CN103777009A - Method using horse radish peroxidase-labeled antibody for detection of swine fever attenuated virus titer - Google Patents
Method using horse radish peroxidase-labeled antibody for detection of swine fever attenuated virus titer Download PDFInfo
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Abstract
A method using a horse radish peroxidase immunological technique for detection of swine fever attenuated virus titer is characterized in that: passage adherent cell MPK cells (Minipig Kidney Cells) are inoculated onto a 96-hole culture plate, an appropriate growth medium is used to maintain the growth of the cells, the MPK cells rapidly proliferate on the 96-hole culture plate, when a single layer of the cells is grown, swine fever virus is inoculated and then is cultured for virus multiplication, after a plurality of days, horse radish peroxidase-labeled antibody and a substrate are added for incubation to make a cell culture developing. By the horse radish peroxidase immunological technique, the virus in the MPK cells can be labeled, polyethylene glycol (PEG) is used to increase the polymerization degree of the virus and the cells so as to promote the infection, the sensitivity of the method is higher than that of a virus titer detection method being specified in pharmacopoeia and using animal experiments, and the method can be used as a method for internal quality control of an enterprise.
Description
Technical field
The present invention relates to a kind of detection method of rapid sensitive, particularly relate to a kind of horseradish peroxidase labeling antibody that uses and detect quickly and efficiently the method for swine fever low virulent strain virus titer as the internal control of the hog cholera vaccine quality of production, belong to veterinary biologics detection technique field.
Background technology
In the method for the detection live vaccines of hog cholera titre of current domestic pharmacopeia regulation, use rabbit as animal used as test, make animal used as test produce hyperthermia immune response by the vaccine liquid of the different extension rates of quiet note, calculate rabbit body infective dose (RID) according to the situation of animal used as test hyperthermia again, thereby determine the contained virus titer of vaccine.In the vaccine development stage, the effect that the detection of virus titer is not limited only to vaccine finished product detects, and also will, through each key link of producing, especially, joining before seedling, need to assess viral titre, to determine content effectively viral in vaccine.In zoopery, each sample needs three groups of animals used as test to detect conventionally, every group of animal used as test is no less than four, therefore each test period at least needs up to a hundred animals used as test to support, only spend in the cost of purchase, transportation and the raising of animal used as test and just need ten thousand yuans of 4-5, in process of the test, also need 4-6 name keeper and experimenter, can spend a large amount of manpower and materials.In addition, the acceptor with animal as test, is difficult to get rid of the individual difference of animal and the impact of experimenter's operation on test, may bring larger error to test.
Summary of the invention
The object of the invention is to find out the zooperal method that substitutes, on the basis using manpower and material resources sparingly, improve conventional efficient, reduce experimental error, the detection method that a kind of weak malicious titre of CSFV of innovation is provided, adopts horseradish peroxidase immunological technique to detect the method for the weak malicious titre of CSFV.
Be different from the use PK-15 cell of describing in pertinent literature as the clone that detects CSFV titre, the present invention has selected the clone identical with production of vaccine, and this is a kind of diverse continuous cell line, miniature pig nephrocyte (MPK).Adapt to domestication through virus, CSFV fast breeding in this clone well, far above using PK-15 cell as the sensitivity detecting with cell; Use this clone can keep the experiment condition mutually unified with research and development link as detecting cell simultaneously.The another kind of method that detects CSFV titre is immunofluorescence technique, but this method is for swine fever low virulent strain relative insensitivity, is difficult to obtain positive findings
[1].
The present invention is owing to adopting horseradish peroxidase immunological technique, sensitivity can reach with body in detect (being zoopery) suitable level, and repeatability is high, experimental result is true, and experimental cost is zooperal 1/20th, only need operating personnel and a reviewing officer to complete, therefore significantly improved the detection efficiency of the weak malicious titre of CSFV, also reduced experimental cost simultaneously, increase the stability of experiment, reduce experimental error, obtained testing result more fast and effectively.
In order to reach object of the present invention, the technical solution that the present invention provides is: this employing horseradish peroxidase immunological technique detects the method for the weak malicious titre of CSFV, be characterized in: pick out the extremely sensitive MPK passage cell of swine fever low virulent strain, this cell is cultured to and covers with individual layer in 96 well culture plates, inoculate afterwards different dilution CSFVs, after continuing to cultivate, add the antibody of horseradish peroxidase-labeled jointly to hatch, finally add substrate to make the cell culture colour developing being infected, account for the percentage calculation virus titer of total hole count by the hole count of colour developing, concrete steps are:
(1) make MPK cell grow up to individual layer in 96 well culture plates;
(2) virus liquid to be measured is carried out to gradient dilution, be inoculated in cell monolayer, maintain Growth of Cells with maintain base;
(3) continue to cultivate after 72～120 hours, by cell culture heat fixation, add horseradish peroxidase labeling antibody to hatch at 37 ℃, then add substrate to hatch under normal temperature;
(4) examine under a microscope the colour developing situation of cell culture, according to the titre of the percentage calculation virus of colour developing hole count.
The MPK cell that the present invention mentions is Minipig Kidney Cell, is go down to posterity attached cell system of one.Through adherent adaptability domestication, obtain adapting to the specificity MPK cell of adherent growth on 96 well culture plates.
Object to better implement the present invention, the cell growth medium using in described step (1), through optimizing, is applicable to the nutrient culture media of MPK Growth of Cells.Every liter of fluid nutrient medium contains the DMEM in high glucose that 5～8 grams of gibco companies produce, 3.5～5.5 grams of M199 that gibco company produces, 1.5～3 grams of NaHCO that Shanghai Hu Shi Chemical Co., Ltd. produces
3, 50～120 milliliters of cow's serums that Taiyuan Run Sheng biomaterial company limited produces; The pH of nutrient culture media is 7.2～7.4.In described step (2), cell culture medium is the maintain base through optimizing.Every liter of fluid nutrient medium contains the DMEM in high glucose that 5～8 grams of gibco companies produce, 3.5～5.5 grams of M199 that gibco company produces, 1.5～3 grams of NaHCO that Shanghai Hu Shi Chemical Co., Ltd. produces
3, 10～40 milliliters of cow's serums that Taiyuan Run Sheng biomaterial company limited produces; The pH of nutrient culture media is 7.2～7.4.
Object to better implement the present invention, the temperature of MPK cell suitable growth in 96 well culture plates is 36.5～37.5 ℃, the pH of suitable cultivation is 7.0～7.5, suitable CO
2concentration is 2%～4%.Before virus inoculation, best incubation time is 96～144 hours, and after virus inoculation, best incubation time is 72～120 hours.
Object to better implement the present invention, the polyglycol (PEG) that the molecular weight that uses Sigma company to produce when the middle virus inoculation of described step (2) is 600～1500 improves the infection rate of virus to cell, and optimum PEG concentration is 1%～4%.
Object to better implement the present invention, in described step (1), the optimum density of cell inoculation is 1～5 × 10
4individual/hole.
Object to better implement the present invention, the HRPO21.2 that the enzyme labelled antibody using is PrioCON company, 3-amino-9-ethyl-carbazole (AEC) DMF that the substrate using is produced for Sigma company forms according to the concentration dissolving configuration of 4mg/ml.
Compared with prior art, the invention has the beneficial effects as follows: a kind of method that adopts horseradish peroxidase immunological technique to detect the weak poison of CSFV is provided, can have fast and effeciently detected the titre of poison a little less than CSFV by this kind of method.Its advantageous feature is concentrated and is:
(1) sensitivity is higher;
(2) detection is comparatively rapid, in the situation that being ready to cell monolayer, within four days, can go out result;
(3) save manpower and cost, significantly improve conventional efficient;
(4) result is easily observed, and easily gets rid of the interference that antigen-antibody non-specific binding is brought.
Accompanying drawing explanation
Fig. 1: use different cells to carry out the result contrast of titre detection;
Fig. 2: use different virus diluted medium to carry out the result contrast of titre detection.
Embodiment
For making advantage and disadvantage of the present invention easier to understand, below by the optimization to experiment condition, the result of comparing animals experiment, in conjunction with specific embodiments, further sets forth the present invention.But these embodiment are only not used in and limit the scope of the invention for the present invention is described; can the details of technical solution of the present invention and form be modified or be replaced lower without departing from the spirit and scope of the present invention, but these modifications and replacement all fall into protection scope of the present invention.
In the following example, NM specific experiment method, carries out according to normal experiment method conventionally.
1. select to connect malicious mode: individual layer connects poison.
2. select cell: PK-15 cell and MPK cell.
3. in the present embodiment, respectively PK-15 cell and MPK cell are pressed to 1～5 × 10 with growth medium
4the density in individual/hole is seeded in 96 porocyte culture plates, every hole 200 μ l.Tissue Culture Plate is placed in constant incubator, and regularization condition is: 36.5～37.5 ℃ of growth temperatures, CO
2concentration is 2%～4%.In 96～144 hours, cell is long for tight adherent individual layer, now can connect poison.
4. virus to be measured is carried out to 10 times of dilutions with the nutrient culture media containing 1%～4%PEG, respectively the virus liquid of different extension rates is inoculated in the culture plate of existing cell monolayer to every hole 100 μ l, each extension rate 8 holes.Virus completed the absorption to cell in 2 hours, now the nutrient solution in culture plate was changed to maintain base.Within 72～120 hours, inner virus reaches expection level in intracellular propagation, can carry out immunoperoxidase experiment.
5. the nutrient culture media in Tissue Culture Plate is discarded, with physiological saline cleaning one time, put into 80 ℃ of thermostatic drying chamber heat fixations 1 hour, after fixing, every hole adds 50 μ l to carry out the HRPO21.2 enzyme labelled antibody of 100 times of dilutions, hatch 1 hour for 37 ℃, with the PBS cleaning containing 0.1%Tween20 5 times, every hole adds 50 μ l AEC substrates again, incubated at room 30min, micro-Microscopic observation.
6. positive hole count is recorded, calculated titre according to the method stipulating in Chinese veterinary pharmacopoeia.Fig. 1 is shown in the contrast of its titre.
1. select to connect malicious mode: individual layer connects poison.
2. select cell: MPK cell.
3. select virus dilution nutrient culture media: containing 1%～4%PEG with do not contain the nutrient culture media of PEG.
4. in the present embodiment, MPK cell is pressed to 1～5 × 10 with growth medium
4the density in individual/hole is seeded in 96 porocyte culture plates, every hole 200 μ l.Tissue Culture Plate is placed in constant incubator, and regularization condition is: 36.5～37.5 ℃ of growth temperatures, CO
2concentration is 2%～4%.After 96～144 hours, cell is long for tight adherent individual layer, now can connect poison.
5. virus to be measured used respectively containing the nutrient culture media of 1%～4%PEG and do not carry out 10 times of dilutions containing the nutrient culture media of PEG, respectively the virus liquid of different extension rates being inoculated in the culture plate of existing cell monolayer to every hole 100 μ l, each extension rate 8 holes.Virus completed the absorption to cell in 2 hours, now the nutrient solution in culture plate was changed to maintain base.After 72～120 hours, virus reaches expection level in intracellular propagation, can carry out immunoperoxidase experiment.
6. the nutrient culture media in Tissue Culture Plate is discarded, with physiological saline cleaning one time, put into 80 ℃ of thermostatic drying chamber heat fixations 1 hour, after fixing, every hole adds 50 μ l to carry out the HRPO21.2 enzyme labelled antibody of 100 times of dilutions, hatch 1 hour for 37 ℃, with the PBS cleaning containing 0.1%Tween20 5 times, every hole adds 50 μ l AEC substrates again, incubated at room 30min, micro-Microscopic observation.
7. positive hole count is recorded, calculated titre according to the method stipulating in Chinese veterinary pharmacopoeia.Fig. 2 is shown in the contrast of its titre.
1. select to connect malicious mode: individual layer connects poison.
2. select cell: MPK cell.
3. in the present embodiment, MPK cell is pressed to 1～5 × 10 with growth medium
4the density in individual/hole is seeded in 96 porocyte culture plates, every hole 200 μ l.Tissue Culture Plate is placed in constant incubator, and regularization condition is: 36.5～37.5 ℃ of growth temperatures, CO
2concentration is 2%～4%.After 96～144 hours, cell is long for tight adherent individual layer, now can connect poison.
4. virus to be measured is diluted to 750RID with the nutrient culture media containing 1%～4%PEG respectively
50, the virus liquid of dilution is inoculated in the culture plate of existing cell monolayer to every hole 100 μ l, inoculation 8 holes.Virus completed the absorption to cell in 2 hours, now the nutrient solution in culture plate was changed to maintain base.After 72～120 hours, virus reaches expection level in intracellular propagation, can carry out immunoperoxidase experiment.
5. the nutrient culture media in Tissue Culture Plate is discarded, with physiological saline cleaning one time, put into 80 ℃ of thermostatic drying chamber heat fixations 1 hour, after fixing, every hole adds 50 μ l to carry out the HRPO21.2 enzyme labelled antibody of 100 times of dilutions, hatch 1 hour for 37 ℃, with the PBS cleaning containing 0.1%Tween20 5 times, every hole adds 50 μ l AEC substrates again, incubated at room 30min, micro-Microscopic observation.
6. observe and show, institute is porose all a colour developing, illustrate that the sensitivity of the method can reach the regulation of veterinary drug allusion quotation to viral effect detection.
The growth medium using in above-described embodiment, through optimizing, is applicable to the nutrient culture media of MPK Growth of Cells.Every liter of DMEM in high glucose that fluid nutrient medium contains 5～8 grams, the M199 of 3.5～5.5 grams, the NaHCO of 1.5～3 grams
3, the cow's serum of 50～120 milliliters, the pH of nutrient culture media is 7.2～7.4.The cell culture medium using in above-described embodiment is the maintain base through optimizing, every liter of DMEM in high glucose that fluid nutrient medium contains 5～8 grams, the M199 of 3.5～5.5 grams, the NaHCO of 1.5～3 grams
3, the cow's serum of 10～40 milliliters; The pH of nutrient culture media is 7.2～7.4.
Claims (8)
1. a method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV, it is characterized in that being to pick out to the extremely sensitive MPK passage cell of swine fever low virulent strain, this cell is cultured to and covers with individual layer in 96 well culture plates, inoculate afterwards different dilution CSFVs, after continuing to cultivate, add the antibody of horseradish peroxidase-labeled jointly to hatch, finally add substrate to make the cell culture colour developing being infected, the percentage calculation virus titer that accounts for total hole count by the hole count of colour developing, concrete steps are:
(1) make MPK cell grow up to individual layer in 96 well culture plates;
(2) virus liquid to be measured is carried out to gradient dilution, be inoculated in cell monolayer, maintain Growth of Cells with maintain base;
(3) continue to cultivate after 72～120 hours, by cell culture heat fixation, add horseradish peroxidase labeling antibody to hatch at 37 ℃, then add substrate to hatch under normal temperature;
(4) examine under a microscope the colour developing situation of cell culture, according to the titre of the percentage calculation virus of colour developing hole count.
2. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1; it is characterized in that the cell growth medium using in step (1) is through optimizing; be applicable to the nutrient culture media of MPK Growth of Cells; wherein every liter of fluid nutrient medium contains the DMEM in high glucose that 5～8 grams of gibco companies produce; 3.5～5.5 grams of M199 that gibco company produces, 1.5～3 grams of NaHCO that Shanghai Hu Shi Chemical Co., Ltd. produces
3, 50～120 milliliters of cow's serums that Taiyuan Run Sheng biomaterial company limited produces, the pH of nutrient culture media is 7.2～7.4.
3. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, is characterized in that in step (2), cell culture medium is the maintain base through optimizing.Wherein every liter of fluid nutrient medium contains the DMEM in high glucose that 5～8 grams of gibco companies produce, 3.5～5.5 grams of M199 that gibco company produces, 1.5～3 grams of NaHCO that Shanghai Hu Shi Chemical Co., Ltd. produces
3, 10～40 milliliters of cow's serums that Taiyuan Run Sheng biomaterial company limited produces, the pH of nutrient culture media is 7.2～7.4.
4. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, the temperature that it is characterized in that MPK cell suitable growth in 96 well culture plates is 36.5～37.5 ℃, the pH of suitable cultivation is 7.0～7.5, suitable CO
2concentration is 2%～4%, and before virus inoculation, best incubation time is 96～144 hours, and after virus inoculation, best incubation time is 72～120 hours.
5. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, the polyglycol (PEG) that the molecular weight that uses Sigma company to produce while it is characterized in that the middle virus inoculation of step (2) is 600～1500 improves the infection rate of virus to cell, and preferred PEG concentration is 1%～4%.
6. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, is characterized in that it is 1～5 × 10 that the middle cell of step (1) is inoculated preferred density
4individual/hole.
7. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, the HRPO21.2 that the enzyme labelled antibody that it is characterized in that use is PrioCON company.
8. a kind of method that adopts horseradish peroxidase immunological technique to detect the weak malicious titre of CSFV according to claim 1, the substrate that it is characterized in that use is that 3-amino-9-ethyl-carbazole (AEC) DMF that Sigma company produces forms according to the concentration dissolving configuration of 4mg/ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106383226A (en) * | 2016-08-19 | 2017-02-08 | 金宇保灵生物药品有限公司 | A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit |
| CN106940371A (en) * | 2017-04-13 | 2017-07-11 | 中国兽医药品监察所 | A kind of pestivirus culture and malicious quantitative detecting method living |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1196394A (en) * | 1997-02-21 | 1998-10-21 | 吉林省卫生防疫站 | Enzyme immuno spot method for determining virus titre |
| WO2003004605A2 (en) * | 2001-07-06 | 2003-01-16 | Geron Corporation | Mesenchymal cells and osteoblasts from human embryonic stem cell |
| CN102191224A (en) * | 2010-03-12 | 2011-09-21 | 复旦大学 | Heavy targeted modification method for herpes simplex viruses and application thereof |
| CN102294029A (en) * | 2010-06-28 | 2011-12-28 | 北京华威特生物科技有限公司 | Preparation method and product of swine fever live vaccine |
| CN102455359A (en) * | 2010-10-19 | 2012-05-16 | 上海生博生物医药科技有限公司 | Kit used for detection of V-type adenovirus titer and application thereof |
-
2012
- 2012-10-18 CN CN201210396578.4A patent/CN103777009B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1196394A (en) * | 1997-02-21 | 1998-10-21 | 吉林省卫生防疫站 | Enzyme immuno spot method for determining virus titre |
| WO2003004605A2 (en) * | 2001-07-06 | 2003-01-16 | Geron Corporation | Mesenchymal cells and osteoblasts from human embryonic stem cell |
| CN1636054A (en) * | 2001-07-06 | 2005-07-06 | 杰龙公司 | Mesenchymal cells and osteoblasts from human embryonic stem cell |
| CN102191224A (en) * | 2010-03-12 | 2011-09-21 | 复旦大学 | Heavy targeted modification method for herpes simplex viruses and application thereof |
| CN102294029A (en) * | 2010-06-28 | 2011-12-28 | 北京华威特生物科技有限公司 | Preparation method and product of swine fever live vaccine |
| CN102455359A (en) * | 2010-10-19 | 2012-05-16 | 上海生博生物医药科技有限公司 | Kit used for detection of V-type adenovirus titer and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王镇等: "猪瘟病毒的形态结构与形态发生", 《微生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106383226A (en) * | 2016-08-19 | 2017-02-08 | 金宇保灵生物药品有限公司 | A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit |
| CN106940371A (en) * | 2017-04-13 | 2017-07-11 | 中国兽医药品监察所 | A kind of pestivirus culture and malicious quantitative detecting method living |
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