CN105759047A - Immunochromatography test strip for rapidly detecting campylobacter jejuni and preparation method thereof - Google Patents
Immunochromatography test strip for rapidly detecting campylobacter jejuni and preparation method thereof Download PDFInfo
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- CN105759047A CN105759047A CN201610257008.5A CN201610257008A CN105759047A CN 105759047 A CN105759047 A CN 105759047A CN 201610257008 A CN201610257008 A CN 201610257008A CN 105759047 A CN105759047 A CN 105759047A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56922—Campylobacter
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
Abstract
The invention provides an immunochromatography test strip for rapidly detecting campylobacter jejuni and a preparation method thereof.According to the immunochromatography test strip for rapidly detecting campylobacter jejuni, a nitrocellulose membrane (NC membrane) is coated with a monoclonal antibody of campylobacter jejuni specific antigen or a polyclonal antibody of campylobacter jejuni specific antigen to serve as a detection line, a secondary antibody IgG serves as a quality control line, a campylobacter jejuni specific antigen monoclonal antibody marked with colloidal gold is combined, membrane chromatography is adopted, and campylobacter jejuni in a specimen is detected.When the immunochromatography test strip is applied for detection, operation is simple, convenient, rapid and fast, no special instrument or equipment is needed for assistance, the result is clear and legible, and the immunochromatography test strip plays an important role in campylobacter jejuni infection diagnosis.
Description
Technical field
The present invention designs medical test field tests, is specifically related to immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni and preparation method thereof.
Technical background
Campylobacter jejuni (campylobacterjejunienteritis) is that Butzler in 1973 etc. separate from Feces of Patients With Diarrhea, has recognized one of its Main Pathogenic Bacteria being mankind's diarrhoea at present.Jejunum campylobacter mattress is a kind of infecting both domestic animals and human disease pathogen mattress, can cause humans and animals that multiple disease occurs, and it is a kind of borne Parasitic Encephalopathy cause of disease mattress, it is believed that be the main cause causing whole world human bacterial property to suffer from diarrhoea, the research of the pathogenesis of jejunum campylobacter mattress is got more and more.Its paathogenic factor includes four aspects such as adhesion, invasion and attack, generation toxin and molecular simulation mechanism, can cause the most serious complication one Guillain-Barre syndrome by molecular simulation mechanism.Jejunum campylobacter mattress can the sickness rate of the campylobacter jejuni enteritis that causes a disease exceedes bacillary dysentery in developed country by generation Cytotonic enterotoxin, cytotoxin and cell lethality expansion toxin, and in developing country, sickness rate is nearly identical to bacillary dysentery.This bacterium is as important food-borne pathogens, breed rapidly under containing micro amount of oxygen environment after entering intestinal along with food, mainly invade jejunum, ileum and colon, invasion and attack intestinal mucosa, cause hyperemia and heamorrhagic lesions, observe that some bacterial strain can produce similar cholera enterotoxin in recent years, cause liquid secretion in enteric cavity to increase.
The detection of current campylobacter jejuni depends on the biochemical identification of GB defined, and its shortcoming is that complex operation, detection cycle are longer, it is impossible to adapt to substantial amounts of sample examination.Immunology detection is quick, accurate, the most stable quick detection means occurred in recent years, but there is no at present both at home and abroad can operate with detection practice quickly detect product.
Summary of the invention
Present invention solves the technical problem that immuno-chromatographic test paper strip of being just to provide a kind of quick detection campylobacter jejuni and preparation method thereof.
The technical scheme is that the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, including plastic bottom board, sample pad, gold-marking binding pad, with detection line and the nitrocellulose filter (NC film) of nature controlling line, adsorptive pads.
Further, described gold-marking binding pad is the glass fibre membrane of the monoclonal antibody of the campylobacter jejuni containing colloid gold label.
Further, described nitrocellulose filter (NC film) is coated with the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen as detecting line and two anti-igg as nature controlling line.
Further, two described anti-igg sheep anti mouses the polyclonal antibody of IgG.
The preparation method of the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, comprises the following steps:
(1) preparation of the polyclonal antibody of the monoclonal antibody of campylobacter jejuni specific antigen or campylobacter jejuni specific antigen: first inducing culture gene engineering bacterium expression campylobacter jejuni PEB1 recombiant protein, PEB1 recombiant protein solution is combined isopyknic adjuvant and injects immune balb/c mice, after detection mice serum is qualified, the splenocyte and the myeloma cell that take mice are merged under 50%PEC4000 effect, set up hybridoma cell strain, then indirect elisa method screening positive cell strain is adopted, then the antibody of positive cell strain secretion is monoclonal antibody or the polyclonal antibody of campylobacter jejuni specific antigen；
(2) with the preparation of detection line and the nitrocellulose filter of nature controlling line: it is 1.4～1.6mg/ml that the monoclonal antibody of campylobacter jejuni specific antigen prepare step (1) or the polyclonal antibody of campylobacter jejuni specific antigen are diluted to concentration, by sheep anti mouse the polyclonal antibody of IgG to be diluted to concentration be 1.8～2.2mg/ml, then it is sprayed on nitrocellulose membrane with a stroke film instrument with the even concentration of 5 μ L/cm, dries in the constant temperature oven of 37 DEG C；
(3) preparation of gold-marking binding pad: first make colloidal gold solution by the mass ratio of 40:1 to addition trisodium citrate Na3C6H5O7 2H2O solution heating in aqueous solution of chloraurate, then adjusting gold colloidal pH value is 7.4～8.0, the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen is added in colloidal gold solution, the quality proportioning of gold colloidal and antibody is 24:1, it is subsequently adding stabilizer (containing 0.05%BSA, pH8.0, 0.01MTris buffer) after stabilized treatment, by the amount uniform adsorption of every square centimeter of 65 μ l on glass fibre membrane, lyophilization, standby；
4) preparation of test strips: by sample pad, gold-marking binding pad, nitrocellulose filter and adsorptive pads are pasted onto on plastic bottom board from top to bottom successively, it is assembled into test strips, the wherein viscous note adsorptive pads in the top of nitrocellulose filter, nitrocellulose filter and adsorptive pads be overlapping 0.2～0.4mm mutually, sticking glass fibrous membrane and sample pad successively in the lower section of nitrocellulose filter, nitrocellulose membrane and glass fibre membrane be overlapping 0.3～0.5mm mutually, glass fibre membrane and sample pad be overlapping 2～4mm mutually, then reagent paper is cut into again the strip of one fixed width, again test strips is arranged in the detection card shell of the flat shelly of strip, obtain the immuno-chromatographic test paper strip quickly detecting campylobacter jejuni of the present invention.
The operation principle of test strips of the present invention: the test strips of the present invention utilizes colloidal gold-labeled method and film chromatography, when in measuring samples containing campylobacter jejuni, the monoclonal antibody of the campylobacter jejuni first containing colloid gold label on glass fibre membrane due to capillary action campylobacter jejuni under the traction of adsorptive pads is combined formation complex, then complex is moved forward by chromatography effect, when being up to detection line, run into the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen, form the gold mark complex of antibody-campylobacter jejuni-antibody, because there being gold grain to deposit at this, it is gathered on detection band, therefore form detection line, naked eyes develop the color result as seen.
Beneficial effects of the present invention is embodied in: have endotoxin can attack small intestinal due to campylobacter jejuni and colorectal mucosa causes acute enteritis, also outbreak of epidemic or the mass food poisoning of diarrhoea can be caused, so utilizing the test strips for detecting campylobacter jejuni prepared by colloidal gold-labeled method and rete analysis method quickly to detect whether to have infected campylobacter jejuni, pathogenic bacterium can be found in time and give treatment in time, in case causing outbreak of epidemic or the mass food poisoning of diarrhoea, and ELISA test strip prepared by the present invention is rapidly and efficiently, simple to operate, be suitable to promote on a large scale.
Detailed description of the invention
Embodiment 1: the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, including plastic bottom board, sample pad, gold-marking binding pad, with detection line and the nitrocellulose filter (NC film) of nature controlling line, adsorptive pads.
Wherein, described gold-marking binding pad is the glass fibre membrane of the monoclonal antibody of the campylobacter jejuni containing colloid gold label.
Wherein, described nitrocellulose filter (NC film) is coated with the monoclonal antibody of campylobacter jejuni specific antigen as detecting line and two anti-igg as nature controlling line.
Wherein, two described anti-igg sheep anti mouses the polyclonal antibody of IgG.
A kind of immuno-chromatographic test paper strip detecting campylobacter jejuni, described plastic bottom board is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.；Adsorptive pads is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., and the glass fibre membrane of the campylobacter jejuni containing colloid gold label is purchased from Thermo Fisher Scientific Inc..The monoclonal antibody of campylobacter jejuni specific antigen is purchased from Amy victory Science and Technology Ltd..
The preparation method of the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, comprises the following steps:
(1) preparation of the monoclonal antibody of campylobacter jejuni specific antigen: first inducing culture gene engineering bacterium expression campylobacter jejuni PEB1 recombiant protein, PEB1 recombiant protein solution is combined isopyknic adjuvant and injects immune balb/c mice, after detection mice serum is qualified, the splenocyte and the myeloma cell that take mice are merged under 50%PEC4000 effect, set up hybridoma cell strain, then adopting indirect elisa method screening positive cell strain, then the antibody of positive cell strain secretion is the monoclonal antibody of campylobacter jejuni specific antigen；
(2) with the preparation of detection line and the nitrocellulose filter of nature controlling line: it is 1.4mg/ml that the monoclonal antibody of campylobacter jejuni specific antigen prepare step (1) or the polyclonal antibody of campylobacter jejuni specific antigen are diluted to concentration, by sheep anti mouse the polyclonal antibody of IgG to be diluted to concentration be 1.8mg/ml, then it is sprayed on nitrocellulose membrane with a stroke film instrument with the even concentration of 5 μ L/cm, dries in the constant temperature oven of 37 DEG C；
(3) preparation of gold-marking binding pad: first make colloidal gold solution by the mass ratio of 40:1 to addition trisodium citrate Na3C6H5O7 2H2O solution heating in aqueous solution of chloraurate, then adjusting gold colloidal pH value is 7.4, the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen is added in colloidal gold solution, the quality proportioning of gold colloidal and antibody is 24:1, it is subsequently adding stabilizer (containing 0.05%BSA, pH8.0, 0.01MTris buffer) after stabilized treatment, by the amount uniform adsorption of every square centimeter of 65 μ l on glass fibre membrane, lyophilization, standby；
4) preparation of test strips: by sample pad, gold-marking binding pad, nitrocellulose filter and adsorptive pads are pasted onto on plastic bottom board from top to bottom successively, it is assembled into test strips, the wherein viscous note adsorptive pads in the top of nitrocellulose filter, nitrocellulose filter and adsorptive pads be overlapping 0.2mm mutually, sticking glass fibrous membrane and sample pad successively in the lower section of nitrocellulose filter, nitrocellulose membrane and glass fibre membrane be overlapping 0.3mm mutually, glass fibre membrane and sample pad be overlapping 2mm mutually, then reagent paper is cut into again the strip of one fixed width, again test strips is arranged in the detection card shell of the flat shelly of strip, obtain the immuno-chromatographic test paper strip quickly detecting campylobacter jejuni of the present invention.
Embodiment 2: the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, including plastic bottom board, sample pad, gold-marking binding pad, with detection line and the nitrocellulose filter (NC film) of nature controlling line, adsorptive pads.
Wherein, described gold-marking binding pad is the glass fibre membrane of the monoclonal antibody of the campylobacter jejuni containing colloid gold label.
Wherein, described nitrocellulose filter (NC film) is coated with the polyclonal antibody of campylobacter jejuni specific antigen as detecting line and two anti-igg as nature controlling line.
Wherein, two described anti-igg sheep anti mouses the polyclonal antibody of IgG.
A kind of immuno-chromatographic test paper strip detecting campylobacter jejuni, described plastic bottom board is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.；Adsorptive pads is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., and the glass fibre membrane of the campylobacter jejuni containing colloid gold label is purchased from Thermo Fisher Scientific Inc..The polyclonal antibody of campylobacter jejuni specific antigen is purchased from Amy victory Science and Technology Ltd..
The preparation method of the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, comprises the following steps:
(1) preparation of the monoclonal antibody of campylobacter jejuni specific antigen: first inducing culture gene engineering bacterium expression campylobacter jejuni PEB1 recombiant protein, PEB1 recombiant protein solution is combined isopyknic adjuvant and injects immune balb/c mice, after detection mice serum is qualified, the splenocyte and the myeloma cell that take mice are merged under 50%PEC4000 effect, set up hybridoma cell strain, then adopting indirect elisa method screening positive cell strain, then the antibody of positive cell strain secretion is the monoclonal antibody of campylobacter jejuni specific antigen；
(2) with the preparation of detection line and the nitrocellulose filter of nature controlling line: it is 1.5mg/ml that the monoclonal antibody of campylobacter jejuni specific antigen prepare step (1) or the polyclonal antibody of campylobacter jejuni specific antigen are diluted to concentration, by sheep anti mouse the polyclonal antibody of IgG to be diluted to concentration be 2mg/ml, then it is sprayed on nitrocellulose membrane with a stroke film instrument with the even concentration of 5 μ L/cm, dries in the constant temperature oven of 37 DEG C；
(3) preparation of gold-marking binding pad: first make colloidal gold solution by the mass ratio of 40:1 to addition trisodium citrate Na3C6H5O7 2H2O solution heating in aqueous solution of chloraurate, then adjusting gold colloidal pH value is 7.7, the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen is added in colloidal gold solution, the quality proportioning of gold colloidal and antibody is 24:1, it is subsequently adding stabilizer (containing 0.05%BSA, pH8.0, 0.01MTris buffer) after stabilized treatment, by the amount uniform adsorption of every square centimeter of 65 μ l on glass fibre membrane, lyophilization, standby；
4) preparation of test strips: by sample pad, gold-marking binding pad, nitrocellulose filter and adsorptive pads are pasted onto on plastic bottom board from top to bottom successively, it is assembled into test strips, the wherein viscous note adsorptive pads in the top of nitrocellulose filter, nitrocellulose filter and adsorptive pads be overlapping 0.3mm mutually, sticking glass fibrous membrane and sample pad successively in the lower section of nitrocellulose filter, nitrocellulose membrane and glass fibre membrane be overlapping 0.4mm mutually, glass fibre membrane and sample pad be overlapping 3mm mutually, then reagent paper is cut into again the strip of one fixed width, again test strips is arranged in the detection card shell of the flat shelly of strip, obtain the immuno-chromatographic test paper strip quickly detecting campylobacter jejuni of the present invention.
Embodiment 3: the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, including plastic bottom board, sample pad, gold-marking binding pad, with detection line and the nitrocellulose filter (NC film) of nature controlling line, adsorptive pads.
Wherein, described gold-marking binding pad is the glass fibre membrane of the monoclonal antibody of the campylobacter jejuni containing colloid gold label.
Wherein, described nitrocellulose filter (NC film) is coated with the monoclonal antibody of campylobacter jejuni specific antigen as detecting line and two anti-igg as nature controlling line.
Wherein, two described anti-igg sheep anti mouses the polyclonal antibody of IgG.
A kind of immuno-chromatographic test paper strip detecting campylobacter jejuni, described plastic bottom board is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.；Adsorptive pads is purchased from Aureal Dongyuan County, Beijing (OriGeneTechnologies) bio tech ltd, nitrocellulose filter is purchased from Thermo Fisher Scientific Inc., and the glass fibre membrane of the campylobacter jejuni containing colloid gold label is purchased from Thermo Fisher Scientific Inc..The monoclonal antibody of campylobacter jejuni specific antigen is purchased from Amy victory Science and Technology Ltd..
The preparation method of the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni, comprises the following steps:
(1) preparation of the monoclonal antibody of campylobacter jejuni specific antigen: first inducing culture gene engineering bacterium expression campylobacter jejuni PEB1 recombiant protein, PEB1 recombiant protein solution is combined isopyknic adjuvant and injects immune balb/c mice, after detection mice serum is qualified, the splenocyte and the myeloma cell that take mice are merged under 50%PEC4000 effect, set up hybridoma cell strain, then adopting indirect elisa method screening positive cell strain, then the antibody of positive cell strain secretion is the monoclonal antibody of campylobacter jejuni specific antigen；
(2) with the preparation of detection line and the nitrocellulose filter of nature controlling line: it is 1.6mg/ml that the monoclonal antibody of campylobacter jejuni specific antigen prepare step (1) or the polyclonal antibody of campylobacter jejuni specific antigen are diluted to concentration, by sheep anti mouse the polyclonal antibody of IgG to be diluted to concentration be 2.2mg/ml, then it is sprayed on nitrocellulose membrane with a stroke film instrument with the even concentration of 5 μ L/cm, dries in the constant temperature oven of 37 DEG C；
(3) preparation of gold-marking binding pad: first make colloidal gold solution by the mass ratio of 40:1 to addition trisodium citrate Na3C6H5O7 2H2O solution heating in aqueous solution of chloraurate, then adjusting gold colloidal pH value is 8.0, the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen is added in colloidal gold solution, the quality proportioning of gold colloidal and antibody is 24:1, it is subsequently adding stabilizer (containing 0.05%BSA, pH8.0, 0.01MTris buffer) after stabilized treatment, by the amount uniform adsorption of every square centimeter of 65 μ l on glass fibre membrane, lyophilization, standby；
4) preparation of test strips: by sample pad, gold-marking binding pad, nitrocellulose filter and adsorptive pads are pasted onto on plastic bottom board from top to bottom successively, it is assembled into test strips, the wherein viscous note adsorptive pads in the top of nitrocellulose filter, nitrocellulose filter and adsorptive pads be overlapping 0.4mm mutually, sticking glass fibrous membrane and sample pad successively in the lower section of nitrocellulose filter, nitrocellulose membrane and glass fibre membrane be overlapping 0.5mm mutually, glass fibre membrane and sample pad be overlapping 4mm mutually, then reagent paper is cut into again the strip of one fixed width, again test strips is arranged in the detection card shell of the flat shelly of strip, obtain the immuno-chromatographic test paper strip quickly detecting campylobacter jejuni of the present invention.
Clinical statistics is tested:
The detection of clinical campylobacter jejuni: collect the cervical mucus of 66 example suspected acute enteritis patients, 24 examples are diagnosed as patient's cervical mucus of acute enteritis, compare with traditional immunofluorescence, the test strips of the present invention has 2 example suspected patients not detect, traditional immunization fluorescence method has 6 examples not detect, the sensitivity of the test strips of the present invention is 97.8%, and immunofluorescence sensitivity is 93.3%, and specificity is all 100%；Confirmed cases detection is all positive, and wherein suspected case immunofluorescence is added up such as following table with ELISA test strip result of the present invention:
| Suspected case | Positive case | Negative case | Sensitivity | Specificity | |
| Immunofluorescence | 66 | 45 | 15 | 90.9% | 100% |
| Test strips | 66 | 49 | 15 | 97% | 100% |
Result of the test shows: the test strip of the present invention has higher sensitivity and specificity, is especially suitable for clinical practice.
Last it is noted that above example is only in order to illustrate technical scheme, it is not intended to limit；Although the present invention being described in detail with reference to previous embodiment, it will be understood by those within the art that: the technical scheme described in previous embodiment still can be modified by it, or wherein portion of techniques feature is carried out equivalent replacement；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 (5)
1. the immuno-chromatographic test paper strip of a quick detection campylobacter jejuni, it is characterised in that it include plastic bottom board, sample pad, gold-marking binding pad, with detection line and the nitrocellulose filter (NC film) of nature controlling line, adsorptive pads.
2. the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni as claimed in claim 1, it is characterised in that described gold-marking binding pad is the glass fibre membrane of the monoclonal antibody of the campylobacter jejuni containing colloid gold label.
3. the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni as claimed in claim 1, it is characterized in that, described nitrocellulose filter (NC film) is coated with the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen as detecting line and two anti-igg as nature controlling line.
4. the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni as claimed in claim 3, it is characterised in that two described anti-igg sheep anti mouses the polyclonal antibody of IgG.
5. the preparation method of the immuno-chromatographic test paper strip of a kind of quick detection campylobacter jejuni as described in Claims 1 to 4 any one, it is characterised in that comprise the following steps:
(1) preparation of the polyclonal antibody of the monoclonal antibody of campylobacter jejuni specific antigen or campylobacter jejuni specific antigen: first inducing culture gene engineering bacterium expression campylobacter jejuni PEB1 recombiant protein, PEB1 recombiant protein solution is combined isopyknic adjuvant and injects immune balb/c mice, after detection mice serum is qualified, the splenocyte and the myeloma cell that take mice are merged under 50%PEC4000 effect, set up hybridoma cell strain, then indirect elisa method screening positive cell strain is adopted, then the antibody of positive cell strain secretion is monoclonal antibody or the polyclonal antibody of campylobacter jejuni specific antigen；
(2) with the preparation of detection line and the nitrocellulose filter of nature controlling line: it is 1.4～1.6mg/ml that the monoclonal antibody of campylobacter jejuni specific antigen prepare step (1) or the polyclonal antibody of campylobacter jejuni specific antigen are diluted to concentration, by sheep anti mouse the polyclonal antibody of IgG to be diluted to concentration be 1.8～2.2mg/ml, then it is sprayed on nitrocellulose membrane with a stroke film instrument with the even concentration of 5 μ L/cm, dries in the constant temperature oven of 37 DEG C；
(3) preparation of gold-marking binding pad: the mass ratio first pressing 40:1 adds trisodium citrate Na in aqueous solution of chloraurate3C6H5O7·2H2Colloidal gold solution is made in the heating of O solution, then adjusting gold colloidal pH value is 7.4～8.0, the monoclonal antibody of campylobacter jejuni specific antigen or the polyclonal antibody of campylobacter jejuni specific antigen is added in colloidal gold solution, the quality proportioning of gold colloidal and antibody is 24:1, is subsequently adding stabilizer (containing 0.05%BSA, pH8.0,0.01MTris buffer) after stabilized treatment, by the amount uniform adsorption of every square centimeter of 65 μ l on glass fibre membrane, lyophilization, standby；
4) preparation of test strips: by sample pad, gold-marking binding pad, nitrocellulose filter and adsorptive pads are pasted onto on plastic bottom board from top to bottom successively, it is assembled into test strips, the wherein viscous note adsorptive pads in the top of nitrocellulose filter, nitrocellulose filter and adsorptive pads be overlapping 0.2～0.4mm mutually, sticking glass fibrous membrane and sample pad successively in the lower section of nitrocellulose filter, nitrocellulose membrane and glass fibre membrane be overlapping 0.3～0.5mm mutually, glass fibre membrane and sample pad be overlapping 2～4mm mutually, then reagent paper is cut into again the strip of one fixed width, again test strips is arranged in the detection card shell of the flat shelly of strip, obtain the immuno-chromatographic test paper strip quickly detecting campylobacter jejuni of the present invention.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110531074A (en) * | 2019-09-06 | 2019-12-03 | 齐鲁工业大学 | A method of detection campylobacter jejuni |
| CN111378717A (en) * | 2019-12-31 | 2020-07-07 | 上海海关动植物与食品检验检疫技术中心 | Nucleic acid immune gold-labeled test strip, kit and method for detecting campylobacter jejuni |
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