CN104181298B - A kind of human parainfluenza virus's IgM antibody test strip and preparation method thereof - Google Patents
A kind of human parainfluenza virus's IgM antibody test strip and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of human parainfluenza virus (HPIVs) IgM antibody test strip and preparation method thereof, this test strips comprises PVC base plate, sample pad, adsorptive pads, be coated with the gold mark pad of HPIVs specificity recombinant antigen, the nitrocellulose filter (NC film) of the nature controlling line of the detection line being coated with mouse-anti people IgM polyclonal antibody and the polyclonal antibody being coated with anti-HPIVs recombinant antigen, immunochromatographic method is used to detect fast the human parainfluenza virus's specific antibody IgM in human serum, so that the antidiastole to human parainfluenza virus's acute HIV infection, Clinical laboratory medicine has certain meaning.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of human parainfluenza virus's IgM antibody test strip and preparation method thereof.
Background technology
Human parainfluenza virus (HPIVs) is the one virus causing children's lower respiratory infection, 4 types (I type is to IV type) can be divided into from HPIVs serology, the infection of the upper respiratory tract of recurrent exerbation (as caught a cold and having a sore throat) can be caused, it also can cause the lower respiratory illness of serious repeated infection (as pneumonia, bronchitis and capillary bronchitis), particularly in the elderly and have in immunodeficiency crowd.
The method diagnosing HPIVs to infect clinically at present has: first pass through tissue cultures, be separated the virus of qualification in cell or direct-detection again and be present in virus in respiratory secretions, comprising methods such as use immunofluorescent test, PCR, enzyme linked immunoassay mensuration, another method detects specific antibody IgM in single serum specimen exactly.
After patient infection HPIVs pathogen, clinical symptoms occurs latter about one week, specific antibody IgM is produced in serum, 10-30 days arrival peaks, 12-26 week disappears, when patient occurs that symptom is gone medical again, IgM antibody has reached higher level, and therefore the IgM antibody positive can be used as the diagnosis index of acute HIV infection.A kind of detection method more advanced at present detects by immunofluorescence technique, but this detection method can only detect corresponding a kind of material at every turn, and susceptibility is poor, and effect is sometimes undesirable.
So check the method for human parainfluenza virus's IgM antibody to have certain Research Significance clinically quickly and efficiently.
Summary of the invention
The technical matters that the present invention solves just is to provide one and checks test strip of human parainfluenza virus in human serum (HPIVs) IgM antibody and preparation method thereof quickly and efficiently.
Technical scheme of the present invention is: a kind of human parainfluenza virus's IgM antibody test strip, and this test strips comprises: the glass fibre membrane of the HPIVs specificity recombinant antigen containing colloid gold label, be coated with the detection line of two anti-IgM and be coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen.
Further, two described anti-IgM are mouse-anti people IgM polyclonal antibody.
Further, the concentration of described HPIVs specificity recombinant antigen is 1mg/mL, and the concentration of described two anti-IgM is 2mg/mL, and the concentration of the polyclonal antibody of described anti-HPIVs recombinant antigen is 1mg/mL.
The preparation method of described human parainfluenza virus's IgM antibody test strip, comprises the steps:
1) the HPIVs specificity recombinant antigen glass fibre membrane of colloid gold label is prepared:
(1) preparation of colloidal gold solution: (volume ratio is the dense HCL of 3:1 and dense HNO with chloroazotic acid
3) soaking container certain hour 20-30min, then clean up with ultrapure water, dry; In container, add 2% chlorauric acid solution of 99.14mL pure water and 0.86mL, heat while stirring, continue to boil 8min after being heated to boiling, add 2.5mL1% trisodium citrate aqueous solution, continue agitating heating 10min, add pure water after cooling to 100mL, obtain claret colloidal gold solution; Wherein, colloid gold particle mean diameter is at 15-25nm;
(2) preparation of gold mark HPIVs specificity recombinant antigen:
A. colloidal gold solution pH is regulated: the colloidal gold solution getting above-mentioned preparation, with the K of 0.1M
2cO
3solution regulates pH to 8.5-9.0;
B. HPIVs specificity recombinant antigen is diluted: with phosphate buffer (PB), HPIVs specificity recombinant antigen being diluted to protein content is 0.2-0.8mg/mL; Wherein, described phosphate buffer is: get KCL, Na
2hPO
4, KH
2pO
41.8g altogether, dissolve with 0.8L distilled water, then use salt acid for adjusting pH, adding water to cumulative volume is that 1L makes;
C. the coupling of collaurum and recombinant antigen: get the dilution 0.2-1.0mL in step b, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, draws out 250 μ L supernatants, discards precipitation, dropwise being joined in step a by supernatant while stirring regulates in the colloidal gold solution of pH, leaves standstill 10min; Add stabilizing agent 10%BSA solution 1mL, make final concentration be 1%, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, leave standstill 10min, abandon supernatant, stay sediment, dissolve with sustained release agent, the centrifugal 10min of 500r/min under 4 DEG C of conditions again, remove aggregation, stay supernatant, again by supernatant centrifugal 30min of 2500r/min under 4 DEG C of conditions, resuspended bottom loose deposits, namely obtains the HPIVs specificity recombinant antigen of colloid gold label;
(3) with three-dimensional planar point film gold spraying instrument instrument HM3055, the HPIVs specificity recombinant antigen dissolution homogeneity having marked collaurum is applied on the plain film of the glass fibre handled well, freeze drying, for subsequent use;
2) preparation is coated with the detection line of two anti-IgM and is coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen:
Two anti-IgM are diluted to 2mg/mL, the polyclonal antibody of anti-HPIVs recombinant antigen is diluted to 1mg/mL; With three-dimensional planar point film gold spraying instrument instrument HM3055, diluted two anti-IgM are evenly sprayed on nitrocellulose filter, obtain detection line; Evenly be sprayed on nitrocellulose filter by the polyclonal antibody of the anti-HPIVs recombinant antigen diluted, obtain nature controlling line, room temperature bag is spent the night, and envelope is for subsequent use;
3) test strips is prepared:
Sample pad is soaked 40min in confining liquid, dries, encapsulate for subsequent use; By PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads combination, on PVC base plate, note thieving paper is glued in the top of nitrocellulose filter, nitrocellulose filter and the mutual overlapping 0.2mm of thieving paper, sticking glass tunica fibrosa and sample pad successively in the below of nitrocellulose filter, mutual overlapping 0.2mm between nitrocellulose filter and glass fibre membrane and between glass fibre membrane and sample pad, obtains product human parainfluenza virus IgM antibody test strip of the present invention.
The principle of work of test strips of the present invention is: adopt colloidal gold immunochromatographimethod technology, when containing HPIVsIgM antibody in measuring samples, the HPIVs specificity recombinant antigen combination of HPIVsIgM antibody colloid gold label first and on glass fibre element film, because chromatography effect compound moves forward along coated film, when running into the mouse-anti people IgM polyclonal antibody on detection line, form antibody-gold indicated weight group antigen-antibody complex, enrichment on detection line, forms red precipitate line.
The present invention can also reach half-quantitative detection object: when prepared by test paper by regulating detection line and nature controlling line encrusting substance concentration, the colour developing depth of detection line and nature controlling line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration.
Beneficial effect of the present invention is embodied in: because the IgM antibody positive can be used as the diagnosis index of acute HIV infection, so with the anti-HPIVsIgM antibody of colloid gold label, by whether there is HPIVsIgM antibody in immunochromatographyassay assay human serum, just fast direct ground connection can judge whether infected HPIVs pathogen.
Embodiment
Embodiment 1: a kind of human parainfluenza virus's IgM antibody test strip, this test strips comprises PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads, wherein said glass fibre membrane is the glass fibre membrane of the HPIVs specificity recombinant antigen containing colloid gold label, and described nitrocellulose filter detection layers is be coated with the detection line of two anti-IgM and be coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen; Described two anti-IgM are mouse-anti people IgM polyclonal antibody; The concentration of described HPIVs specificity recombinant antigen is 1mg/mL, and the concentration of described two anti-IgM is 2mg/mL, and the concentration of the polyclonal antibody of described anti-HPIVs recombinant antigen is 1mg/mL.
A kind of formation of human parainfluenza virus's IgM antibody test strip: PVC offset plate is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, Aureal Dongyuan County, adsorptive pads Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., HPIVs specificity recombinant antigen, polyclonal antibody equal purchased from American Aureal company of Dongyuan County (OriGene) of mouse-anti people IgM polyclonal antibody and anti-HPIVs recombinant antigen, the glass fibre membrane of the class parainfluenza virus specificity recombinant antigen containing colloid gold label is purchased from Thermo Fisher Scientific Inc..
A kind of preparation method of human parainfluenza virus's IgM antibody test strip is:
1) the HPIVs specificity recombinant antigen glass fibre membrane of colloid gold label is prepared:
(1) preparation of colloidal gold solution: (volume ratio is the dense HCL of 3:1 and dense HNO with chloroazotic acid
3) soaking container certain hour 20min, then clean up with ultrapure water, dry; In container, add 2% chlorauric acid solution of 99.14mL pure water and 0.86mL, heat while stirring, continue to boil 8min after being heated to boiling, add 2.5mL1% trisodium citrate aqueous solution, continue agitating heating 10min, add pure water after cooling to 100mL, obtain claret colloidal gold solution; Wherein, colloid gold particle mean diameter is at 15nm;
(2) preparation of gold mark HPIVs specificity recombinant antigen:
A. colloidal gold solution pH is regulated: the colloidal gold solution getting above-mentioned preparation, with the K of 0.1M
2cO
3solution regulates pH to 8.5;
B. HPIVs specificity recombinant antigen is diluted: with phosphate buffer (PB), HPIVs specificity recombinant antigen being diluted to protein content is 0.2mg/mL; Wherein, described phosphate buffer is: get KCL, Na
2hPO
4, KH
2pO
41.8g altogether, dissolve with 0.8L distilled water, then use salt acid for adjusting pH, adding water to cumulative volume is that 1L makes;
C. the coupling of collaurum and recombinant antigen: get the dilution 0.2mL in step b, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, draws out 250 μ L supernatants, discards precipitation, dropwise being joined in step a by supernatant while stirring regulates in the colloidal gold solution of pH, leaves standstill 10min; Add stabilizing agent 10%BSA solution 1mL, make final concentration be 1%, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, leave standstill 10min, abandon supernatant, stay sediment, dissolve with sustained release agent, the centrifugal 10min of 500r/min under 4 DEG C of conditions again, remove aggregation, stay supernatant, again by supernatant centrifugal 30min of 2500r/min under 4 DEG C of conditions, resuspended bottom loose deposits, namely obtains the HPIVs specificity recombinant antigen of colloid gold label;
(3) with three-dimensional planar point film gold spraying instrument instrument HM3055, be applied to by the HPIVs specificity recombinant antigen dissolution homogeneity having marked collaurum on the plain film of the glass fibre handled well, room temperature bag is spent the night, for subsequent use;
2) preparation is coated with the detection line of two anti-IgM and is coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen:
Two anti-IgM are diluted to 2mg/mL, the polyclonal antibody of anti-HPIVs recombinant antigen is diluted to 1mg/mL; With three-dimensional planar point film gold spraying instrument instrument HM3055, diluted two anti-IgM are evenly sprayed on nitrocellulose filter, obtain detection line; Evenly be sprayed on nitrocellulose filter by the polyclonal antibody of the anti-HPIVs recombinant antigen diluted, obtain nature controlling line, room temperature bag is spent the night, for subsequent use;
3) test strips is prepared:
Sample pad is soaked 40min in confining liquid, dries, encapsulate for subsequent use, described confining liquid be containing the pH of bovine serum albumin be 8.5 phosphate cleansing solution; By PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads combination, on PVC base plate, note thieving paper is glued in the top of nitrocellulose filter, nitrocellulose filter and the mutual overlapping 0.2mm of thieving paper, sticking glass tunica fibrosa and sample pad successively in the below of nitrocellulose filter, mutual overlapping 0.2mm between nitrocellulose filter and glass fibre membrane and between glass fibre membrane and sample pad, obtains product human parainfluenza virus IgM antibody test strip.
Embodiment 2: a kind of human parainfluenza virus's IgM antibody test strip, this test strips comprises PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads, wherein said glass fibre membrane is the glass fibre membrane of the HPIVs specificity recombinant antigen containing colloid gold label, and described nitrocellulose filter detection layers is be coated with the detection line of two anti-IgM and be coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen; Described two anti-IgM are mouse-anti people IgM polyclonal antibody; The concentration of described HPIVs specificity recombinant antigen is 1mg/mL, and the concentration of described two anti-IgM is 2mg/mL, and the concentration of the polyclonal antibody of described anti-HPIVs recombinant antigen is 1mg/mL.
The formation of human parainfluenza virus's IgM antibody test strip: PVC offset plate is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, Aureal Dongyuan County, adsorptive pads Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., HPIVs specificity recombinant antigen, polyclonal antibody equal purchased from American Aureal company of Dongyuan County (OriGene) of mouse-anti people IgM polyclonal antibody and anti-HPIVs recombinant antigen, the glass fibre membrane of the class parainfluenza virus specificity recombinant antigen containing colloid gold label is purchased from Thermo Fisher Scientific Inc..
A kind of preparation method of human parainfluenza virus's IgM antibody test strip is:
1) the HPIVs specificity recombinant antigen glass fibre membrane of colloid gold label is prepared:
(1) preparation of colloidal gold solution: (volume ratio is the dense HCL of 3:1 and dense HNO with chloroazotic acid
3) soaking container certain hour 25min, then clean up with ultrapure water, dry; In container, add 2% chlorauric acid solution of 99.14mL pure water and 0.86mL, heat while stirring, continue to boil 8min after being heated to boiling, add 2.5mL1% trisodium citrate aqueous solution, continue agitating heating 10min, add pure water after cooling to 100mL, obtain claret colloidal gold solution; Wherein, colloid gold particle mean diameter is at 20nm;
(2) preparation of gold mark HPIVs specificity recombinant antigen:
A. colloidal gold solution pH is regulated: the colloidal gold solution getting above-mentioned preparation, with the K of 0.1M
2cO
3solution regulates pH to 8.7;
B. HPIVs specificity recombinant antigen is diluted: with phosphate buffer (PB), HPIVs specificity recombinant antigen being diluted to protein content is 0.5mg/mL; Wherein, described phosphate buffer is: get KCL, Na
2hPO
4, KH
2pO
41.8g altogether, dissolve with 0.8L distilled water, then use salt acid for adjusting pH, adding water to cumulative volume is that 1L makes;
C. the coupling of collaurum and recombinant antigen: get the dilution 0.6mL in step b, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, draws out 250 μ L supernatants, discards precipitation, dropwise being joined in step a by supernatant while stirring regulates in the colloidal gold solution of pH, leaves standstill 10min; Add stabilizing agent 10%BSA solution 1mL, make final concentration be 1%, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, leave standstill 10min, abandon supernatant, stay sediment, dissolve with sustained release agent, the centrifugal 10min of 500r/min under 4 DEG C of conditions again, remove aggregation, stay supernatant, again by supernatant centrifugal 30min of 2500r/min under 4 DEG C of conditions, resuspended bottom loose deposits, namely obtains the HPIVs specificity recombinant antigen of colloid gold label;
(3) with three-dimensional planar point film gold spraying instrument instrument HM3055, be applied to by the HPIVs specificity recombinant antigen dissolution homogeneity having marked collaurum on the plain film of the glass fibre handled well, room temperature bag is spent the night, for subsequent use;
2) preparation is coated with the detection line of two anti-IgM and is coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen:
Two anti-IgM are diluted to 2mg/mL, the polyclonal antibody of anti-HPIVs recombinant antigen is diluted to 1mg/mL; With three-dimensional planar point film gold spraying instrument instrument HM3055, diluted two anti-IgM are evenly sprayed on nitrocellulose filter, obtain detection line; Evenly be sprayed on nitrocellulose filter by the polyclonal antibody of the anti-HPIVs recombinant antigen diluted, obtain nature controlling line, room temperature bag is spent the night, for subsequent use;
3) test strips is prepared:
Sample pad is soaked 40min in confining liquid, dries, encapsulate for subsequent use, described confining liquid be containing the pH of bovine serum albumin be 8.7 phosphate cleansing solution; By PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads combination, on PVC base plate, note thieving paper is glued in the top of nitrocellulose filter, nitrocellulose filter and the mutual overlapping 0.2mm of thieving paper, sticking glass tunica fibrosa and sample pad successively in the below of nitrocellulose filter, mutual overlapping 0.2mm between nitrocellulose filter and glass fibre membrane and between glass fibre membrane and sample pad, obtains product human parainfluenza virus IgM antibody test strip.
Embodiment 3: a kind of human parainfluenza virus's IgM antibody test strip, this test strips comprises PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads, wherein said glass fibre membrane is the glass fibre membrane of the HPIVs specificity recombinant antigen containing colloid gold label, and described nitrocellulose filter detection layers is be coated with the detection line of two anti-IgM and be coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen; Described two anti-IgM are mouse-anti people IgM polyclonal antibody; The concentration of described HPIVs specificity recombinant antigen is 1mg/mL, and the concentration of described two anti-IgM is 2mg/mL, and the concentration of the polyclonal antibody of described anti-HPIVs recombinant antigen is 1mg/mL.
A kind of formation of human parainfluenza virus's IgM antibody test strip: PVC offset plate is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, Aureal Dongyuan County, adsorptive pads Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., HPIVs specificity recombinant antigen, polyclonal antibody equal purchased from American Aureal company of Dongyuan County (OriGene) of mouse-anti people IgM polyclonal antibody and anti-HPIVs recombinant antigen, the glass fibre membrane of the class parainfluenza virus specificity recombinant antigen containing colloid gold label is purchased from Thermo Fisher Scientific Inc..
A kind of preparation method of human parainfluenza virus's IgM antibody test strip is:
1) the HPIVs specificity recombinant antigen glass fibre membrane of colloid gold label is prepared:
(1) preparation of colloidal gold solution: (volume ratio is the dense HCL of 3:1 and dense HNO with chloroazotic acid
3) soaking container certain hour 30min, then clean up with ultrapure water, dry; In container, add 2% chlorauric acid solution of 99.14mL pure water and 0.86mL, heat while stirring, continue to boil 8min after being heated to boiling, add 2.5mL1% trisodium citrate aqueous solution, continue agitating heating 10min, add pure water after cooling to 100mL, obtain claret colloidal gold solution; Wherein, colloid gold particle mean diameter is at 25nm;
(2) preparation of gold mark HPIVs specificity recombinant antigen:
A. colloidal gold solution pH is regulated: the colloidal gold solution getting above-mentioned preparation, with the K of 0.1M
2cO
3solution regulates pH to 9.0;
B. HPIVs specificity recombinant antigen is diluted: with phosphate buffer (PB), HPIVs specificity recombinant antigen being diluted to protein content is 0.8mg/mL; Wherein, described phosphate buffer is: get KCL, Na
2hPO
4, KH
2pO
41.8g altogether, dissolve with 0.8L distilled water, then use salt acid for adjusting pH, adding water to cumulative volume is that 1L makes;
C. the coupling of collaurum and recombinant antigen: get the dilution 1.0mL in step b, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, draws out 250 μ L supernatants, discards precipitation, dropwise being joined in step a by supernatant while stirring regulates in the colloidal gold solution of pH, leaves standstill 10min; Add stabilizing agent 10%BSA solution 1mL, make final concentration be 1%, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, leave standstill 10min, abandon supernatant, stay sediment, dissolve with sustained release agent, the centrifugal 10min of 500r/min under 4 DEG C of conditions again, remove aggregation, stay supernatant, again by supernatant centrifugal 30min of 2500r/min under 4 DEG C of conditions, resuspended bottom loose deposits, namely obtains the HPIVs specificity recombinant antigen of colloid gold label;
(3) with three-dimensional planar point film gold spraying instrument instrument HM3055, be applied to by the HPIVs specificity recombinant antigen dissolution homogeneity having marked collaurum on the plain film of the glass fibre handled well, room temperature bag is spent the night, for subsequent use;
2) preparation is coated with the detection line of two anti-IgM and is coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen:
Two anti-IgM are diluted to 2mg/mL, the polyclonal antibody of anti-HPIVs recombinant antigen is diluted to 1mg/mL; With three-dimensional planar point film gold spraying instrument instrument HM3055, diluted two anti-IgM are evenly sprayed on nitrocellulose filter, obtain detection line; Evenly be sprayed on nitrocellulose filter by the polyclonal antibody of the anti-HPIVs recombinant antigen diluted, obtain nature controlling line, room temperature bag is spent the night, for subsequent use;
3) test strips is prepared:
Sample pad is soaked 40min in confining liquid, dries, encapsulate for subsequent use, described confining liquid be containing the pH of bovine serum albumin be 9.0 phosphate cleansing solution; By PVC base plate, nitrocellulose filter detection layers, sample pad, glass fibre element film and adsorptive pads combination, on PVC base plate, note thieving paper is glued in the top of nitrocellulose filter, nitrocellulose filter and the mutual overlapping 0.2mm of thieving paper, sticking glass tunica fibrosa and sample pad successively in the below of nitrocellulose filter, mutual overlapping 0.2mm between nitrocellulose filter and glass fibre membrane and between glass fibre membrane and sample pad, obtains product human parainfluenza virus IgM antibody test strip.
Detection method: get blood and separation of serum by routine clinical, get 150-200ul serum, be added drop-wise in the sample pad of the test strips of preparation in embodiment 1,2 or 3 with suction pipe, 10-15 minute sentence read result: as contained HPIVsIgM antibody in sample, then it is through colloidal gold pad, can be combined with gold mark HPIVs recombinant antigen, and then the mouse-anti people IgM polyclonal antibody on tested survey line is caught, assemble and form red precipitation, being the positive, otherwise being then negative; No matter in sample whether containing antibody to be detected, gold mark recombinant antigen all can with nature controlling line on wrap the anti-HPIVs recombinant antigen of quilt polyclonal antibody form red precipitate line, i.e. nature controlling line.Line like this does not occur, illustrates that test strips lost efficacy.
The present invention can also reach half-quantitative detection object: when prepared by test paper by regulating detection line and nature controlling line encrusting substance concentration, the colour developing depth of detection line and nature controlling line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration.
Clinical statistics is tested:
Clinical patients human parainfluenza virus IgM antibody detects: in epidemic season, detect 47 routine patient suspected's serum samples, the confirmed cases 13 parts of accepting for medical treatment, the human parainfluenza virus detection method immunofluorescence technique conventional with tradition compares, and test strips method of the present invention has 2 routine suspected case IgM antibody to fail to detect.Its sensitivity is 95.7%, and specificity is 100%.Testing result is as shown in table 1.
Table 1: immunofluorescence technique and ELISA test strip result of the present invention are added up
| Suspected case | Positive case | Negative case | Sensitivity | Specificity | |
| Immunofluorescence technique | 47 | 31 | 12 | 91.5％ | 100％ |
| Test strips | 47 | 31 | 14 | 95.7％ | 100％ |
Test findings shows: test strip of the present invention has higher sensitivity and specificity, is very applicable to clinical practice.
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 (1)
1. human parainfluenza virus's IgM antibody test strip, it is characterized in that, this test strips comprises: the glass fibre membrane of the HPIVs specificity recombinant antigen containing colloid gold label, be coated with the detection line of anti-IgM and be coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen;
Described test strips is prepared according to the following steps:
1) the HPIVs specificity recombinant antigen glass fibre membrane of colloid gold label is prepared:
(1) preparation of colloidal gold solution: with chloroazotic acid soaking container certain hour, then clean up with ultrapure water, dry; In container, add 2% chlorauric acid solution of 99.14mL pure water and 0.86mL, heat while stirring, continue to boil 8min after being heated to boiling, add 2.5mL1% trisodium citrate aqueous solution, continue agitating heating 10min, add pure water after cooling to 100mL, obtain claret colloidal gold solution;
(2) preparation of gold mark HPIVs specificity recombinant antigen:
A. regulate colloidal gold solution pH: the colloidal gold solution getting above-mentioned preparation, use K
2cO
3solution regulates pH to 8.5-9.0;
B. HPIVs specificity recombinant antigen is diluted: with phosphate buffer PB, HPIVs specificity recombinant antigen being diluted to protein content is 0.2-0.8mg/mL;
C. the coupling of collaurum and recombinant antigen: get the dilution in step b, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, Aspirate supernatant, discards precipitation, is dropwise joined in step a by supernatant while stirring and regulates in the colloidal gold solution of pH, leaves standstill; Add stabilizing agent, the centrifugal 30min of 2500r/min under 4 DEG C of conditions, leave standstill, abandon supernatant, stay sediment, resuspended with sustained release agent, namely obtain the HPIVs specificity recombinant antigen of colloid gold label;
(3) with three-dimensional planar point film gold spraying instrument instrument HM3055, the HPIVs specificity recombinant antigen dissolution homogeneity having marked collaurum is applied on the glass fibre membrane handled well, freeze drying, for subsequent use;
2) preparation is coated with the detection line of anti-IgM and is coated with the nitrocellulose filter of nature controlling line of polyclonal antibody of anti-HPIVs recombinant antigen:
Anti-IgM is diluted to 2mg/mL, the polyclonal antibody of anti-HPIVs recombinant antigen is diluted to 1mg/mL; With three-dimensional planar point film gold spraying instrument instrument HM3055, the anti-IgM diluted evenly is sprayed on nitrocellulose filter, obtains detection line; Evenly be sprayed on nitrocellulose filter by the polyclonal antibody of the anti-HPIVs recombinant antigen diluted, obtain nature controlling line, room temperature bag is spent the night, and envelope is for subsequent use;
3) test strips is prepared:
Sample pad is soaked 40min in confining liquid, dries, encapsulate for subsequent use; PVC base plate, nitrocellulose filter, sample pad, glass fibre membrane and thieving paper are combined, on PVC base plate, thieving paper is pasted in the top of nitrocellulose filter, nitrocellulose filter and the mutual overlapping 0.2mm of thieving paper, sticking glass tunica fibrosa and sample pad successively in the below of nitrocellulose filter, mutual overlapping 0.2mm between nitrocellulose filter and glass fibre membrane and between glass fibre membrane and sample pad, obtains human parainfluenza virus's IgM antibody test strip.
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| CN107656048A (en) * | 2017-09-15 | 2018-02-02 | 刘哲 | A kind of immuno-chromatographic test paper strip and its application of half-quantitative detection antigen or antibody |
| CN108802381B (en) * | 2018-06-13 | 2021-03-12 | 河南中泽生物工程有限公司 | Bovine viral diarrhea virus identification and detection test strip and preparation method thereof |
| CN110501493A (en) * | 2019-08-28 | 2019-11-26 | 郑州安图生物工程股份有限公司 | A kind of detection kit of hemadsorption virus type 1's IgM antibody |
| CN111458505A (en) * | 2020-04-07 | 2020-07-28 | 浙江理工大学绍兴生物医药研究院有限公司 | Detection paper and method for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus |
| CN111505281A (en) * | 2020-04-17 | 2020-08-07 | 南昌大学 | Sensitization type immunochromatography kit for sensitively detecting novel coronavirus antibody |
| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| CN112986578B (en) * | 2021-02-04 | 2023-03-10 | 南昌大学 | Test strip for screening serum specific Ig E and preparation method thereof |
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| CN101846631A (en) * | 2010-06-04 | 2010-09-29 | 江南大学 | Method for quickly testing melamine content in milk powder by using gold nanoparticle colorimetric method |
| CN103983776A (en) * | 2014-05-27 | 2014-08-13 | 国家纳米科学中心 | Colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies |
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| CN101846631A (en) * | 2010-06-04 | 2010-09-29 | 江南大学 | Method for quickly testing melamine content in milk powder by using gold nanoparticle colorimetric method |
| CN103983776A (en) * | 2014-05-27 | 2014-08-13 | 国家纳米科学中心 | Colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies |
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