CN102393455B - Preparation method of immunogold rapid test paper for vibrio parahaemolyticus - Google Patents
Preparation method of immunogold rapid test paper for vibrio parahaemolyticus Download PDFInfo
- Publication number
- CN102393455B CN102393455B CN201110248547.XA CN201110248547A CN102393455B CN 102393455 B CN102393455 B CN 102393455B CN 201110248547 A CN201110248547 A CN 201110248547A CN 102393455 B CN102393455 B CN 102393455B
- Authority
- CN
- China
- Prior art keywords
- rabbit
- vibrio parahaemolytious
- antibody
- vibrio
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000012360 testing method Methods 0.000 title claims abstract description 67
- 241000607272 Vibrio parahaemolyticus Species 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 68
- 238000001514 detection method Methods 0.000 claims abstract description 43
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 229940031551 inactivated vaccine Drugs 0.000 claims abstract description 5
- 229960005486 vaccine Drugs 0.000 claims abstract description 5
- 241000607598 Vibrio Species 0.000 claims description 36
- 239000010931 gold Substances 0.000 claims description 25
- 229910052737 gold Inorganic materials 0.000 claims description 25
- 238000002347 injection Methods 0.000 claims description 23
- 239000007924 injection Substances 0.000 claims description 23
- 241000894006 Bacteria Species 0.000 claims description 20
- 238000007920 subcutaneous administration Methods 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 13
- 239000002671 adjuvant Substances 0.000 claims description 13
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 13
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 13
- 230000036039 immunity Effects 0.000 claims description 12
- 239000000084 colloidal system Substances 0.000 claims description 10
- 239000005018 casein Substances 0.000 claims description 9
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 9
- 235000021240 caseins Nutrition 0.000 claims description 9
- 239000003365 glass fiber Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 9
- 210000003462 vein Anatomy 0.000 claims description 9
- 241000283707 Capra Species 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
- 238000001990 intravenous administration Methods 0.000 claims description 4
- 238000010253 intravenous injection Methods 0.000 claims description 4
- 238000010241 blood sampling Methods 0.000 claims description 3
- 230000009849 deactivation Effects 0.000 claims description 3
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 230000028993 immune response Effects 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 13
- 206010016952 Food poisoning Diseases 0.000 description 5
- 208000019331 Foodborne disease Diseases 0.000 description 5
- 238000013019 agitation Methods 0.000 description 5
- 239000010408 film Substances 0.000 description 5
- 244000061458 Solanum melongena Species 0.000 description 4
- 230000004520 agglutination Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000607142 Salmonella Species 0.000 description 3
- 108010088160 Staphylococcal Protein A Proteins 0.000 description 3
- 206010047400 Vibrio infections Diseases 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 241000607594 Vibrio alginolyticus Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000002547 anomalous effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011059 hazard and critical control points analysis Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000588919 Citrobacter freundii Species 0.000 description 1
- 241000193155 Clostridium botulinum Species 0.000 description 1
- 208000002881 Colic Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 244000000028 waterborne pathogen Species 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of immunogold rapid test paper for vibrio parahaemolyticus. The method comprises the following steps of: (a) preparing a vibrio parahaemolyticus antigen; (b) preparing a vibrio parahaemolyticus inactivated vaccine; (c) preparing a polyvalent antiserum with a vaccine immune experimental rabbit; (d) purifying the antiserum; (e) marking with colloidal gold; and (f) preparing an immunogold test strip. A detection technology in which the immunogold rapid test paper for vibrio parahaemolyticus is adopted has the advantages of easiness, sensitivity, rapidness, specificity and the like; the customs clearance speed of port imported and exported goods can be increased greatly; and meanwhile, the test paper has the advantages of low price, no need of instrument, easiness and rapidness for operationing and easiness in judging results.
Description
Technical field
The present invention relates to detection technique field, be specifically related to a kind of preparation method of immunogold rapid test paper for vibrio parahaemolyticus.
Background technology
Vibrio parahemolyticus all belongs to vibrionaceae vibrio bacterial, is by food, water-borne pathogen.Vibrio parahemolyticus has similar biological characteristics and same pathogenic, all can cause diarrhoea and food poisoning.
Vibrio parahaemolytious equals nineteen fifty-three by Fujino the earliest and obtains from Japan's food poisoning patient first separation, claim again Halophiles, this bacterium is because of containing growing in the nutrient culture media of salt denseer (more than 3% ~ 4%), and irreproducible and gain the name in salt-free nutrient culture media.Vibrio parahaemolytious is distributed in all over the world, is conventionally present in bay and region, shore line and marine product, people Duo Yin is edible pollute this bacterium again without the marine product of well processed as crab class, oyster! Shrimp etc. and cause food poisoning.The food poisoning that vibrio parahaemolytious causes, the ratio accounting in food posioning is very high, and its harm is only second to salmonella, Escherichia coli, staphylococcus and clostridium botulinum.China's Coastal Areas has the report that causes food poisoning because of vibrio parahemolyticus every year.Cardinal symptom after people is poisoning comprises: diarrhea (being watery stool or blood sample just), abdominal cramps, feel sick, vomiting and headache, the report that also once had fever and felt cold, but less.
At present, the U.S. and European Union to import aquatic products all Compulsory Feature carry out the detection of vibrio parahaemolytious, assay is negative can be open to the custom, otherwise the aquatic products of outlet will all automatically be detained or destroy on the spot.The outlet of Zhe Gei China aquatic products is provided with technology barriers.Therefore, outlet aquatic product manufactory is conscientiously implemented HACCP plan, strengthens the detection to vibrio parahaemolytious, and quarantine departments strengthens factory implement the supervision of HACCP plan situation and the check of outlet aquatic products is just seemed and is even more important.
At present, the domestic detection for vibrio parahemolyticus has had corresponding national standard and industry standard, and method is roughly divided into following process: (1) front increasing bacterium; (2) selective enrichment; (3) selectivity is cultivated; (4) biochemical identification; (5) Kahagawa phenomenon and Serologic test.Checkout procedure needs 5d-6d could report positive findings.The method of inspection is loaded down with trivial details, wastes time and energy, and not only affects the work efficiency of inspection body, and makes Product transport and the storage time lengthening of production division, and production cost strengthens.
Collaurum fast diagnose test paper method is the novel vitro diagnostic techniques having grown up on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis since 20th century the nineties.Simple to operate, only sample drop to be checked must be added in sample cell, 10 a few minutes just can, according to color judged result, reach the object of detection excessively.The method does not need special instrument, and those skilled in the art can complete, and testing cost is low, can greatly reduce workload, is adapted to the needs of port fast detecting.Therefore, set up as early as possible a set of fast detecting vibrio parahemolyticus accurately, collaurum fast diagnose test paper method reliable, science is imperative.This for strengthen importing and exporting the quick test quarantine of vibrios in aquatic products, the aspects such as the investigation of aquiculture disease and control, nuisanceless aquatic products detection and production and aquatic products hygienic quality supervision and inspection all tool be of great significance.
In view of this, the invention provides the preparation method of the immunogold rapid test paper of a kind of vibrio parahaemolytious,, detection method fast easy to set up.
Summary of the invention
The present invention is intended to take vibrio parahemolyticus as material, and how anti-preparation is, utilizes advanced collaurum production technology to develop and detects vibrio parahemolyticus mark detection test paper.To achieve these goals, solution of the present invention is as follows:
The preparation method of immunogold rapid test paper for vibrio parahaemolyticus, comprises the following steps:
A) preparation of vibrio parahaemolytious antigen: with peptone water medium by vibrio parahaemolytious overnight incubation;
B) make vibrio parahaemolytious inactivated vaccine;
C) with vaccine immunity experimental rabbit, prepare polyvalent antiserum: adopt the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit; obtain respectively the anti-vibrio parahaemolytious antibody of rabbit prepared by subcutaneous multi-point injection (referred to as rabbit anti-1; lower with) and vein directly inject the anti-vibrio parahaemolytious antibody of rabbit (anti-2 referred to as rabbit, lower together) of preparation;
D) antiserum purifying;
E) colloid gold label: the rabbit anti-serum collaurum of purifying is carried out to mark;
F) preparation of Immuno gold test strips: first the rabbit of purifying in step d) anti-2 is sprayed on miillpore filter detection line, goat anti-rabbit antibody is sprayed on control line, again the rabbit of the colloid gold label of preparing in step e) anti-1 is sprayed on glass fibre membrane, is then assembled into Immuno gold test strips;
Described step b), makes vibrio parahaemolytious inactivated vaccine, is specially: first through 37 ℃ of deactivation 24h of formaldehyde of 0.3%, then through cultivate detect really without viable bacteria after as antigen;
Described step c), prepares polyvalent antiserum with vaccine immunity experimental rabbit, adopts the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, is specially:
1) intravenous immune: injection in every 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting while reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection immunity of Freund's adjuvant: after Freund's complete adjuvant mixes with bacterium liquid equal-volume, the subcutaneous multi-point injection 4mL of rabbit nape portion, after 2 weeks, with incomplete Freund's adjuvant and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion, after 2 weeks, 5-7 days intravenous injection 4mL before blood sampling, bloodletting while reaching 1:1280 or 1:2560 when tiring.
Described step f), the preparation of Immuno gold test strips, is specially:
Anti-1 with colloid gold label rabbit, set up the method for colloidal gold immunochromatographimethod technology vibrio parahaemolyticus; Application miillpore filter is made the immunoassay technology of carrier, first rabbit anti-2 is sprayed on miillpore filter detection line, goat anti-rabbit antibody is sprayed on control line, and the rabbit anti-1 of colloid gold label is sprayed on glass fibre membrane, after dry, add confining liquid to mix, after drying liquid, be assembled into and detect vibrio parahaemolytious; By gold grain, amplify immune response system again, reaction result is shown on solid phase carrier miillpore filter.
Above-mentioned confining liquid formula adopts: contain 1%(W/V) casein and 0.05%(V/V) concentration of Tween-20 is the PBS solution of 0.01 mol/L, the pH value of PBS solution is 7.2.
The preparation method of immunogold rapid test paper for vibrio parahaemolyticus, when prepared immunogold rapid test paper is used for Site Detection, test strips is kept flat on the table, at application of sample end, add the sample of 100uL, first sample dissolves the gold mark rabbit anti-1 being sprayed on glass fibre membrane, then solution moves toward NC film, and when running into the rabbit anti-2 of detection line (T), the standard of judgement detection paper result is as follows:
In sample, contain vibrio parahaemolytious: on detection line and control line (C), all manifest a purplish red colo(u)r streak (forming sandwich reaction, i.e. the anti-1-vibrio parahaemolytious-rabbit of gold mark rabbit anti-2);
In sample, do not contain vibrio parahaemolytious: only on control line, occur a purplish red colo(u)r streak;
Test paper is invalid: on detection line and control line, all do not occur a purplish red colo(u)r streak.
Beneficial effect of the present invention is as follows:
Adopt the easy and simple to handle, quick of this method immunogold rapid test paper for vibrio parahaemolyticus, do not need special instruments and equipment, the aubergine response line on detection line is bright in colour, be easy to judgement, and testing result can be preserved.By applying this detection of test strips method to 150 duplicate samples, result test strips method positive rate is 2%, and the separated Positive rate of bacterium is 2%, and both reach 100% by coincidence rate.The vibrio parahaemolytious antigen immune originality that the present invention selects is good, and the antibody agglutination titer that immune rabbit makes reaches 2
11-2
12, with colloid gold label, form test strip for detection of vibrio parahaemolytious, there is high specificity, sensitivity is high, simple to operate, consuming time short, and result is easy to judgement, and with vibrio alginolyticus, Escherichia coli, salmonella, Kazakhstan Vickers bacterium with cross reaction does not occur.The successful development of the method, can simple and quick detection vibrio parahaemolytious.
Adopt the detection technique of immunogold rapid test paper for vibrio parahaemolyticus of the present invention to have advantages of simple, sensitive, quick, special, can greatly facilitate the speed of being open to the custom of port cargoes imported and exported, simultaneously because this Test paper is cheap, do not need instrument and equipment, easy and simple to handle fast, result easily judges.This has important practical significance for prevention and control China Safety of Aquatic Products.Have no at present and utilize immune colloidal gold technique to detect the report of importing and exporting vibrio parahemolyticus in aquatic animal and products thereof both at home and abroad, this technology is expected to provide a kind of rapid sensitive, easy reliable method for the detection of entry and exit vibrio parahemolyticus, is particularly suitable for port quarantine department and implements to test fast to put needs.
Embodiment
1, first the present embodiment prepares vibrio parahaemolytious inactivated bacterial liquid
The nutrient solution of formalin-inactivated vibrio parahaemolytious with 0.3%, after testing without survival bacterium, by this bacterium with content 1,600,000,000 bacterium/every milliliter of immunizing rabbit, adopt the direct injecting immune of vein and two kinds of modes of subcutaneous multi-point injection immunity, by document, adopt antigen absorption process sealing mixed antibody to react with reporting to the leadship after accomplishing a task of other enteric bacillis, low temperature is preserved.
2, the preparation of the anti-vibrio parahaemolytious antibody of rabbit
The bacterium liquid of deactivation is adopted to intravenous injection and Freund's adjuvant immunity, and concrete immune programme for children is as follows:
1) intravenous immune: injection in every 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting while reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection immunity of Freund's adjuvant: after Freund's complete adjuvant mixes with bacterium liquid equal-volume, the subcutaneous multi-point injection 4mL of rabbit nape portion, after 2 weeks, use incomplete Freund's adjuvant and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion, 5-7 days intravenous injection 4mL before blood sampling after 2 weeks, bloodletting while reaching 1:1280 or 1:2560 when tiring;
3) agglutination titer: adopt slide aggegation experiment to detect the anti-vibrio parahaemolytious antibody titer of rabbit, after the anti-vibrio parahaemolytious antibody purification of rabbit, the concentration of adjuvant immunity is 28.72 mg/mL, and slide agglutination test is tired as 1:2
11, the concentration of intravenous immune is 28.72 mg/mL, slide agglutination test is tired as 1:2
7;
3, the preparation of the anti-vibrio parahaemolytious antibody colloidal gold of rabbit
1) preparation of colloidal gold solution: prepare by trisodium citrate reduction method, getting 1% (W/V) gold chloride, 1 mL joins in 99 mL ultrapure waters, gained chlorauric acid solution concentration is 0.01 (V/V) %, with water-bath magnetic agitation pot, be heated to boiling, 1% trisodium citrate aqueous solution that adds rapidly 2 mL under magnetic stirring apparatus constantly stirs with certain rotating speed, continues agitating heating approximately 15 min and to solution colour, stablizes constant.The collaurum that this method makes is vinicolor, through ultraviolet spectrophotometer, surveys its absorption peak at 520 nm places, and the colloid gold particle diameter obtaining is about 20 nm left and right, and cooling rearmounted 4 ℃ of refrigerators keep in Dark Place;
2) anti-1 mark of rabbit: get the colloidal gold solution that needs preparation amount, with 0.1 mol/L K
2cO
3adjust its pH to 8.5 with the HCl of 0.1 mol/L.Then under magnetic agitation, dropwise add the rabbit antibody standard gold of adjuvant immunity for the anti-1(of rabbit of purifying, every mL colloidal gold solution adds anti-1 16.7ug of rabbit), continue to add 5% (W/V) PEG(MW 6000 after magnetic agitation 30 min) to final concentration 0.1% (W/V), continue again to stir 15 min, finally place 4 ℃ of refrigerators, hold over night.By the good centrifugal 10min of anti-1 collaurum 4000 r/min of rabbit of mark, get centrifugal 30 min of supernatant 15000 r/min next day, and careful suction abandoned supernatant.Resuspended damping fluid for precipitation (containing 1% casein, 0.05% (W/V) PEG6000,0.05% (V/V) Tween-20, the PBS damping fluid of 0.01 mol/L pH7.2) returns to 1/20th of original volume, puts 4 ℃ of refrigerators standby;
4, the making of reaction test strips
Reaction test paper is made the test strips that size is 5.0 cm-6.0cm * 0.4cm-0.6 cm left and right; specifically consist of: the bottom is PVC offset plate; the upper end of test strips is thieving paper; lower end is glass fibre membrane and the sample pad that combines collaurum, and centre is nitrocellulose filter (NC film), and wherein top end has been sprayed quality control reagent; as goat anti-rabbit antibody; it is anti-2 that lower side has been sprayed rabbit, test strips surface coverage thin film, shield.
When prepared immunogold rapid test paper is used for Site Detection, test strips is kept flat on the table, at application of sample end, add the sample of 100uL, first sample dissolves the gold mark rabbit anti-1 being sprayed on glass fibre membrane, then solution moves toward NC film, when running into the rabbit anti-2 of detection line (T), if contain vibrio parahaemolytious in sample, form sandwich reaction, gold is marked the anti-1-vibrio parahaemolytious-rabbit of rabbit anti-2, on detection line and control line (C), all manifest a purplish red colo(u)r streak, if do not contain vibrio parahaemolytious in sample, only on control line, there is a purplish red colo(u)r streak, if all there is not a purplish red colo(u)r streak in detection line and control line, this test strips is invalid test strips so.
embodiment 1
The anti-vibrio parahaemolytious antibody labeling of rabbit, gets the colloidal gold solution that needs preparation amount, with 0.1 mol/L K
2cO
3adjust its pH to 8.5 with the HCl of 0.1 mol/L.Then under magnetic agitation, dropwise add the anti-vibrio parahaemolytious antibody of rabbit of purifying (with the rabbit antibody standard gold of adjuvant immunity, every mL colloidal gold solution adds the anti-vibrio parahaemolytious antibody of rabbit 16.7ug), continue to add 5%PEG(MW 6000 after magnetic agitation 30 min) to final concentration 0.1%, continue again to stir 15 min, finally place 4 ℃ of refrigerators, hold over night.By the good centrifugal 10min of anti-vibrio parahaemolytious antibody colloidal gold 4000 r/min of rabbit of mark, get centrifugal 30 min of supernatant 15000 r/min next day, and careful suction abandoned supernatant.Resuspended damping fluid for precipitation (containing 1% casein, 0.05% PEG6000,0.05% Tween-20, the PBS damping fluid of 0.01 mol/L pH7.2) returns to 1/20th of original volume, puts 4 ℃ of refrigerators standby.
embodiment 2
Reaction test strips consists of: size is 5.0 cm ~ 6.0cm * 0.4cm ~ 0.6 cm left and right; specifically consist of: the bottom is PVC offset plate; the upper end of test strips is thieving paper; lower end is glass fibre membrane and the sample pad that combines collaurum, and centre is nitrocellulose filter (NC film), and wherein top end has been sprayed quality control reagent; as goat anti-rabbit igg or SPA; it is anti-2 that lower side has been sprayed rabbit, test strips surface coverage thin film, shield.
embodiment 3
The formulation selection experiment of confining liquid:
The NC film that is coated with T, C line seals with following 8 kinds of confining liquids respectively:
(1) PBS of the 0.01mol/L pH7.2 of 1%BSA, 0.5%Tween-20
(2) PBS of the 0.01 mol/L pH7.2 of 0.5%BSA, 0.5%Tween-20
(3) PBS of the 0.01 mol/L pH7.2 of 1%BSA, 0.05%Tween-20
(4) PBS of the 0.01 mol/L pH7.2 of 0.5%BSA, 0.05%Tween-20;
(5) PBS of the 0.01 mol/L pH7.2 of 1%casein, 0.5%Tween-20;
The PBS of the 0.01 mol/L pH7.2 of (6) 0.5% casein, 0.5%Tween-20;
The PBS of 0.01 mol/L pH7.2 of (7) 1% casein, 0.05% Tween-20;
The PBS of 0.01 mol/L pH7.2 of (8) 0.5% casein, 0.05%Tween-20.
Through experiment test repeatedly, obtain result and be: adopt with the PBS sealing effect of the 0.01 mol/L pH7.2 containing 1% casein, 0.05%Tween-20 preferably, spot colour developing is clear, and background is more shallow.
embodiment 4
The antibody concentration that is coated with rabbit anti-2 by detection line is 0,24.32,48.64,72.96,97.28,6 concentration gradients of 121.6mg/ml, when the volume of collaurum rabbit anti-1 is mark 1/5,1/10,1/15 of collaurum volume and 1/20 o'clock, be assembled into square formation test strips, then with the vibrio parahaemolytious bacteria suspension of 2 times of serial dilutions, detect.Anti-2 concentration of result detection line rabbit are that the above detection line colour developing of 72.96mg/ml is clearer; When the volume of collaurum rabbit anti-1 is mark 1/15 o'clock of collaurum volume, detection line, control line colour developing were clear and background color is more shallow.Therefore finally determine that the concentration of the detection line rabbit anti-2 of test strips is 72.96mg/ml, anti-1 dilution of collaurum rabbit for labelled precursor amass 1/15, sensitivity can reach 2.6 * 10
3cfu/ml.
Same, control line is coated with staphylococcal protein A, concentration is 0,2,4,6,8,6 concentration gradients of 10mg/ml, then reacts by anti-1 concentration of definite above collaurum rabbit, finally show that the least concentration of the coated staphylococcal protein A of control line is 6mg/ml.
Control line is coated with goat anti-rabbit igg, and concentration is 6 concentration gradients of 2,3,4,5,6,7 mg/ml, then reacts by anti-1 concentration of definite above collaurum rabbit, finally show that the least concentration of the coated staphylococcal protein A of control line is 4mg/ml.
embodiment 5
The stability test experiment of test strips:
Follow-on test 6 months, test period, detecting the control line (C) of vibrio parahaemolytious bacteria suspension appearance and the color of detection line (T) does not all have ANOMALOUS VARIATIONS, and this explanation test strips is stable strong.
After testing, the immunogold rapid test paper for vibrio parahaemolyticus that adopts the present invention to prepare has good specificity and stability, specific as follows:
The specificity of immunogold rapid test paper for vibrio parahaemolyticus:
Detect the 7 strain bacterial strains such as vibrio parahaemolytious, vibrio alginolyticus, comma bacillus, mermaid vibrios, Maxwell vibrios, Citrobacter freundii, salmonella, the control line (C) and the detection line (T) that only have vibrio parahaemolytious, all there is aubergine, show positive findings; And all the other 6 strain bacterial strains only have control line (C) to occur aubergine, there is not aubergine in detection line (T), shows negative findings.
The stability of immunogold rapid test paper for vibrio parahaemolyticus
Follow-on test 6 months, test period, detecting the control line (C) of vibrio parahaemolytious bacteria suspension appearance and the color of detection line (T) does not all have ANOMALOUS VARIATIONS, and this explanation test strips is stable strong.
The variation that is appreciated that a lot of details is possible, but therefore this do not run counter to scope and spirit of the present invention, and the suitable variation that any person of an ordinary skill in the technical field does it all should be considered as not departing from the category of patent of the present invention.
Claims (1)
1. the preparation method of immunogold rapid test paper for vibrio parahaemolyticus, is characterized in that, comprises the following steps:
A) preparation of vibrio parahaemolytious antigen: with peptone water medium by vibrio parahaemolytious overnight incubation;
B) make vibrio parahaemolytious inactivated vaccine;
C) with vaccine immunity experimental rabbit, prepare polyvalent antiserum: adopt the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, obtain respectively the anti-vibrio parahaemolytious antibody of rabbit that the anti-vibrio parahaemolytious antibody of rabbit prepared by subcutaneous multi-point injection and vein are directly injected preparation;
D) antiserum purifying;
E) colloid gold label: the anti-vibrio parahaemolytious antibody of rabbit prepared by the subcutaneous multi-point injection of purifying carries out mark with collaurum;
F) preparation of Immuno gold test strips: the anti-vibrio parahaemolytious antibody of rabbit of first vein of purifying in step d) directly being injected to preparation is sprayed on miillpore filter detection line, goat anti-rabbit antibody is sprayed on control line, the anti-vibrio parahaemolytious antibody of rabbit of again being prepared by the subcutaneous multi-point injection of the colloid gold label of preparing in step e) is sprayed on glass fibre membrane, is then assembled into Immuno gold test strips; Described step b), makes vibrio parahaemolytious inactivated vaccine, is specially: first through 37 ℃ of deactivation 24h of formaldehyde of 0.3%, then through cultivate detect really without viable bacteria after as antigen; Described step c), prepares polyvalent antiserum with vaccine immunity experimental rabbit, adopts the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, is specially:
1) intravenous immune: injection in every 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting while reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection immunity of Freund's adjuvant: after Freund's complete adjuvant mixes with bacterium liquid equal-volume, the subcutaneous multi-point injection 4mL of rabbit nape portion, after 2 weeks, with incomplete Freund's adjuvant and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion, after 2 weeks, 5-7 days intravenous injection 4mL before blood sampling, bloodletting while reaching 1:1280 or 1:2560 when tiring; Described step f), the preparation of Immuno gold test strips, is specially:
The anti-vibrio parahaemolytious antibody of rabbit of preparing with the subcutaneous multi-point injection of colloid gold label, sets up the method for colloidal gold immunochromatographimethod vibrio parahaemolyticus; Application miillpore filter is made the immune detection of carrier, the anti-vibrio parahaemolytious antibody of rabbit of first vein directly being injected to preparation is sprayed on miillpore filter detection line, goat anti-rabbit antibody is sprayed on control line, the anti-vibrio parahaemolytious antibody of rabbit prepared by the subcutaneous multi-point injection of colloid gold label is sprayed on glass fibre membrane, after dry, add confining liquid on miillpore filter, after drying liquid, be assembled into and detect vibrio parahaemolytious test paper; By gold grain, amplify immune response system again, reaction result is shown on solid phase carrier miillpore filter; Described confining liquid formula adopts: contain 1%(W/V) casein and 0.05%(V/V) concentration of Tween-20 is the PBS solution of 0.01 mol/L, the pH value of PBS solution is 7.2; When prepared immunogold rapid test paper is used for Site Detection, test strips is kept flat on the table, at application of sample end, add the sample of 100uL, first sample dissolves the anti-vibrio parahaemolytious antibody of gold mark rabbit being sprayed on glass fibre membrane, then solution moves toward miillpore filter, when running into the anti-vibrio parahaemolytious antibody of rabbit of detection line, the standard of judgement detection paper result is as follows:
In sample, contain vibrio parahaemolytious: on detection line and control line, all manifest a purplish red colo(u)r streak;
In sample, do not contain vibrio parahaemolytious: only on control line, occur a purplish red colo(u)r streak;
Test paper is invalid: on detection line and control line, all do not occur a purplish red colo(u)r streak.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110248547.XA CN102393455B (en) | 2011-08-26 | 2011-08-26 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110248547.XA CN102393455B (en) | 2011-08-26 | 2011-08-26 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102393455A CN102393455A (en) | 2012-03-28 |
| CN102393455B true CN102393455B (en) | 2014-01-29 |
Family
ID=45860814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110248547.XA Expired - Fee Related CN102393455B (en) | 2011-08-26 | 2011-08-26 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102393455B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103134933B (en) * | 2013-02-05 | 2015-07-01 | 江西中德生物工程有限公司 | Test strip used for detecting vibrio parahemolytocus and purpose thereof |
| CN103773851A (en) * | 2013-12-10 | 2014-05-07 | 天津出入境检验检疫局动植物与食品检测中心 | Preparation and application of nucleic acid sidewise-flow test strip detection kit for detecting Bibrio parahemolyticus |
| CN104316690B (en) * | 2014-11-03 | 2015-12-30 | 上海理工大学 | Vibrio parahemolyticus immune colloid gold Rapid detection test strip |
| CN105445465A (en) * | 2015-11-16 | 2016-03-30 | 珠海国际旅行卫生保健中心 | Fluorescent nano-immunochromatography kit for quickly detecting vibrio parahaemolyticus and preparation method |
| JP6367783B2 (en) * | 2015-11-26 | 2018-08-01 | 田中貴金属工業株式会社 | Immunochromatographic analyzer for detection of varicella-zoster virus |
| CN106336458A (en) * | 2016-07-29 | 2017-01-18 | 浙江省海洋水产养殖研究所 | Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof |
| CN106645718B (en) * | 2017-03-11 | 2018-12-07 | 中国海洋大学 | A kind of pathogenicity vibrio parahaemolytious quick detection test paper |
| CN107356749A (en) * | 2017-08-06 | 2017-11-17 | 潘金文 | Noxious pollutant immunochromatography quick detection kit in a kind of aquatic products |
| CN110836975B (en) * | 2019-11-29 | 2023-05-02 | 扬州大学 | Colloidal gold plastic package detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101000345A (en) * | 2006-11-28 | 2007-07-18 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunological chromatographic test paper for testing and its preparation method |
| JP2007327777A (en) * | 2006-06-06 | 2007-12-20 | Sekisui Chem Co Ltd | Biological measuring method |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| CN101561436A (en) * | 2009-06-05 | 2009-10-21 | 中国检验检疫科学研究院 | Colloidal gold test paper for listeria monocytogenes, preparation method thereof and application thereof |
| CN101609095A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of colloidal gold immunochromatographimethod method and colloid gold immune test strip of new fast quantifying and detecting Listeria monocytogenes |
| CN101819209A (en) * | 2010-05-15 | 2010-09-01 | 厦门出入境检验检疫局检验检疫技术中心 | Method for detecting foot and mouth disease (FMD) non-structural protein 3AB antibody by using dot immuno-gold filtration assay (DIGFA) |
| CN102135540A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of staphylococcus aureus and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| CN102135537A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Shigella detection method, gold labeling quick diagnosis kit for same and preparation method thereof |
-
2011
- 2011-08-26 CN CN201110248547.XA patent/CN102393455B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007327777A (en) * | 2006-06-06 | 2007-12-20 | Sekisui Chem Co Ltd | Biological measuring method |
| CN101000345A (en) * | 2006-11-28 | 2007-07-18 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunological chromatographic test paper for testing and its preparation method |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| CN101561436A (en) * | 2009-06-05 | 2009-10-21 | 中国检验检疫科学研究院 | Colloidal gold test paper for listeria monocytogenes, preparation method thereof and application thereof |
| CN101609095A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of colloidal gold immunochromatographimethod method and colloid gold immune test strip of new fast quantifying and detecting Listeria monocytogenes |
| CN101819209A (en) * | 2010-05-15 | 2010-09-01 | 厦门出入境检验检疫局检验检疫技术中心 | Method for detecting foot and mouth disease (FMD) non-structural protein 3AB antibody by using dot immuno-gold filtration assay (DIGFA) |
| CN102135540A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of staphylococcus aureus and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| CN102135537A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Shigella detection method, gold labeling quick diagnosis kit for same and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 志贺氏菌多克隆抗体制备及ELISA检测方法的建立;秦巧玲;《中国优秀硕士学位论文全文数据库》;20090228;第15页 * |
| 秦巧玲.志贺氏菌多克隆抗体制备及ELISA检测方法的建立.《中国优秀硕士学位论文全文数据库》.2009, |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102393455A (en) | 2012-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102393455B (en) | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus | |
| CN103558388B (en) | Double-antibody sandwich method for detecting salmonella typhimurium in food based on monoclonal antibodies | |
| CN107167598B (en) | A kind of kit of the quick rugged Cronobacter sakazakii of detection slope and its application | |
| CN104792991A (en) | Specific double antibody sandwich method for detecting salmonella in food based on monoclonal antibody | |
| CN101363850A (en) | Melamine rapid immune detecting kit | |
| CN107202884A (en) | Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit | |
| CN102943113A (en) | Loop-mediated isothermal amplification detection primer groups of Escherichia coli 0157, detection method and reagent kit | |
| CN202794185U (en) | Test paper strip for detecting aflatoxin M1 | |
| CN101566632B (en) | Method for ELISA rapid detection of thermoduric bacteria | |
| CN203069594U (en) | Colloidal gold test strip for detecting erythrocin | |
| CN102455357B (en) | Kit and method for detecting beta-lactamase | |
| CN103134927B (en) | For detecting method and the kit of Shigella | |
| CN202486141U (en) | Reagent kit for detecting beta-lactam antibiotic and melamine | |
| CN103513035A (en) | Test strip and method for detecting aflatoxin M1 | |
| CN201852842U (en) | Kit for detecting beta-lactamase | |
| CN105891412B (en) | A kind of citrobacter freundii test strip and preparation method thereof | |
| Xue et al. | Rapid immunochromatographic assay for Escherichia coli O157: H7 in bovine milk using IgY labeled by Fe3O4/Au composite nanoparticles | |
| CN203178285U (en) | Test paper for rapidly detecting residual cyproheptadine | |
| CN103105494B (en) | A kind of kit and method detecting beta-lactam antibiotic and melamine | |
| CN109738638A (en) | Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli | |
| CN101498726A (en) | Colloidal gold test paper card for detecting enrofloxacin medicament residue | |
| CN202720233U (en) | Test strip for detecting tylosin and tilmicosin | |
| CN104655842B (en) | Quantitative detection method for halomonas strains by adopting indirect enzyme-linked immunosordent assay | |
| CN100449315C (en) | Streptomycin immune chromatographic semiquantitative test paper detecting method | |
| CN200986548Y (en) | Streptomycin quick semi-quantitative determination reagent paper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170823 Address after: 361000 China (Fujian) free trade test zone, Xiamen District, 2165 Hong Kong Road Patentee after: XIAMEN BAINENG INSPECTION TECHNOLOGY SERVICES CO.,LTD. Address before: 361000 2165, Jian Gang Road, Haicang District, Fujian, Xiamen Patentee before: THE INSPECTION AND QUARANTINE TECHNOLOGY CENTER, XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140129 |