosis is seemed to particularly important.
At present, medically pertussal inspection is had to several different methods, comprise white blood cell count(WBC) method, cell culture method, fluoresent antibody staining, Serological testing etc., cell culture method, by separated bacterium colony, is observed colonial morphology, can only be as tentative diagnosis; Fluoresent antibody staining is to use Nasopharyngeal swabs smear, adds fluorescently-labeled antiserum, under fluorescent microscope, check, and early stage patient 75～80% positives, advantage is can quick diagnosis, but shortcoming is poor specificity, has false positive, just as auxiliary, cultivates clinically; Serological testing is do paired sera agglutination test and mend in conjunction with test, if antibody titer is risen progressively, can make a definite diagnosis, and recent useful enzyme linked immunosorbent assay is surveyed IgM, IgG and IgA antibody, helpful to early diagnosis; In addition, the form that the detection method of Bordetella pertussis or its constituent is cultivated as spot hybridization, PCR method, histocyte, the vigor of enzyme etc., still in laboratory or the clinical observation stage, are not also generally applied.
Bordetella pertussis tentative diagnosis be take separation cultivation as main, latent period, (catarrhal period) separated positive rate can reach 91.5%, and convalescence only has an appointment 26%, make a definite diagnosis is to make the aggegation of serum slide or immunofluorescence dyeing with isolate and I phase immune serum, so be mainly confined to the I phase phase, detect, detectability is shorter.So, be badly in need of working out a kind of not only quick but also longer Bordetella pertussis detection method of detectability.
Summary of the invention
For above-mentioned present situation, the technical matters that the present invention solves is just to provide a kind of detection method that can fast detecting Bordetella pertussis, and detectability is long, from ill latent period, spasm period until convalescence all can effectively detect.
Technical scheme of the present invention is: a kind of pertussis diagnostic kit, percolating device is concentrated in the detector probe, Bordetella pertussis capsular antigen, the immunity of spot gold that comprise nano gold mark mouse-anti people IgA antibody, and nitrocellulose filter and adsorptive pads three parts that described concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form.
Further, described is 0.92-1.86 μ g/mL for soaking the concentration of the nano gold mark mouse-anti people IgA antibody of detector probe.
Further, described Bordetella pertussis capsular antigen concentration is 0.85～1.65 μ g/mL.
Wherein, described Bordetella pertussis capsular antigen is adopted with the following method and is prepared:
(1) bacterium is separated cultivates: with potato blood glycerin agar medium culture Bordetella pertussis, 5-7, after day, stops cultivating separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacteria, during first separated cultivation, nutritional requirement is higher, so need could grow with potato blood glycerin agar nutrient culture media (being Bao～Jin Shi nutrient culture media), after 37 ℃ are cultivated for 2～3 days, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture is even muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme is processed bacterial suspension: at every milliliter, containing in the suspension of 200,000,000 bacterial cells, add 100 μ g to 1mg lysozymes, 27-35 ℃, insulation 15min, broken bacterial cell;
(3) antigen extracts: with hydrogen, as solvent, the phosphate of 0.03-0.05mol/L, as damping fluid, extracts, and temperature is controlled at below 5 ℃, and pH is controlled within the scope of 5.0-7.0;
(4) antigen isolation and purification: carry out separation by ion exchange process, wherein, described exchanger is ion-exchange gel, preferably DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen is concentrated: adopt freezing method to concentrate, the antigen of first being prepared by step (4) is cooled to solid, and then slowly dissolve, the pure ice crystal containing antigen does not float on liquid level, antigen concentrates in lower floor's solution, removes upper strata ice cube, again with dilution, regulates concentration to 0.85～1.65 μ g/mL, obtain antigen concentrate ,-20～-4 ℃ of preservations; Freezing method is the concentrated relatively effective method that to obtain of protein one class biomacromolecule, utilizes the difference of solvent and solute dissolved click and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: will put into the Bordetella pertussis capsular antigen solution that regulates concentration on nitrocellulose filter, standing 20-30min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, takes out, and low temperature saves backup.
Wherein, the preparation method of the detector probe of described nano gold mark mouse-anti people IgA antibody comprises the steps:
(1) prepare nm of gold system: chloroazotic acid soaking container, through ultrapure water, rinse again, dry, adding mass concentration is 1% gold chloride, with ultrapure water, dissolves, ratio between described gold chloride and ultrapure water is: (97.5-98.8): (3.85-4.05), agitating heating is boiled, and adding mass concentration is 1% trisodium citrate, continues to be heated with stirring to form claret solution, stir coolingly, obtain nano-Au solution; Described nano Au particle particle diameter is 14-18nm;
(2) prepare the detector probe of nano gold mark mouse-anti people IgA antibody: above-mentioned nano-Au solution is regulated to its pH to 7.6-8.5 with solution of potassium carbonate, by mouse-anti people IgA antibody dilution to 0.3-0.75mg/ml, 12000rpm, centrifugal 40min under 4 ℃ of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, fully mix combination, add and the isopyknic confining liquid of aforementioned mixed liquor, room temperature continues to hatch 20～28min, 14000rpm, centrifugal 15～25min under 4 ℃ of conditions, remove supernatant, the resuspended precipitation of dilution washing are once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Wherein, described plastics capsule can be various shape, and there is the approximately square hole of 0.5～0.8cm of the length of side in the central authorities of lid, and the position square groove corresponding with square hole on lid arranged at box bottom, big or small equally large with the square hole on lid; Adsorptive pads is positioned over the groove of box, and nitrocellulose filter is placed between adsorptive pads and lid, closely closes lid, makes nitrocellulose filter be adjacent to adsorptive pads, and described water accepting layer is stacked together and is formed by 3-5 layer adsorptive pads.Because the relative nitrocellulose filter of area of water accepting layer is less, can make the antibody-antigen-Jin labeling antibody compound reacting concentrate to middle part gathering, and play inspissation, further strengthen color developing effect.
Detection principle of the present invention is: use the immunity of spot gold to concentrate percolation test, take nitrocellulose filter as carrier, and be coated with in the percolating device of antigen, drip successively sample to be measured, Immuno gold and cleansing solution, because nitrocellulose filter adheres on absorbent material, the quick combination of antigen when therefore sample to be measured is flowed through percolating device and on film, be combined with golden labeling antibody again, form antibody-antigen-Jin labeling antibody compound, the relative nitrocellulose filter of area of water accepting layer is less, antibody-antigen-Jin labeling antibody compound that can make to react is concentrated to middle part and is assembled, and play inspissation, further strengthen color developing effect, reach fast detecting object (general 5min left and right completes), positive reaction presents punctation on film.
Beneficial effect of the present invention is: kit is simple and easy to preparation, easy to detect, fast, highly sensitive, compares its detectability of other detection methods larger, from ill latent period, spasm period until convalescence all can be imitated detection, is applicable to very much clinical practice.
Embodiment
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By below and the description of claim, feature of the present invention, object and advantage will be more obvious.
Embodiment 1:
A kind of pertussis diagnostic kit, percolating device is concentrated in the detector probe, Bordetella pertussis capsular antigen, the immunity of spot gold that comprise nano gold mark mouse-anti people IgA antibody, and nitrocellulose filter and adsorptive pads three parts that described concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form; Described is 0.92 μ g/mL for soaking the concentration of the nano gold mark mouse-anti people IgA antibody of detector probe; Described Bordetella pertussis capsular antigen concentration is 0.85 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacterium is separated cultivates: with potato blood glycerin agar medium culture Bordetella pertussis, after 5 days, stop cultivating separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacteria, during first separated cultivation, nutritional requirement is higher, so need could grow with potato blood glycerin agar nutrient culture media (being Bao～Jin Shi nutrient culture media), after 37 ℃, 2 days cultivate, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture is even muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme is processed bacterial suspension: at every milliliter, containing in the suspension of 200,000,000 bacterial cells, add 100 μ g to 1mg lysozymes, and 27 ℃, insulation 15min, broken bacterial cell;
(3) antigen extracts: with hydrogen, as solvent, the phosphate of 0.03mol/L, as damping fluid, extracts, and temperature is controlled at below 5 ℃, and pH is 5.0;
(4) antigen isolation and purification: carry out separation by ion exchange process, wherein, described exchanger is ion-exchange gel, preferably DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen is concentrated: adopt freezing method to concentrate, the antigen of first being prepared by step (4) is cooled to solid, and then slowly dissolve, the pure ice crystal containing antigen does not float on liquid level, antigen concentrates in lower floor's solution, removes upper strata ice cube, again with dilution, regulates concentration to 0.85 μ g/mL, obtain antigen concentrate ,-20 ℃ of preservations; Freezing method is the concentrated relatively effective method that to obtain of protein one class biomacromolecule, utilizes the difference of solvent and solute dissolved click and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: will put into the Bordetella pertussis capsular antigen solution that regulates concentration on nitrocellulose filter, standing 20min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, takes out, and low temperature saves backup.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) prepare nm of gold system: chloroazotic acid soaking container, through ultrapure water, rinse again, dry, adding mass concentration is 1% gold chloride, with ultrapure water, dissolves, ratio between described gold chloride and ultrapure water is: 97.5:3.85, agitating heating is boiled, and adding mass concentration is 1% trisodium citrate, continues to be heated with stirring to form claret solution, stir coolingly, obtain nano-Au solution; Described nano Au particle particle diameter is 14nm;
(2) prepare the detector probe of nano gold mark mouse-anti people IgA antibody: above-mentioned nano-Au solution is regulated to its pH to 7.6 with solution of potassium carbonate, by mouse-anti people IgA antibody dilution to 0.3mg/ml, 12000rpm, centrifugal 40min under 4 ℃ of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, fully mix combination, add and the isopyknic confining liquid of aforementioned mixed liquor, room temperature continues to hatch 20min, 14000rpm, centrifugal 15min under 4 ℃ of conditions, remove supernatant, the resuspended precipitation of dilution washing are once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: nitrocellulose filter and adsorptive pads three parts that concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form; Plastics capsule can be various shape, and there is the square hole of the about 0.5cm of the length of side in the central authorities of lid, and the position square groove corresponding with square hole on lid arranged at box bottom, big or small equally large with the square hole on lid; Adsorptive pads is positioned over the groove of box, and nitrocellulose filter is placed between adsorptive pads and lid, closely closes lid, makes nitrocellulose filter be adjacent to adsorptive pads, and described water accepting layer is stacked together and is formed by 3 layers of adsorptive pads.Because the relative nitrocellulose filter of area of water accepting layer is less, can make the antibody-antigen-Jin labeling antibody compound reacting concentrate to middle part gathering, and play inspissation, further strengthen color developing effect.
Embodiment 2:
A kind of pertussis diagnostic kit, percolating device is concentrated in the detector probe, Bordetella pertussis capsular antigen, the immunity of spot gold that comprise nano gold mark mouse-anti people IgA antibody, and nitrocellulose filter and adsorptive pads three parts that described concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form; Described is 1.39 μ g/mL for soaking the concentration of the nano gold mark mouse-anti people IgA antibody of detector probe; Described Bordetella pertussis capsular antigen concentration is 1.25 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacterium is separated cultivates: with potato blood glycerin agar medium culture Bordetella pertussis, after 6 days, stop cultivating separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacteria, during first separated cultivation, nutritional requirement is higher, so need could grow with potato blood glycerin agar nutrient culture media (being Bao～Jin Shi nutrient culture media), after 37 ℃, 2 days cultivate, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture is even muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme is processed bacterial suspension: at every milliliter, containing in the suspension of 200,000,000 bacterial cells, add 100 μ g to 1mg lysozymes, and 31 ℃, insulation 15min, broken bacterial cell;
(3) antigen extracts: with hydrogen, as solvent, the phosphate of 0.05mol/L, as damping fluid, extracts, and temperature is controlled at below 5 ℃, and it is 6.0 that pH controls;
(4) antigen isolation and purification: carry out separation by ion exchange process, wherein, described exchanger is ion-exchange gel, preferably DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen is concentrated: adopt freezing method to concentrate, the antigen of first being prepared by step (4) is cooled to solid, and then slowly dissolve, the pure ice crystal containing antigen does not float on liquid level, antigen concentrates in lower floor's solution, removes upper strata ice cube, again with dilution, regulates concentration to 1.25 μ g/mL, obtain antigen concentrate ,-12 ℃ of preservations; Freezing method is the concentrated relatively effective method that to obtain of protein one class biomacromolecule, utilizes the difference of solvent and solute dissolved click and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: will put into the Bordetella pertussis capsular antigen solution that regulates concentration on nitrocellulose filter, standing 25min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, takes out, and low temperature saves backup.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) prepare nm of gold system: chloroazotic acid soaking container, through ultrapure water, rinse again, dry, adding mass concentration is 1% gold chloride, with ultrapure water, dissolves, ratio between described gold chloride and ultrapure water is: 98.15:3.95, agitating heating is boiled, and adding mass concentration is 1% trisodium citrate, continues to be heated with stirring to form claret solution, stir coolingly, obtain nano-Au solution; Described nano Au particle particle diameter is 16nm;
(2) prepare the detector probe of nano gold mark mouse-anti people IgA antibody: above-mentioned nano-Au solution is regulated to its pH to 8.05 with solution of potassium carbonate, by mouse-anti people IgA antibody dilution to 0.52mg/ml, 12000rpm, centrifugal 40min under 4 ℃ of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, fully mix combination, add and the isopyknic confining liquid of aforementioned mixed liquor, room temperature continues to hatch 24min, 14000rpm, centrifugal 20min under 4 ℃ of conditions, remove supernatant, the resuspended precipitation of dilution washing are once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: nitrocellulose filter and adsorptive pads three parts that concentrated percolating device has added Bordetella pertussis pod membrane (K) antigen by plastics capsule, point form; Plastics capsule can be various shape, and there is the square hole of the about 0.65cm of the length of side in the central authorities of lid, and the position square groove corresponding with square hole on lid arranged at box bottom, big or small equally large with the square hole on lid; Adsorptive pads is positioned over the groove of box, and nitrocellulose filter is placed between adsorptive pads and lid, closely closes lid, makes nitrocellulose filter be adjacent to adsorptive pads, and described water accepting layer is stacked together and is formed by 4 layers of adsorptive pads.Because the relative nitrocellulose filter of area of water accepting layer is less, can make the antibody-antigen-Jin labeling antibody compound reacting concentrate to middle part gathering, and play inspissation, further strengthen color developing effect.
Embodiment 3:
A kind of pertussis diagnostic kit, percolating device is concentrated in the detector probe, Bordetella pertussis capsular antigen, the immunity of spot gold that comprise nano gold mark mouse-anti people IgA antibody, and nitrocellulose filter and adsorptive pads three parts that described concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form; Described is 1.86 μ g/mL for soaking the concentration of the nano gold mark mouse-anti people IgA antibody of detector probe; Described Bordetella pertussis capsular antigen concentration is 1.65 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacterium is separated cultivates: with potato blood glycerin agar medium culture Bordetella pertussis, 5-7, after day, stops cultivating separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacteria, during first separated cultivation, nutritional requirement is higher, so need could grow with potato blood glycerin agar nutrient culture media (being Bao～Jin Shi nutrient culture media), after 37 ℃, 3 days cultivate, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture is even muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme is processed bacterial suspension: at every milliliter, containing in the suspension of 200,000,000 bacterial cells, add 100 μ g to 1mg lysozymes, and 35 ℃, insulation 15min, broken bacterial cell;
(3) antigen extracts: with hydrogen, as solvent, the phosphate of 0.05mol/L, as damping fluid, extracts, and temperature is controlled at below 5 ℃, and it is 7.0 that pH controls;
(4) antigen isolation and purification: carry out separation by ion exchange process, wherein, described exchanger is ion-exchange gel, preferably DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen is concentrated: adopt freezing method to concentrate, the antigen of first being prepared by step (4) is cooled to solid, and then slowly dissolve, the pure ice crystal containing antigen does not float on liquid level, antigen concentrates in lower floor's solution, removes upper strata ice cube, again with dilution, regulates concentration to 1.65 μ g/mL, obtain antigen concentrate ,-4 ℃ of preservations; Freezing method is the concentrated relatively effective method that to obtain of protein one class biomacromolecule, utilizes the difference of solvent and solute dissolved click and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: will put into the Bordetella pertussis capsular antigen solution that regulates concentration on nitrocellulose filter, standing 30min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, takes out, and low temperature saves backup.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) prepare nm of gold system: chloroazotic acid soaking container, through ultrapure water, rinse again, dry, adding mass concentration is 1% gold chloride, with ultrapure water, dissolves, ratio between described gold chloride and ultrapure water is: 98.8:4.05, agitating heating is boiled, and adding mass concentration is 1% trisodium citrate, continues to be heated with stirring to form claret solution, stir coolingly, obtain nano-Au solution; Described nano Au particle particle diameter is 18nm;
(2) prepare the detector probe of nano gold mark mouse-anti people IgA antibody: above-mentioned nano-Au solution is regulated to its pH to 8.5 with solution of potassium carbonate, by mouse-anti people IgA antibody dilution to 0.75mg/ml, 12000rpm, centrifugal 40min under 4 ℃ of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, fully mix combination, add and the isopyknic confining liquid of aforementioned mixed liquor, room temperature continues to hatch 28min, 14000rpm, centrifugal 25min under 4 ℃ of conditions, remove supernatant, the resuspended precipitation of dilution washing are once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: nitrocellulose filter and adsorptive pads three parts that concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form; Plastics capsule can be various shape, and there is the square hole of the about 0.8cm of the length of side in the central authorities of lid, and the position square groove corresponding with square hole on lid arranged at box bottom, big or small equally large with the square hole on lid; Adsorptive pads is positioned over the groove of box, and nitrocellulose filter is placed between adsorptive pads and lid, closely closes lid, makes nitrocellulose filter be adjacent to adsorptive pads, and described water accepting layer is stacked together and is formed by 5 layers of adsorptive pads.Because the relative nitrocellulose filter of area of water accepting layer is less, can make the antibody-antigen-Jin labeling antibody compound reacting concentrate to middle part gathering, and play inspissation, further strengthen color developing effect.
Detect test:
1. test specimen: sample to be measured (sample adopts nasopharynx examination or the sampling of cough plate method), positive, negative sample.
2. detection method: the testing sample of collecting is done to 1:10 dilution with PBS, with pipettor, 150 these dilute samples of μ l are added drop-wise in the kit of the embodiment of the present invention 1 preparation, after diafiltration 30s, continue to drip 150 μ lPBS damping fluids and rinse, diafiltration 30s; The 75 μ l gold that instil are marked mouse-anti people IgA antibody test probes, and diafiltration 30s, drips 150 μ lPBS damping fluids and rinse; The colour developing of observation spot.Positive and negative sample contrast parallel detection are as quality control.
3. testing result: the testing result of 30 positive and 30 routine normal specimens is shown: spot gold of the present invention immunity concentrates the sensitivity of percolation to be respectively 96%; Specificity is 100%.
Finally it should be noted that: above embodiment only, in order to technical scheme of the present invention to be described, is not intended to limit; Although the present invention is had been described in detail with reference to previous embodiment, those of ordinary skill in the art is to be understood that: its technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of embodiment of the present invention technical scheme.
Claims (7)
1. a pertussis diagnostic kit, percolating device is concentrated in the detector probe, Bordetella pertussis capsular antigen, the immunity of spot gold that it is characterized in that comprising nano gold mark mouse-anti people IgA antibody, and nitrocellulose filter and adsorptive pads three parts that described concentrated percolating device has added Bordetella pertussis capsular antigen by plastics capsule, point form.
2. a kind of pertussis diagnostic kit as claimed in claim 1, is characterized in that: described is 0.92-1.86 μ g/mL for soaking the concentration of the nano gold mark mouse-anti people IgA antibody of detector probe.
3. a kind of pertussis diagnostic kit as claimed in claim 1, is characterized in that: described Bordetella pertussis capsular antigen concentration is 0.85～1.65 μ g/mL.
4. a kind of pertussis diagnostic kit as described in claim 1～3 any one, is characterized in that: the nitrocellulose filter that described point has added Bordetella pertussis capsular antigen is adopted with the following method and prepared:
(1) bacterium is separated cultivates: with potato blood glycerin agar medium culture Bordetella pertussis, 5-7, after day, stops cultivating separated bacterium colony;
(2) lysozyme is processed bacterial suspension: in the suspension of bacterial cell, add lysozyme, 27-35 ℃, insulation 15min, broken bacterial cell;
(3) antigen extracts: with hydrogen, as solvent, the phosphate of 0.03-0.05mol/L, as damping fluid, extracts, and temperature is controlled at below 5 ℃, and pH is controlled at 5.0-7.0;
(4) antigen isolation and purification: carry out separation by ion exchange process, wherein, described exchanger is ion-exchange gel, preferably DEAE-sephadex or CM-sephadex;
(5) antigen is concentrated: adopt freezing method to concentrate, the antigen of first being prepared by step (4) is cooled to solid, and then slowly dissolve, the pure ice crystal containing antigen does not float on liquid level, antigen concentrates in lower floor's solution, removes upper strata ice cube, again with dilution, regulates concentration to 0.85～1.65 μ g/mL, obtain antigen concentrate ,-20～-4 ℃ of preservations;
(6) antigen coated nitrocellulose filter: will put into the Bordetella pertussis capsular antigen solution that regulates concentration on nitrocellulose filter, standing 20-30min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, takes out, and low temperature saves backup.
5. a kind of pertussis diagnostic kit as described in claim 1～3 any one, is characterized in that: the preparation method of the detector probe of described nano gold mark mouse-anti people IgA antibody comprises the steps:
(1) prepare nm of gold system: chloroazotic acid soaking container, through ultrapure water, rinse again, dry, adding mass concentration is 1% gold chloride, with ultrapure water, dissolves, ratio between described gold chloride and ultrapure water is: (97.5-98.8): (3.85-4.05), agitating heating is boiled, and adding mass concentration is 1% trisodium citrate, continues to be heated with stirring to form claret solution, stir coolingly, obtain nano-Au solution, (2) prepare the detector probe of nano gold mark mouse-anti people IgA antibody: above-mentioned nano-Au solution is regulated to its pH to 7.6-8.5 with solution of potassium carbonate, by mouse-anti people IgA antibody dilution to 0.3-0.75mg/ml, 12000rpm, centrifugal 40min under 4 ℃ of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, fully mix combination, add and the isopyknic confining liquid of aforementioned mixed liquor, room temperature continues to hatch 20～28min, 14000rpm, centrifugal 15～25min under 4 ℃ of conditions, remove supernatant, the resuspended precipitation of dilution washing are once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
6. a kind of pertussis diagnostic kit as claimed in claim 1, it is characterized in that described plastics capsule can be various shape, there is the approximately square hole of 0.5～0.8cm of the length of side in the central authorities of lid, the one position square groove corresponding with square hole on lid arranged at box bottom, big or small equally large with the square hole on lid; Adsorptive pads is positioned over the groove of box, and nitrocellulose filter is placed between adsorptive pads and lid.
7. a kind of pertussis diagnostic kit as claimed in claim 6, is characterized in that described water accepting layer is stacked together and is formed by 3-5 layer adsorptive pads.
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