CN104198704B - A kind of pertussis diagnostic kit and preparation method - Google Patents
A kind of pertussis diagnostic kit and preparation method Download PDFInfo
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- CN104198704B CN104198704B CN201410443710.1A CN201410443710A CN104198704B CN 104198704 B CN104198704 B CN 104198704B CN 201410443710 A CN201410443710 A CN 201410443710A CN 104198704 B CN104198704 B CN 104198704B
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Abstract
The invention discloses a kind of pertussis diagnostic kit and preparation method, this kit comprises detector probe, Bordetella pertussis capsular antigen, the concentrated percolating device of spot gold immunity of nano gold mark mouse-anti people IgA antibody.Described concentrated percolating device has added the nitrocellulose filter of the detector probe of nano gold mark mouse-anti people IgA antibody by plastic caddy, point and water accepting layer three part forms, adsorptive pads is positioned over the groove of box, nitrocellulose filter is placed between adsorptive pads and lid, and water accepting layer is stacked together by 3-5 layer adsorptive pads and forms.Kit of the present invention is on the basis of nm of gold amplifying technique, Immuno gold spot antigen-antibody reaction produced by concentrated percolating device is again concentrated and is amplified, improve detection sensitivity, to expand detectability, carry out effect detection from ill latent period, spasm period until convalescence all can have.
Description
Technical field
The present invention relates to technical field of medical examination, particularly relate to a kind of pertussis diagnostic kit and preparation method.
Background technology
Pertussis is a kind of Acute respiratory infectious disease 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 even about 3 months several weeks, therefore claims pertussis.Pertussal cough has been found that and can cause subconjunctival hemorrhage, fracture of rib, the retention of urine, hernia, faint after cough, and vertebral artery interlayer, violent cough can cause pleura to break, cause pneumothorax, especially concerning children's, harm is very serious, the length of stage of spasmodic cough and the sooner or later relevant for the treatment of, the treated as soon as possible of diagnosis ability as early as possible, so seem particularly important to pertussal quick diagnosis.
At present, medically have multiple method to pertussal inspection, comprise white blood cell count(WBC) method, cell culture method, fluoresent antibody staining, Serological testing etc., cell culture method, by being separated bacterium colony, observes colonial morphology, can only as tentative diagnosis; Fluoresent antibody staining uses Nasopharyngeal swabs smear, adds fluorescently-labeled antiserum, checks under fluorescent microscope, and early stage patient 75 ~ 80% is positive, and advantage is can quick diagnosis, but shortcoming is poor specificity, has false positive, just cultivates as auxiliary clinically; Serological testing does paired sera agglutination test and mends binding tests, can make a definite diagnosis if antibody titer is risen progressively, and recent useful enzyme linked immunosorbent assay surveys IgM, IgG and IgA antibody, helpful to early diagnosis; In addition, the detection method of Bordetella pertussis or its constituent is still in laboratory or clinical observation stage as spot hybridization, PCR method, the form of histocyte cultivation, the vigor etc. of enzyme, is not also generally applied.
Bordetella pertussis tentative diagnosis is to be separated cultivation, be separated positive rate latent period (catarrhal period) and can 91.5% be reached, and convalescence only has an appointment 26%, make a definite diagnosis is make serum slide agglutination or immunofluorescence dyeing with isolate and I phase immune serum, so primary limitation detected in I phase phase, detectability is shorter.So, be badly in need of working out a kind of not only fast but also the longer Bordetella pertussis detection method of detectability.
Summary of the invention
For above-mentioned present situation, the technical matters that the present invention solves just is to provide a kind of detection method that can detect Bordetella pertussis fast, 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, comprise the detector probe of nano gold mark mouse-anti people IgA antibody, Bordetella pertussis capsular antigen, the immunity of spot gold concentrate percolating device, described concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms.
Further, the described concentration for the nano gold mark mouse-anti people IgA antibody of soaking detector probe is 0.92-1.86 μ g/mL.
Further, described Bordetella pertussis capsular antigen concentration is 0.85 ~ 1.65 μ g/mL.
Wherein, described Bordetella pertussis capsular antigen is adopted and is prepared with the following method:
(1) bacteria distribution is cultivated: cultivate Bordetella pertussis with potato blood triglyceride agar medium, after 5-7 day, stops cultivating, is separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacterium, when first separation is cultivated, nutritional requirement is higher, so potato blood triglyceride agar medium (i.e. Bao ~ Jin Shi nutrient culture media) need be used to grow, after within 2 ~ 3 days, cultivating through 37 DEG C, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture in evenly muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme process bacterial suspension: add 100 μ g to 1mg lysozymes, 27-35 DEG C in every milliliter of suspension containing 200,000,000 bacterial cells, insulation 15min, disrupt bacteria cell;
(3) Antigen extraction: with hydrogen as solvent, the phosphate of 0.03-0.05mol/L, as damping fluid, extracts, and temperature controls below 5 DEG C, and pH controls within the scope of 5.0-7.0;
(4) antigen isolation and purification: be separated by ion exchange process, wherein, described exchanger is ion-exchange gel, preferred DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen concentrates: adopt freezing method to concentrate, first antigen prepared by step (4) is cooled to solid, and then slowly dissolve, pure ice crystal not containing antigen floats on liquid level, antigen then concentrates in lower floor's solution, removes upper strata ice cube, again regulates concentration to 0.85 ~ 1.65 μ g/mL with dilution, obtain antigen concentrate ,-20 ~-4 DEG C of preservations; Freezing method is that protein one class biomacromolecule is so concentrated that to compare effective method, utilizes the difference of solvent and solute dissolves point and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: nitrocellulose filter will be put into the Bordetella pertussis capsular antigen solution regulating concentration, leave standstill 20-30min, treat that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, take out, Cord blood is for subsequent use.
Wherein, the preparation method of the detector probe of described nano gold mark mouse-anti people IgA antibody comprises the steps:
(1) nm of gold system is prepared: chloroazotic acid soaking container, again through ultrapure water, dry, add the gold chloride that mass concentration is 1%, dissolve with ultrapure water, ratio between described gold chloride and ultrapure water is: (97.5-98.8): (3.85-4.05), agitating heating is boiled, and adds the trisodium citrate that mass concentration is 1%, continues to be heated with stirring to form claret solution, stir cooling, obtain nano-Au solution; Described nano Au particle particle diameter is 14-18nm;
(2) detector probe of nano gold mark mouse-anti people IgA antibody is prepared: above-mentioned nano-Au solution solution of potassium carbonate is regulated its pH to 7.6-8.5, mouse-anti people IgA antibody is diluted to 0.3-0.75mg/ml, 12000rpm, centrifugal 40min under 4 DEG C of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, abundant mixing combines, add confining liquid isopyknic with aforementioned mixed liquor, room temperature continues to hatch 20 ~ 28min, 14000rpm, centrifugal 15 ~ 25min under 4 DEG C of conditions, remove supernatant, the resuspended precipitation of dilution is also washed once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Wherein, described plastic caddy can be various shape, and there are the square hole of the length of side about 0.5 ~ 0.8cm in the central authorities of lid, and have the square groove that a position is corresponding with the square hole on lid bottom box, size is 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 by 3-5 layer adsorptive pads and forms.Because the area of water accepting layer is less relative to nitrocellulose filter, the antibody-antigene-Jin labeling antibody compound reacted can be made to concentrate and to assemble to middle part, and play inspissation, strengthen color developing effect further.
Cleaning Principle of the present invention is: use the immunity of spot gold to concentrate percolation test, take nitrocellulose filter as carrier, and bag is by the percolating device of antigen, drip sample to be measured successively, Immuno gold and cleansing solution, because nitrocellulose filter adheres on absorbent material, therefore the antigen of sample to be measured when flowing through percolating device on film is combined fast, be combined with golden labeling antibody again, form antibody-antigene-Jin labeling antibody compound, the area of water accepting layer is less relative to nitrocellulose filter, antibody-antigene-Jin labeling antibody the compound reacted can be made to concentrate assemble to middle part, and play inspissation, further enhancing color developing effect, reach quick testing goal (general about 5min 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, carries out effect detection, be very applicable to clinical practice from ill latent period, spasm period until convalescence all can have.
Embodiment
The details of various aspects of the present invention is able to detailed description by chapters and sections subsequently.By hereafter 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, comprise the detector probe of nano gold mark mouse-anti people IgA antibody, Bordetella pertussis capsular antigen, the immunity of spot gold concentrate percolating device, described concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms; The described concentration for the nano gold mark mouse-anti people IgA antibody of soaking detector probe is 0.92 μ g/mL; Described Bordetella pertussis capsular antigen concentration is 0.85 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacteria distribution is cultivated: cultivate Bordetella pertussis with potato blood triglyceride agar medium, after 5 days, stops cultivating, is separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacterium, when first separation is cultivated, nutritional requirement is higher, so potato blood triglyceride agar medium (i.e. Bao ~ Jin Shi nutrient culture media) need be used to grow, after cultivating through 37 DEG C, 2 days, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture in evenly muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme process bacterial suspension: add 100 μ g to 1mg lysozymes, 27 DEG C in every milliliter of suspension containing 200,000,000 bacterial cells, insulation 15min, disrupt bacteria cell;
(3) Antigen extraction: with hydrogen as solvent, the phosphate of 0.03mol/L, as damping fluid, extracts, and temperature controls below 5 DEG C, and pH is 5.0;
(4) antigen isolation and purification: be separated by ion exchange process, wherein, described exchanger is ion-exchange gel, preferred DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen concentrates: adopt freezing method to concentrate, first antigen prepared by step (4) is cooled to solid, and then slowly dissolve, pure ice crystal not containing antigen floats on liquid level, antigen then concentrates in lower floor's solution, removes upper strata ice cube, again regulates concentration to 0.85 μ g/mL with dilution, obtain antigen concentrate ,-20 DEG C of preservations; Freezing method is that protein one class biomacromolecule is so concentrated that to compare effective method, utilizes the difference of solvent and solute dissolves point and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: nitrocellulose filter will be put into the Bordetella pertussis capsular antigen solution regulating concentration, leaves standstill 20min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, and take out, Cord blood is for subsequent use.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) nm of gold system is prepared: chloroazotic acid soaking container, again through ultrapure water, dry, add the gold chloride that mass concentration is 1%, dissolve with ultrapure water, ratio between described gold chloride and ultrapure water is: 97.5:3.85, agitating heating is boiled, and adds the trisodium citrate that mass concentration is 1%, continues to be heated with stirring to form claret solution, stir cooling, obtain nano-Au solution; Described nano Au particle particle diameter is 14nm;
(2) detector probe of nano gold mark mouse-anti people IgA antibody is prepared: above-mentioned nano-Au solution solution of potassium carbonate is regulated its pH to 7.6, mouse-anti people IgA antibody is diluted to 0.3mg/ml, 12000rpm, centrifugal 40min under 4 DEG C of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, abundant mixing combines, add confining liquid isopyknic with aforementioned mixed liquor, room temperature continues to hatch 20min, 14000rpm, centrifugal 15min under 4 DEG C of conditions, remove supernatant, the resuspended precipitation of dilution is also washed once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms; Plastic caddy can be various shape, and the central authorities of lid have the length of side to be about the square hole of 0.5cm, and have the square groove that a position is corresponding with the square hole on lid bottom box, size is 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 by 3 layers of adsorptive pads and forms.Because the area of water accepting layer is less relative to nitrocellulose filter, the antibody-antigene-Jin labeling antibody compound reacted can be made to concentrate and to assemble to middle part, and play inspissation, strengthen color developing effect further.
Embodiment 2:
A kind of pertussis diagnostic kit, comprise the detector probe of nano gold mark mouse-anti people IgA antibody, Bordetella pertussis capsular antigen, the immunity of spot gold concentrate percolating device, described concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms; The described concentration for the nano gold mark mouse-anti people IgA antibody of soaking detector probe is 1.39 μ g/mL; Described Bordetella pertussis capsular antigen concentration is 1.25 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacteria distribution is cultivated: cultivate Bordetella pertussis with potato blood triglyceride agar medium, after 6 days, stops cultivating, is separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacterium, when first separation is cultivated, nutritional requirement is higher, so potato blood triglyceride agar medium (i.e. Bao ~ Jin Shi nutrient culture media) need be used to grow, after cultivating through 37 DEG C, 2 days, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture in evenly muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme process bacterial suspension: add 100 μ g to 1mg lysozymes, 31 DEG C in every milliliter of suspension containing 200,000,000 bacterial cells, insulation 15min, disrupt bacteria cell;
(3) Antigen extraction: with hydrogen as solvent, the phosphate of 0.05mol/L, as damping fluid, extracts, and temperature controls below 5 DEG C, it is 6.0 that pH controls;
(4) antigen isolation and purification: be separated by ion exchange process, wherein, described exchanger is ion-exchange gel, preferred DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen concentrates: adopt freezing method to concentrate, first antigen prepared by step (4) is cooled to solid, and then slowly dissolve, pure ice crystal not containing antigen floats on liquid level, antigen then concentrates in lower floor's solution, removes upper strata ice cube, again regulates concentration to 1.25 μ g/mL with dilution, obtain antigen concentrate ,-12 DEG C of preservations; Freezing method is that protein one class biomacromolecule is so concentrated that to compare effective method, utilizes the difference of solvent and solute dissolves point and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: nitrocellulose filter will be put into the Bordetella pertussis capsular antigen solution regulating concentration, leaves standstill 25min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, and take out, Cord blood is for subsequent use.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) nm of gold system is prepared: chloroazotic acid soaking container, again through ultrapure water, dry, add the gold chloride that mass concentration is 1%, dissolve with ultrapure water, ratio between described gold chloride and ultrapure water is: 98.15:3.95, agitating heating is boiled, and adds the trisodium citrate that mass concentration is 1%, continues to be heated with stirring to form claret solution, stir cooling, obtain nano-Au solution; Described nano Au particle particle diameter is 16nm;
(2) detector probe of nano gold mark mouse-anti people IgA antibody is prepared: above-mentioned nano-Au solution solution of potassium carbonate is regulated its pH to 8.05, mouse-anti people IgA antibody is diluted to 0.52mg/ml, 12000rpm, centrifugal 40min under 4 DEG C of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, abundant mixing combines, add confining liquid isopyknic with aforementioned mixed liquor, room temperature continues to hatch 24min, 14000rpm, centrifugal 20min under 4 DEG C of conditions, remove supernatant, the resuspended precipitation of dilution is also washed once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis pod membrane (K) antigen by plastic caddy, point and adsorptive pads three part forms; Plastic caddy can be various shape, and the central authorities of lid have the length of side to be about the square hole of 0.65cm, and have the square groove that a position is corresponding with the square hole on lid bottom box, size is 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 by 4 layers of adsorptive pads and forms.Because the area of water accepting layer is less relative to nitrocellulose filter, the antibody-antigene-Jin labeling antibody compound reacted can be made to concentrate and to assemble to middle part, and play inspissation, strengthen color developing effect further.
Embodiment 3:
A kind of pertussis diagnostic kit, comprise the detector probe of nano gold mark mouse-anti people IgA antibody, Bordetella pertussis capsular antigen, the immunity of spot gold concentrate percolating device, described concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms; The described concentration for the nano gold mark mouse-anti people IgA antibody of soaking detector probe is 1.86 μ g/mL; Described Bordetella pertussis capsular antigen concentration is 1.65 μ g/mL.
The preparation of described Bordetella pertussis capsular antigen:
(1) bacteria distribution is cultivated: cultivate Bordetella pertussis with potato blood triglyceride agar medium, after 5-7 day, stops cultivating, is separated bacterium colony; Because Bordetella pertussis is obligate aerobic bacterium, when first separation is cultivated, nutritional requirement is higher, so potato blood triglyceride agar medium (i.e. Bao ~ Jin Shi nutrient culture media) need be used to grow, after cultivating through 37 DEG C, 3 days, visible tiny, circular, smooth, protruding, silver gray, opaque bacterium colony, have fuzzy zone of hemolysis around, Liquid Culture in evenly muddy growth, and has a small amount of viscous precipitate;
(2) lysozyme process bacterial suspension: add 100 μ g to 1mg lysozymes, 35 DEG C in every milliliter of suspension containing 200,000,000 bacterial cells, insulation 15min, disrupt bacteria cell;
(3) Antigen extraction: with hydrogen as solvent, the phosphate of 0.05mol/L, as damping fluid, extracts, and temperature controls below 5 DEG C, it is 7.0 that pH controls;
(4) antigen isolation and purification: be separated by ion exchange process, wherein, described exchanger is ion-exchange gel, preferred DEAE-sephadex or CM-sephadex; Ion-exchange gel not only exchanging equivalent is high, and chromatography condition is gentle, simple to operate;
(5) antigen concentrates: adopt freezing method to concentrate, first antigen prepared by step (4) is cooled to solid, and then slowly dissolve, pure ice crystal not containing antigen floats on liquid level, antigen then concentrates in lower floor's solution, removes upper strata ice cube, again regulates concentration to 1.65 μ g/mL with dilution, obtain antigen concentrate ,-4 DEG C of preservations; Freezing method is that protein one class biomacromolecule is so concentrated that to compare effective method, utilizes the difference of solvent and solute dissolves point and reaches away the object of most of solvent;
(6) antigen coated nitrocellulose filter: nitrocellulose filter will be put into the Bordetella pertussis capsular antigen solution regulating concentration, leaves standstill 30min, treats that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, and take out, Cord blood is for subsequent use.
The preparation of the detector probe of described nano gold mark mouse-anti people IgA antibody:
(1) nm of gold system is prepared: chloroazotic acid soaking container, again through ultrapure water, dry, add the gold chloride that mass concentration is 1%, dissolve with ultrapure water, ratio between described gold chloride and ultrapure water is: 98.8:4.05, agitating heating is boiled, and adds the trisodium citrate that mass concentration is 1%, continues to be heated with stirring to form claret solution, stir cooling, obtain nano-Au solution; Described nano Au particle particle diameter is 18nm;
(2) detector probe of nano gold mark mouse-anti people IgA antibody is prepared: above-mentioned nano-Au solution solution of potassium carbonate is regulated its pH to 8.5, mouse-anti people IgA antibody is diluted to 0.75mg/ml, 12000rpm, centrifugal 40min under 4 DEG C of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, abundant mixing combines, add confining liquid isopyknic with aforementioned mixed liquor, room temperature continues to hatch 28min, 14000rpm, centrifugal 25min under 4 DEG C of conditions, remove supernatant, the resuspended precipitation of dilution is also washed once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
Described concentrated percolating device preparation: concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms; Plastic caddy can be various shape, and the central authorities of lid have the length of side to be about the square hole of 0.8cm, and have the square groove that a position is corresponding with the square hole on lid bottom box, size is 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 by 5 layers of adsorptive pads and forms.Because the area of water accepting layer is less relative to nitrocellulose filter, the antibody-antigene-Jin labeling antibody compound reacted can be made to concentrate and to assemble to middle part, and play inspissation, strengthen color developing effect further.
Detection experiment:
1. test specimen: sample to be measured (sample adopts nasopharynx swab or the sampling of cough plate method), positive, negative sample.
2. 150 these dilute samples of μ l to be added drop-wise in kit prepared by the embodiment of the present invention 1 with pipettor, after diafiltration 30s, to continue dropping 150 μ lPBS wash buffer, diafiltration 30s by detection method: the testing sample PBS collected is done 1:10 dilution; Instil 75 μ l gold mark mouse-anti people IgA antibody detector probe, and diafiltration 30s, drips 150 μ lPBS wash buffer; Observation spot development.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%.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in previous embodiment, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments 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 (2)
1. a pertussis diagnostic kit, it is characterized in that comprising the detector probe of nano gold mark mouse-anti people IgA antibody, Bordetella pertussis capsular antigen, the immunity of spot gold concentrate percolating device, described concentrated percolating device has added the nitrocellulose filter of Bordetella pertussis capsular antigen by plastic caddy, point and adsorptive pads three part forms;
The concentration of the detector probe of described nano gold mark mouse-anti people IgA antibody is 0.92-1.86 μ g/mL;
Described Bordetella pertussis capsular antigen concentration is 0.85 ~ 1.65 μ g/mL;
The nitrocellulose filter that described point has added Bordetella pertussis capsular antigen is adopted and is prepared with the following method:
(1) bacteria distribution is cultivated: cultivate Bordetella pertussis with potato blood triglyceride agar medium, after 5-7 day, stops cultivating, is separated bacterium colony;
(2) lysozyme process bacterial suspension: add lysozyme in the suspension of bacterial cell, 27-35 DEG C, insulation 15min, disrupt bacteria cell;
(3) Antigen extraction: with hydrogen as solvent, the phosphate of 0.03-0.05mol/L, as damping fluid, extracts, and temperature controls below 5 DEG C, and pH controls at 5.0-7.0;
(4) antigen isolation and purification: be separated by ion exchange process, wherein, described exchanger is DEAE-sephadex or CM-sephadex;
(5) antigen concentrates: adopt freezing method to concentrate, first antigen prepared by step (4) is cooled to solid, and then slowly dissolve, pure ice crystal not containing antigen floats on liquid level, antigen then concentrates in lower floor's solution, removes upper strata ice cube, again regulates concentration to 0.85 ~ 1.65 μ g/mL with dilution, obtain antigen concentrate ,-20 ~-4 DEG C of preservations;
(6) antigen coated nitrocellulose filter: nitrocellulose filter will be put into the Bordetella pertussis capsular antigen solution regulating concentration, leave standstill 20-30min, treat that nitrocellulose filter fully adsorbs Bordetella pertussis capsular antigen, take out, Cord blood is for subsequent use.
2. a kind of pertussis diagnostic kit as claimed in claim 1, 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) nm of gold system is prepared: chloroazotic acid soaking container, again through ultrapure water, dry, add the gold chloride that mass concentration is l%, dissolve with ultrapure water, described gold chloride with between ultrapure water according to (97.5-98.8): the ratio of (3.85-4.05) mixes, agitating heating is boiled, and adds the trisodium citrate that mass concentration is 1%, continues to be heated with stirring to form claret solution, stir cooling, obtain nano-Au solution, (2) detector probe of nano gold mark mouse-anti people IgA antibody is prepared: above-mentioned nano-Au solution solution of potassium carbonate is regulated its pH to 7.6-8.5, mouse-anti people IgA antibody is diluted to 0.3-0.75mg/ml, 12000rpm, centrifugal 40min under 4 DEG C of conditions, obtain supernatant, be added drop-wise to rapidly in nano-Au solution, abundant mixing combines, add confining liquid isopyknic with aforementioned mixed liquor, room temperature continues to hatch 20 ~ 28min, 14000rpm, centrifugal 15 ~ 25min under 4 DEG C of conditions, remove supernatant, the resuspended precipitation of dilution is also washed once, obtain the detector probe of nano gold mark mouse-anti people IgA antibody.
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| CN201410443710.1A CN104198704B (en) | 2014-09-02 | 2014-09-02 | A kind of pertussis diagnostic kit and preparation method |
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| CN104745689A (en) * | 2015-01-30 | 2015-07-01 | 湖北永邦医疗科技有限公司 | Primers, probe and kit used for detecting bordetella pertussis |
| CN105067809A (en) * | 2015-07-27 | 2015-11-18 | 青岛汉唐生物科技有限公司 | ELISA percolation method for rapidly detecting pathogen antibody, kit for detection, and preparation method of kit |
| CN105548541B (en) * | 2016-01-11 | 2017-11-07 | 王好玉 | A kind of avian influenza virus detection kit |
| CN105785042A (en) * | 2016-04-06 | 2016-07-20 | 陈秀海 | Detection kit for epidemic hemorrhagic fever virus |
| CN108303550A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of the spot gold diafiltration kit and its quantitative detecting method of detection adiponectin |
| CN109239340B (en) * | 2018-10-24 | 2024-06-25 | 合肥职业技术学院 | Gel immunodiffusion tester |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
| CN202854142U (en) * | 2012-11-09 | 2013-04-03 | 宁波天润生物药业有限公司 | Pertussis IgG antibody test card |
| CN103439493A (en) * | 2013-08-08 | 2013-12-11 | 南京大渊生物技术工程有限责任公司 | Aptamer percolated biochip and preparation method thereof |
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| JP5805943B2 (en) * | 2010-12-08 | 2015-11-10 | 株式会社北里大塚バイオメディカルアッセイ研究所 | Method for producing pertussis Fim3 and method for detecting pertussis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
| CN202854142U (en) * | 2012-11-09 | 2013-04-03 | 宁波天润生物药业有限公司 | Pertussis IgG antibody test card |
| CN103439493A (en) * | 2013-08-08 | 2013-12-11 | 南京大渊生物技术工程有限责任公司 | Aptamer percolated biochip and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 熊平源等.斑点金免疫渗滤实验.《医学免疫学实验教程》.2011, * |
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