CN107287167B - The hybridoma of the anti-A rotavirus monoclonal antibody of mouse and its monoclonal antibody of secretion can be secreted - Google Patents
The hybridoma of the anti-A rotavirus monoclonal antibody of mouse and its monoclonal antibody of secretion can be secreted Download PDFInfo
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Abstract
The present invention relates to the monoclonal antibodies for the hybridoma and its secretion that can secrete the anti-A rotavirus monoclonal antibody of mouse.The purposes for detecting the test strip of A rotavirus in sample, test strip comprising the monoclonal antibody etc. are prepared the invention further relates to the monoclonal antibody.The monoclonal antibody specificity of the present invention is strong, high sensitivity, stability are good, available for the A rotavirus in quickly and accurately sample survey.
Description
Technical field
The present invention relates to the miscellaneous of the anti-A rotavirus monoclonal antibody of mouse, its purposes and the secretion monoclonal antibody
Hand over oncocyte.
Background technology
A rotavirus (Group A Rotavirus, RV) is the single main cause for causing global severe childhood diarrhea, and
And human and animal's acute gastrointestinal disease can be caused.The virus infects intestinal epithelial cell first, in turn results in cell and injures,
Cause diarrhea, it will threat to life when situation is serious.Global almost each about 5 years old child once infected RV at least one
It is secondary.It compares with the pathogen of other initiation enterogastritis, such as high fever, vomiting, the vomiting serious symptoms with diarrhea of RV initiations
Frequency higher.It shows according to investigations, each annual in the whole world has 600,000 children to die of RV infection, accounts for global less than 5 years old children
The 5% of total death toll, the death for having more than 85% are to be happened at the developing countries such as Africa and Asia.In the U.S., because obtaining stomach
Enteritis and in the affected children ranges be hospitalized 30%-70% be because RV infects, and in China, the caused acute stomach and intestine of virus infection
Inflammation accounts for 30%-40%, and each year, therefore there are about 30,000 by dead less than 5 years old children.RV infection has not only seriously jeopardized infant's
Health and life quality, and huge financial burden is brought to family and society, RV infection become one it is urgently to be resolved hurrily
Important public hygiene problem.By the end of current, the specific drug of RV infection can not be treated effectively still on the market, therefore study fast
Fast and accurate RV early detections method has far-reaching significance the prevention of the virus.
At present in the detection technique of rotavirus, dependable with function is satisfactory to be still the World Health Organization
The immunological technique that WHO recommends, such as ELISA double antibody sandwich methods.The detection object of most of immunological technique is Feces of Patients
Wait the RV antigens in samples, the combination dependent on RV antigens and monoclonal antibody.In addition, the blood red egg being difficult to avoid that in sample
In vain, the ingredients such as bilirubin, chyle will also tend to the combination of interference monoclonal antibody and antigen, so as to laboratory or clinical sample
The detection of product causes undesirable influence.Therefore, the characteristics such as the potency of the monoclonal antibody used in detection, can be to the special of detection
Property with sensitivity cause very big influence.
It is more demanding to preservation condition, operating condition based on the test strip of monoclonal antibody.In laboratory or clinic
In practical operation, particularly in poor remote districts of experiment/medical condition etc., the business water of preservation condition and operating personnel
It is flat to be often unable to reach standard requirement sometimes.For example, reagent by multigelation or may freeze due to power failure or misoperation
Overlong time.In hot backward areas (such as Africa), reagent may be accidentally placed in the environment of high temperature thermal acceleration.This
In the case of, conventional test strips often occur character variation, testing result is caused to be not sufficiently stable reliably, this be also people for a long time with
To expect to solve the problems, such as.
Invention content
Hybridoma prepared by the present invention can secret out of the anti-A rotavirus monoclonal antibody of mouse of high-titer.This
When the monoclonal antibody of invention is applied to the test strips such as colloidal gold platform of RV in quick sample survey, high specificity, sensitivity
Height, stability are good.
The present invention is using classical authentic monoclonal antibody technology.The limited public affairs of safe biotechnology (are opened in Hangzhou with RV antigens
Department) 2 Balb/C mouse are immunized, choosing the preferable mouse of antibody titer after serum screening takes spleen to be used for cell fusion, after fusion
The hybridoma cell strain of 2 plants of energy anti-A rotavirus monoclonal antibodies of stably excreting mouse is cloned, filtered out through limiting dilution assay,
Then by preparing mouse ascites, the anti-A rotavirus monoclonal antibody of mouse is obtained after purified, and be prepared into detection kit
Detect clinical sample.Secreted by the hybridoma cell strain for secreting the anti-A rotavirus monoclonal antibody of mouse that we prepare
The specific binding of high-titer can occur with purpose antigen for monoclonal antibody.It is fast using double antibody sandwich method on colloidal gold platform
In fast qualitative detection excrement during RV, high specificity, high sensitivity, stability are good.
Description of the drawings
Fig. 1 illustrates the anti-A rotavirus monoclonal antibody 1 of mouse of the present invention and the anti-A rotavirus monoclonal of mouse resists
Body 2 and the antibody of the anti-A rotavirus monoclonal antibody 3 of two kind of commercialized mouse and the anti-A rotavirus monoclonal antibody 4 of mouse
The result of bioactivity.Using LOG (dilution) as abscissa, antibody OD values are as ordinate.
Fig. 2 illustrates showing for the colloidal gold test of the monoclonal antibody of the anti-A rotavirus of mouse using the present invention
It is intended to.1-PVC bottom plates, 2- sample pads, 3- colloidal gold pads, 4- coated films, 5- markers coating area (T lines), 6- Quality Controls object coating
Area's (C lines), 7- water absorption pads.
Fig. 3 illustrates test strip of the present invention and explains schematic diagram for the positive, feminine gender, the null result when detecting.
Embodiment
Include following embodiment herein, for becoming apparent from, being explicitly described the technical side of the present invention in an exemplary manner
Case.Those skilled in the art should be appreciated that according to disclosure herein to be permitted in disclosed specific embodiment
It is change but still obtain similar or similar as a result, without departing from thought and range of the invention more.The specific reality of the present invention
The mode of applying is only used for explaining the present invention, and is not intended to limit the present invention by any mode.
Embodiment 1
The preparation and screening of hybridoma cell strain
(1) RV mice immunized with antigen
By RV antigens (be purchased from Hangzhou Kitgen Biotechnology Co., Ltd.) normal saline dilution to 2.0mg/ml, with Freund
Freund's complete adjuvant isometric mixing (purchased from Sigma companies, article No. SLBF-9338V), is oil emulsion with the emulsification of 1ml syringes, directly
Do not disperse to stop emulsifying to the oil emulsion instilled in water.The lotion is subcutaneously applied with the dosage four limbs armpits of 100 μ l/ only
Give BALB/c mouse (purchased from Chengdu up to large Experimental Animal Center, 6 week old are female, 2).Enhance after 14 days immune for the first time and exempt from
Epidemic disease takes antigen to be emulsified after isometric mixing (purchased from Sigma companies, article No. SLBM9367V) with incomplete Freund's adjuvant, immunizing agent
It measures as 50 μ l/ only.Enhance week about later and be immunized once, immune every time to adopt tail blood before, separation serum uses indirect ELISA
Method measures potency.After 5 times immune, 2 mice serum potency are all higher than 1:106, you can for merging.3 days before fusion, RV is taken to resist
Original normal saline dilution is to 2.0mg/ml, then mixes tail vein supplementary immunization in equal volume with physiological saline, and dosage is 50 μ l/
Only.
(2) preparation of hybridoma cell line
1. the preparation of feeder cells
Feeder cells are made with normal 10 week old BALB/c mouse peritoneal macrophages.1 day before fusion, BALB/c takes blood
Neck is drawn to put to death, 0.1% bromogeramine impregnates 1 minute, is transferred to 75% alcohol and impregnates 1 minute.With cutting under sterile working in super-clean bench
Knife abdominal cut skin, exposure peritonaeum, with syringe Intraperitoneal injection RPMI1640 basic culture solution 3ml, after taken out with dropper, then
RPMI1640 basic culture solutions 7ml is added in rinse repeatedly.Flushing liquor is recycled, 1000rpm is centrifuged and stayed precipitation in 5 minutes, with having added in
The RPMI1640 culture solutions of 20% newborn bovine serum are resuspended, and adjustment cell concentration is 3-5 × 105A/ml, 96 orifice plates of addition, 100
μ l/ holes, 37 DEG C, 5%CO2Culture.
2. the preparation of immune spleen cell
After mouse supplementary immunization three days, spleen is aseptically taken out, is placed in plate, RPMI1640 basic culture solutions
Rinse primary, shred, grind, filter after the splenocyte that is disperseed, 1000rpm, which is centrifuged, stays precipitation for 5 minutes, and RPMI1640 is basic
Culture solution is resuspended, and the dilution of 3% acetic acid counts.
3. the preparation of myeloma cell
Murine myeloma cell Sp2/0 (being purchased from Cell Bank of Chinese Academy of Sciences) is cultivated after 8-anaguanine screens to right
In number growth period, take 6 big bottle (75cm2) cell suspension is made, 1000rpm is centrifuged 5 minutes and is stayed precipitation, is cultivated with RPMI1640 bases
Liquid is resuspended, counts, by 0.5-2 × 105The cell concentration of a/ml carries out sub-bottle culture, and (ordinary circumstance was replaced primary per 1-2 days
Complete 1640 culture mediums of 15-30ml).
4. cell fusion and HAT selection culture hybridomas
Myeloma cell and immune spleen cell are pressed 1:8 ratio mixing, RPMI1640 is used in 50ml conical centrifuge tubes
Basic culture solution is washed 1 time, and 1000rpm is centrifuged 5 minutes and stayed precipitation.By cell mixing, it is slowly added to the PEG4000 of 1.2ml50%
Fusion, fusion adds in 30ml RPMI1640 basic culture solutions after 1 minute terminate cell fusion.700rpm/min is centrifuged 5 minutes
It is resuspended in afterwards in 1640 culture mediums containing 1%HAT and 20% newborn bovine serum, averagely instills 30 set of 96 porocyte culture plates.
37 DEG C, 5%CO2Culture, next day is added to be expired containing 1%HAT and 1640 culture mediums to the hole of 20% newborn bovine serum.5 days later half
Culture medium is changed, culture medium is partly changed again after 7 days.
5. the screening of positive cell strain
With 0.06M pH9.6 carbonate buffer solutions dilution RV antigens (Hangzhou Kitgen Biotechnology Co., Ltd.) to 5 μ g/
Ml is coated with 100 μ l in 96 hole elisa Plates, for detecting cells and supernatant per hole.It is positioned in refrigerator and stays overnight for 2-8 DEG C, second
It abandons liquid in hole, and ELISA washing lotions board-washing three times, pats dry, with 150 μ of PBS of the 0.01M pH7.2 containing 10% calf serum
L/ holes, 37 DEG C are closed 2 hours, are patted dry, Vacuum Package is for use.The 9th day after splenocyte fusion, 100 μ l of cell conditioned medium are taken in above-mentioned
In 96 hole detection plates, 37 DEG C are incubated 40 minutes, and ELISA board-washings add in 5000 times of diluted horseradish peroxidase marks after machine-washing five times
The 100 μ L of sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of note, after 37 DEG C of incubations are same as above board-washing in 30 minutes, often
Hole adds in 100 μ L developing solutions (containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphorus acid bufferings
Liquid), 37 DEG C are incubated 10 minutes, and 50 μ L 2M sulfuric acid solutions are added in per hole and terminate reaction, survey 450nm absorption values.Mouse during merging
Serum is diluted to 100 times as positive control, and 1640 complete culture solutions of RPMI are as negative control, negative control OD value ＜ 0.2,
Positive control OD values ＞ 1.8 is effective for detecting system, for the positive, otherwise is the moon during sample OD value >=2 × negative control OD value
Property.Secretory antibody positive cell hole is cloned with 1 cells/well on 96 well culture plates with limiting dilution assay, screening positive Kong Yi
Continuously clone four times of upper method, reach 100% monoclonal, are transferred to 24 holes and continue to cultivate, and are transferred to when cell covers with 80% thin
Born of the same parents' bottle expands culture, and sub-bottle is passed on when cell covers with cell bottle 80%, during cell growth to the logarithmic phase of passage, with appropriate nothing
Serum RPMI-1640 culture medium cell dispersion bottle inner cells collect cell suspension in conical centrifuge tube, record cell suspension body
Product V, takes appropriate cell suspension to carry out cell count, cell suspension density (a/ml) is obtained, after remaining 1000rpm centrifuges 5min
Supernatant is abandoned, the cell number of precipitation is calculated according to cell density and centrifugation precursor, appropriate frozen stock solution is added in into cell precipitation
Cell density is adjusted to 4-8 × 106Then a/ml is sub-packed in sterile cryopreservation tube, every cryopreservation tube refinement cytosol 0.5ml.Carefully
Born of the same parents' fusion obtains the hybridoma cell strain RVAb-1 of 2 plants of energy anti-A rotavirus monoclonal antibodies of stably excreting mouse with hybridizing altogether
Tumor cell strain RVAb-2, and the antibody secreted respectively can be matched, and with double antibody sandwich method detection RV antigens March 30 in 2017
It is deposited in China typical culture collection center (China, Wuhan, Wuhan University) day, preserving number is respectively CCTCC NO：
C201743、CCTCC NO：C201744.
Embodiment 2
The preparation of monoclonal antibody
The BALB/c mouse of 6-8 weeks health is chosen, (Tianjin section is close europeanized for every mouse peritoneal injection 0.5mL atoleine
Learn reagent Co., Ltd), every mouse peritoneal injection 0.5~1 × 10 after 7 days6A hybridoma cell strain RVAb-1 or hybridoma
Cell strain RVAb-2.Inoculating cell generates ascites after 7-10 days, the health status and sign of ascites of close observation mouse are as treating ascites
It is as more as possible, and before mouse is dying, put to death mouse, sucked ascites in test tube with dropper, two plants of cells collect respectively 8mL with
10mL ascites.Supernatant is taken after the ascites 4000rpm centrifugations 10min of collection, sample is taken to be put in -20 DEG C of refrigerators respectively and is preserved.Ascites
Purify after sulfate of ammoniac saturation precipitation, then with Protein A affinity chromatography, two plants of monoclonal antibodies that SDS-PAGE is detected are pure
Degree is all higher than 90%.
Embodiment 3
Bioactivity
(1) Hybridoma Cell Culture supernatant bioactivity
With 0.06M pH9.6 carbonate buffer solutions dilution RV antigens (Hangzhou Kitgen Biotechnology Co., Ltd.) to 5 μ g/
Ml is coated with 100 μ l in 96 hole elisa Plates per hole.It is positioned in refrigerator and stays overnight for 2-8 DEG C, abandon liquid in hole within second day, ELISA is washed
Trigger is washed three times, is patted dry, and with the PBS of the 0.01M pH7.2 containing 10% calf serum, 150 μ l/ holes, 37 DEG C of closings abandon liquid in 2 hours
Body pats dry, for detecting Hybridoma Cell Culture supernatant potency, titer of ascites, antibody titer after purification.Hybridoma is trained
It is former times supernatant culture solution to support the first hole of supernatant bioactivity, from the second hole to the 4th hole with the PBS buffer solution of 0.01M pH7.2
10 times dilute step by step, and the 5th hole to the tenth hole is diluted step by step with 2 times.Mice serum is diluted to 100 times when 11-holes are to merge
Make positive control, negative control is made in the 12nd hole with 1640 complete culture solutions of RPMI, is 100 μ l per hole sample volume.37 DEG C incubate
It educates 40 minutes, ELISA board-washings add in sheep anti-mouse igg (Beijing of 5000 times of diluted horseradish peroxidase labels after machine-washing five times
Bioisystech Co., Ltd of Zhong Shan Golden Bridge) 100 μ L, after 37 DEG C of incubations are ibid washed for 30 minutes, 100 μ L are added in per hole and contain 0.1% (M/
V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 10 minutes, and 50 μ L are added in per hole
2M sulfuric acid solutions terminate reaction, survey 450nm absorption values.Negative control OD value ＜ 0.2, positive control OD values ＞ 1.8 are detection system
System is effective, on the contrary for feminine gender for the positive when OD value >=2 × negative control OD value.It is dilute corresponding to the minimum positive hole of detected value
The ratio of releasing is Hybridoma Cell Culture supernatant potency, and hybridoma cell strain RVAb-1 culture supernatants potency is more than 1:1024, it is miscellaneous
Tumor cell strain RVAb-2 culture supernatants potency is handed over to be more than 1:512.
(2) titer of ascites detects
ELISA detection method is the same as described in (1) above part.Dilution process is different, specially：First hole is former times abdomen
Water is diluted with the PBS buffer solution of 0.01M pH7.2 from the second hole to seven apertures in the human head 10, step by step again from octal to 11-holes with 2
It dilutes step by step again.Dilution ratio corresponding to the minimum positive hole of detected value is titer of ascites, hybridoma cell strain RVAb-1 with
Titer of ascites prepared by hybridoma cell strain RVAb-2 is all higher than 1:5×105。
(3) antibody titer detects
First by the anti-A rotavirus monoclonal antibody of two plants of mouse of the present invention, (Sichuan mikey biology new material technology is limited
Company) 1mg/ml is uniformly diluted to the PBS buffer solution of 0.01M pH7.2, after dilute initial first hole of 100 times of conducts again, from
Second hole starts to dilute step by step for 3 times to 11-holes work, is 100 μ l per hole injection volume.37 DEG C are incubated 50 minutes, and ELISA is washed
Trigger washes after five times sheep anti-mouse igg (the Sichuan mikey biological material skill for adding in 12000 times of diluted horseradish peroxidase labels
Art Co., Ltd) 100 μ L, 37 DEG C are incubated after 1h ibid wash, and 100 μ L are added in per hole containing 0.1% (M/V) o-phenylenediamine, and 0.1%
(V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 15 minutes, and 50 μ L 2M sulfuric acid solutions are added in per hole and are terminated
Reaction detects 450nm absorption values.Negative control OD value ＜ 0.2, positive control OD values ＞ 1.8 are effective for detecting system.Antibody is imitated
Valency criterion：With LOG (dilution) for abscissa, make curve, curvilinear equation y=min+ by ordinate of antibody OD values
(max-min)/(1+10^ ((logEC50-x) × Hillslope)), by sigmaplot data processing software matched curves,
Take middle titer=10logEC50.As a result show that the middle titer of the anti-A rotavirus monoclonal antibody of two plants of mouse of the present invention is equal
More than 6 × 104.Hangzhou Kitgen Biotechnology Co., Ltd. that in kind control test has been commercialized can double-antibody sandwich
Method detects the anti-A rotavirus monoclonal antibody of pairing mouse of RV antigens, and by fitting, middle titer is 6-8 × 103, it is aobvious
It writes and is below two plants of pairing antibody that hybridoma of the present invention obtains.Table 1 and Fig. 1 lists titration result.
1 antibody titer of table detects
Embodiment 4
The preparation and use of test strip
(1) preparation of test strip
The anti-A groups of colyliforms disease of mouse that the hybridoma cell strain RVAb-1 and hybridoma cell strain RVAb-2 of the present invention secretes respectively
Malicious monoclonal antibody 12 energy of A rotavirus monoclonal antibody anti-with mouse should be in colloidal gold platform fast qualitative detection excrement
RV.The test strip is by being included in the sample pad, colloidal gold pad, coating that sequentially fixed and end interconnects on PVC bottom plates
Film, water absorption pad composition, the colloidal gold pad are coated with the colloidal gold composite of the anti-A rotavirus antigen monoclonal antibody 2 of mouse,
Being coated with of being spaced from sequentially is distributed on the coated film on membrane body along the direction from sample pad to water absorption pad
Marker coating area (T lines) and Quality Control the object coating area (C lines) of the anti-A rotavirus antigen monoclonal antibody 1 of mouse.Label
Concentration, coating concentration, a stroke film spacing preferred embodiment see the table below 2,3,4.In the test strips of the present invention, Quality Control coating object is selected from anti-mouse
IgG, anti-rabbit IgG, rabbit igg, biotin, Avidin or albumen for being combined with biotin or Avidin etc., preferably anti-mouse IgG.Figure
2 schematic diagram for the test strips, 1- bottom plates, 2- sample pads, 3- colloidal gold pads, 4- coated films are coated with the anti-A rotavirus of mouse
The colloidal gold composite of antigen monoclonal antibody 2 (RVAb-2 secretions), 5- markers coating area (T lines) are coated with the anti-A groups of wheels of mouse
Shape virus antigen monoclonal antibody 1 (RVAb-1 secretions), 6- Quality Controls object coating area (C lines), 7- water absorption pads.Fig. 3 is of the invention
Negative, positive, null result explains schematic diagram.
2 label concentration of table
Table 3 is coated with concentration
Table 4 draws film spacing
Alternative solution:Anti- mouse IgG by anti-rabbit IgG, rabbit igg, biotin, Avidin and can be combined with biotin or affine
The albumen of element substitutes.
(2) antijamming capability of test strip of the present invention is detected
By negative with being separately added into hemoglobin, bilirubin, chyle in positive reference product, then this is prepared by said program
The test strip of invention simultaneously examines above processed reference material, is respectively repeated 10 times detection, result colour developing is consistent.As a result table
Bright hemoglobin, bilirubin, chyle are on the test strip of the present invention without influence.Safe biological skill is opened using Hangzhou with similarity condition
Art Co., Ltd can double antibody sandwich method detection RV antigens mouse anti-A rotavirus pairing Antibody preparation test strip,
The judgement of its testing result is correct, but there is the phenomenon that inconsistent of developing the color, and shows that hemoglobin, bilirubin, chyle open Thailand to Hangzhou
Bioisystech Co., Ltd can double antibody sandwich method detection RV antigens mouse anti-A rotavirus pairing Antibody preparation test paper
Item has certain influence, and testing result is listed in the following table 5.
5 antijamming capability of table detects
(3) sensitivity technique
The test strip prepared using the above scheme examines 100 positives and 100 negative samples respectively.Table 6
Listing test strip prepared by the monoclonal antibody of the present invention, (the safe biology medicine company share in Beijing ten thousand has with commercialization test strips
Limit company) comparative experiments, inspection result shows the anti-A rotavirus monoclonal antibody 1 of mouse and the anti-A groups of colyliforms of mouse of the present invention
Its sensitivity of test strip and specificity prepared by viral monoclonal antibodies 2 is above commercialization test strips, is listed in table 6
Testing result.
Table 6 is positive to be examined with negative sample
(4) Detection of Stability
By the anti-A rotavirus monoclonal antibody 1 of mouse and the anti-A rotavirus monoclonal antibody 2 of mouse of the present invention with
It is handled under the conditions of lower：A-20 DEG C of multigelation 3 times；B-20 DEG C of multigelation 4 times；C-20 DEG C of multigelation 5 times；D-20 DEG C of preservation
12 months；37 DEG C of e thermal accelerations 7 days；37 DEG C of f thermal accelerations 20 days, other conditions are constant, are prepared into Test paper as stated above
Item examines 10 parts of positive reference product and 10 parts of negative reference product respectively, and coincidence rate is 100%.Show two strain antibodies of the present invention
It has good stability, and good using test strip stability, the accuracy of Antibody preparation of the present invention.It is handled under similarity condition
Commercialized B producers (Hangzhou Kitgen Biotechnology Co., Ltd.) can double antibody sandwich method detection RV antigens anti-A groups of colyliforms
Virus pairing antibody, and test strip is prepared, this 10 parts of positive reference product and 10 parts of negative reference product are detected respectively.As a result it shows
Show its multigelation 4 to 5 times or -20 DEG C preserve 12 months or thermal acceleration and partly start to degrade after 7 days, inspection result and reference
Product not in full conformity with.Testing result is listed in table 7.
7 stability test of table
It can be seen that monoclonal antibody produced by the present invention has still maintained very high steady under conditions of changeable, harsh
It is qualitative.This characteristic ensure that when the test strip for detecting the RV in sample is made, and test strip obtained can be with
Suitable for kinds of experiments room and clinical condition, and its stabilization is still maintained after Cord blood, multigelation, thermal acceleration
Reliability.
Claims (12)
1. the hybridoma of the anti-A rotavirus monoclonal antibody of mouse can be secreted, preserving number is CCTCC NO：C201743.
2. the anti-A rotavirus monoclonal antibody 1 of mouse, is secreted by hybridoma according to claim 1.
3. the hybridoma of the anti-A rotavirus monoclonal antibody of mouse can be secreted, preserving number is CCTCC NO：C201744.
4. the anti-A rotavirus monoclonal antibody 2 of mouse, is secreted by hybridoma according to claim 3.
5. the monoclonal antibody according to claim 2 or 4 prepares the Test paper for detecting A rotavirus in sample
The purposes of item.
6. purposes according to claim 5, wherein the sample is excrement.
7. for detecting the test strip of A rotavirus in sample, it includes the Dan Ke according to claim 2 and 4
Grand antibody.
8. test strip according to claim 7, wherein the sample is excrement.
9. test strip according to claim 7 or 8 is colloid metal type test strips.
10. test strip according to claim 9, the sample that sequentially fixed and end interconnects on bottom plate
Pad, colloidal gold pad, coated film, water absorption pad are coated with the anti-A groups of colyliforms of mouse according to claim 2 in the colloidal gold pad
The colloidal gold composite of viral monoclonal antibodies 1, along the direction from sample pad to water absorption pad in membrane body on the coated film
On the detection zone T being spaced from and quality control region C is sequentially distributed with, wherein the detection zone T is coated with according to claim 4 institute
The anti-A rotavirus monoclonal antibody 2 of mouse stated.
11. test strip according to claim 10, wherein the quality control region C is coated with anti-mouse IgG, rabbit igg, biology
Element, Avidin or the albumen for being combined with biotin or Avidin.
12. test strip according to claim 11, wherein the quality control region C is coated with anti-mouse IgG.
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