CN102719406A - Monoclonal antibody resistant to nephropathogenic avian infectious bronchitis virus strain DS10 - Google Patents
Monoclonal antibody resistant to nephropathogenic avian infectious bronchitis virus strain DS10 Download PDFInfo
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Abstract
The invention provides a monoclonal antibody resistant to a nephropathogenic avian infectious bronchitis virus strain DS10 and belongs to the technical field of biology. The invention further provides a hybridoma cell strain 2G4 capable of secreting the monoclonal antibody resistant to the nephropathogenic avian infectious bronchitis virus strain DS10, and the preservation number of the hybridoma cell strain is CGMCC No.6172. The hybridoma cell strain 2G4 is capable of efficiently and stably secreting the monoclonal antibody resistant to the nephropathogenic IBV (infectious bronchitis virus) strain DS10, and the titer reaches up to 1:102000. After authenticated, a single reaction zone can appear when specific binding is performed between the monoclonal antibody and the nephropathogenic IBV strain DS10, and the fact proves that the monoclonal antibody resistant to the nephropathogenic infectious bronchitis virus strain DS10 is the specific antibody of the nephropathogenic infectious bronchitis virus strain DS10.
Description
Technical field
The invention belongs to biological technical field, relate to the antibody engineering technology, be specifically related to the anti-monoclonal antibody of having a liking for kidney type avian infectious bronchitis virus DS10 strain.
Background technology
Chicken infectious bronchitis (Avian Infectious Bronchitis; IB) be IBV (Infectious Bronchitis Virus by coronavirus genus; IBV) a kind of acute, the height contagious disease of the chicken that causes are principal character to cause chicken respiratory infection, ephritis, egg drop reduction, can cause that young chicken death rate is up to 40%; The chicken that grows up infects back egg productivity decline 10%-50%, brings great financial loss for world's poultry husbandry.
Avian infectious bronchitis virus (IBV) has multiple serotype and many tissue tropisms; Since 1931 report breathing pattern IB the earliest; Reported infectious bronchitiss such as kidney type, visible peristalsis visible intestinal peristalsis, reproductive tract type, glandular stomach type subsequently, kind surplus the serotype of having reported so far reaches 30, new in recent years serotype and variant still constantly occur; Brought greatly difficulty for EPDML research and epidemic prevention, also caused serious economy loss to poultry husbandry.Vaccinated flock of China and non-vaccinated flock all are prone to have a liking for the infection of kidney type IBV, and that has wherein reported has the kidney of having a liking for type avian infectious bronchitis virus DS10 strain, a HF2 strain etc.
One of control key of having a liking for kidney type chicken infectious bronchitis is to set up the method for quick diagnosis; The method of present domestic diagnosis IB remains isolation identification, serological test and the diagnosis of molecular biology method (RT-PCR) etc. of virus; But these methods or waste time and energy, otherwise operation is too loaded down with trivial details, and laboratory condition requires high; Be inappropriate for the diagnosis of a large amount of clinical samples, be difficult to apply.
Summary of the invention
The purpose of this invention is to provide a kind of can efficiently the secretion and resist the hybridoma cell strain 2G4 that has a liking for kidney type IBV DS10 strain monoclonal antibody.
Another object of the present invention provides that hybridoma cell strain 2G4 excretory is anti-has a liking for kidney type IBV DS10 strain monoclonal antibody, and this monoclonal antibody can specific identification be had a liking for kidney type avian infectious bronchitis virus DS10 strain.
A purpose more of the present invention provides can be quick, easy, detects the test kit of having a liking for kidney type avian infectious bronchitis virus exactly.
The present invention provides a kind of anti-hybridoma cell strain 2G4 that has a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody that secretes; This hybridoma cell strain is preserved in (address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Its preserving number is CGMCC No.6172; Preservation date is: on May 28th, 2012, the anti-hybridoma 2G4 that has a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody of classification called after secretion.
The present invention provides that said hybridoma cell strain excretory is anti-has a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody.
The present invention also provides a kind of test kit of having a liking for kidney type avian infectious bronchitis virus that is used to detect, and said test kit comprises the said anti-kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody of having a liking for.
It is simple and feasible that the present invention has a liking for the antigenic purifying mode of kidney type avian infectious bronchitis virus DS10 strain (be abbreviated as and have a liking for kidney type IBV DS10 strain); Hybridoma cell strain 2G4 excretory is anti-has a liking for kidney type IBV DS10 strain monoclonal antibody high specificity, and the preparation method is simple; Hybridoma cell strain 2G4 can be efficiently, stably excreting is anti-has a liking for kidney type IBV DS10 strain monoclonal antibody; Tire up to being 1:102000; Through identify this monoclonal antibody can with have a liking for kidney type IBV DS10 strain specificity and combine; A single reaction zone occurs, prove that anti-to have a liking for kidney type IBV DS10 strain monoclonal antibody be to have a liking for the specific antibody of kidney type IBV DS10 strain.Have a liking for kidney type IBV DS10 strain monoclonal antibody because this hybridoma cell strain 2G4 can secrete to resist efficiently, make follow-up purification step simple and efficient.It is provided by the invention that anti-to have a liking for kidney type IBV DS10 strain monoclonal antibody be that the fast monitored of having a liking for the clinical quick diagnosis of kidney type IBV and having a liking for kidney type IBV vaccine immunity provides guarantee.Adopt the anti-test kit of having a liking for kidney type IBV DS10 strain Monoclonal Antibody of the present invention can be quick, easy, detect avian infectious bronchitis virus exactly.
Description of drawings
Fig. 1 representes that the indirect ELISA experiment detects the specificity result of hybridoma cell strain 2G4 excretory monoclonal antibody.Among Fig. 1, ordinate zou is the OD that hybridoma cell strain 2G4 excretory monoclonal antibody and different virus reaction back ELIASA detect
450Value, AIV (H9) is bird flu (H9 hypotype) virus, IBDV is an infections chicken cloacal bursa virus; EDSV is a chicken egg drop syndrome virus, and NDV is a newcastle disease virus, and IBV (DS10) is for having a liking for kidney type IBV DS10 strain virus; Negative negative SPF allantoic fluid, PBS is the contrast of PBS damping fluid.
Embodiment
Embodiment one has a liking for breeding, the purifying of kidney type IBV DS10 strain
1, has a liking for kidney type IBV DS10 strain
Have a liking for kidney type IBV DS10 strain, on June 25th, 2010 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preserving number is: CGMCC No.3957.
2, have a liking for the breeding of kidney type IBV DS10 strain
Get-70 ℃ and frozen have a liking for kidney type IBV DS10 standard seed culture of viruses in 4 ℃ of thawings; Press 1:100 times of virus dilution with the PBS damping fluid then; (egg is planted by logical laboratory animal Science and Technology Ltd. available from Beijing Cimmeria dimension to be inoculated in 11 age in days SPF chicken embryos; This laboratory hatching) in the allantoic cavity, inoculum size is 0.1 mL/embryo.Postvaccinal SPF chicken embryo is put 37 ℃ of cultivations.Discard embryo dead in 24 hours.Cultivate after 35 hours, results dead germ and dead germ not, and put it into after 4 ℃ of refrigerators are refrigerated to whole embryo death, collect dead germ allantoic fluid (contain and have a liking for kidney type IBV DS10), check aseptic packing afterwards frozen subsequent use in-70 ℃ of refrigerators.
3, have a liking for the antigenic purifying of kidney type IBV DS10 strain
(1) get allantoic fluid (method prepares gained in the title 2 in the present embodiment) that-70 ℃ of frozen containing have a liking for kidney type IBV DS10 after 4 ℃ of thawings, centrifugal 20 min under 4700 * g, 4 ℃ of conditions get supernatant; (2) with the supernatant that obtains in the step (1) with 14000 * g, 4 ℃ of centrifugal 15 min of condition, get supernatant; (3) supernatant that step (2) is obtained is centrifugal 1.5 h under 54000 * g, 4 ℃ of conditions; Abandon supernatant; In deposition, add an amount of TEN damping fluid, and make its dispersion, dissolving, gained purified virus solution with aseptic dropper piping and druming deposition; Kidney type IBV DS10 strain deactivation purifying antigen (being called for short the deactivation purifying antigen) is had a liking in conduct after adding 4 ℃ of deactivation 24 h of beta-propiolactone (final concentration 0.5 ‰), and-70 ℃ of preservations are subsequent use.
The foundation of embodiment two hybridoma cell strains
1, mouse immune
With the deactivation purifying antigen of the having a liking for kidney type IBV DS10 strain female BALB/c mouse (available from Yangzhou University's Experimental Animal Center) in immune 6 ages in week, immunity is 4 times altogether.First immunisation adds the emulsion of equal-volume Freund's complete adjuvant (available from Sigma company) with the deactivation purifying antigen, immunizing dose be 200 μ L/ only; Second and third time immunity all adds the emulsion of isopyknic Freund's incomplete adjuvant (available from Sigma company) with the deactivation purifying antigen, immunizing dose is 100 μ L/ only (between first, second and third time immunity 14 days at interval); Immune for the third time back 7 days; The tail vein blood separation of serum, with Microhemagglutination suppress (HI) experiment (Qian Xiaojia etc. antigenic preparation of avian infectious bronchitis virus (M41) HI and Preliminary Applications [J]. Anhui agricultural sciences, 2007; 35 (30): 9545-9546; 954.) detect the serum antibody level, to choose the highest mouse of serum antibody titer and adopt the deactivation purifying antigen from tail vein injection booster immunization (being the 4th immunity), immunizing dose is 50 μ L/.Acquisition is had a liking for serum HI after kidney type IBV DS10 deactivation purifying antigen is exempted from BALB/c mouse three and is tired and reach 1:256, and-20 ℃ of preservations are subsequent use as positive reference serum after this serum packing.
2, the preparation of feeder cell
Carry out 24h before the cytogamy, with one about 8 ages in week non-immune BALB/c mouse pluck eyeball blood sampling separation of serum, this serum is subsequent use as negative reference serum; Mouse after putting to death is placed 75% alcohol-pickled 10 min.Change Bechtop afterwards over to; Mouse is lain on the back be fixed on the dissection plate that disinfects; Mention skin of abdomen and with aseptic eye scissors it is cut off in the position of belly above that with aseptic nipper, the exposed through blunt dissection mouse peritoneum is drawn HAT nutrient solution (the Sigma company that 10 mL contain 20% calf serum with aseptic glass syringe; H0262) after being injected in the mouse peritoneal; It is motionless that the right hand is held syringe, and left hand is massaged the mouse web portion both sides lightly with aseptic nipper folder cotton ball soaked in alcohol, and right hand syringe is inhaled repeatedly and blown till the HAT nutrient solution yellowing that contains 20% calf serum of inhalation syringe then.Inject aseptic plate to the nutrient solution of sucking-off, place microscopically to carry out cell counting, adjustment nutrient solution volume makes cell density be about 10
5Individual/mL.Using pipettor then is 10 with cell concn
5The nutrient solution of individual/mL is added in 5 96 porocyte culture plates, and every hole 100 μ L place 37 ℃, and are in the incubator of 5% CO2, subsequent use as feeder cell.
3, cytogamy
Cytogamy according to ordinary method (Liu Xiu is of ancient India, the application (first version) [M] of monoclonal antibody on agricultural. Hefei: Anhui science and technology press, 1994:18-30.) carry out.Get the splenocyte and the SP2/0 myeloma cell (ATCC that carry out the BALB/c mouse of the 4th immunity after three days in the present embodiment step 1; Central Plains, Beijing is closed and is gathered economy and trade ltd) in the ratio mixing of 5:1 in 50 mL graduated centrifuge tubes; DMEM basic medium (available from Sigma company) with 20-30 mL washs 2 times, and is centrifugal under 1000 rpm, 10 min conditions then, abandons supernatant; Touch the pipe end with palm, make sedimentation cell loose evenly; This fusion pipe is moved into preheating and rotation gently in 37 ℃ of water-baths, in 60 s, 1 mL is preheated to 37 ℃ 50% PEG-4000 (giving birth to emerging biotechnology company available from Nanjing) and is added drop-wise on the sedimentation cell in the fusion pipe along tube wall; In 5 min, 25 mL are preheated to 37 ℃ DMEM basic medium and are added drop-wise in the fusion pipe concrete grammar along tube wall: the 1st min adds 1 mL, and the 2nd min adds 4 mL, and 3-5 min adds 20 mL, and the limit edged rotates centrifuge tube gently; Then fusion pipe is left standstill 10 min at 37 ℃, centrifugal 5 min abandon supernatant under 1000 rpm, add 50 mL and contain HAT (Sigma company, DMEM substratum re-suspended cell H0262); Add by every hole 0.1mL and to have cultivated in 96 well culture plates of feeder cell (seeing embodiment two steps 2), put 37 ℃, 5% CO
2Cultivate in the incubator.Observation of cell upgrowth situation after 5 days, and swap out with the fresh DMEM substratum that contains HAT and to cultivate 1/2 substratum in the plate hole; After 10 days, with containing HT (Sigma company, the swap out substratum of the former HAT of containing of DMEM substratum H0137); Observe the fused cell growing state, treat that it is distributed to hole floorage 1/5 when above, the sucking-off cell culture supernatant carries out antibody test.
4, hybridoma screening
Antibody through detecting in each fused cell culture supernatant liquid screens hybridoma.When the detection hole is positive, explain that the hybridoma in this hole is secreted the anti-kidney type IBV DS10 strain monoclonal antibody of having a liking for.
Adopt following method to detect the antibody (indirect ELISA detection) in the fused cell culture supernatant liquid: to get the deactivation purifying antigen and dilute with volume ratio 1:200 with carbonate buffer solution; Every hole adds the deactivation purifying antigen after the 100 μ L dilution in 96 porocyte plates, and 4 ℃ encapsulate and spend the night; Discard liquid,,, clap dried at last with thieving paper 5 min/ time with PBST damping fluid (the PBS damping fluid that contains 0.5% Tween-20) washing three times; With 1% BSA-PBST (the PBST damping fluid that contains 1% bovine serum albumin (BSA) (available from Sigma company)), 100 μ L/holes, 37 ℃ of sealing 1 h; Again with PBST damping fluid washing 3 times, 5 min/ time, last clap dried; Negative reference serum (preparation of embodiment two steps 2) that doubly dilutes with cell culture supernatant (acquisition of present embodiment step 3), with 0.5% BSA-PBST (containing mass concentration is the PBST damping fluid of 0.5% BSA) 1:100 and positive reference serum (preparation of embodiment two steps 1) add in the respective aperture (first ranked first row hole add the PBS damping fluid as blank); 100 μ L/ holes; Behind 37 ℃ of effect 1.5 h; Discard liquid with PBST damping fluid washing 3 times; 5 min/ time, the last bat done; Add the sheep anti-mouse igg (available from Beijing gold China fir bio tech ltd) of pressing the HRP mark of 1:3500 dilution with 0.5% BSA-PBST, 100 μ L/ holes are behind 37 ℃ of effect 1 h; Discard liquid; With PBST damping fluid washing 3 times, 5 min/ time, last clap dried; Add substrate colour developing liquid (Biosharp company, TMB-S-002), 100 μ L/ holes, 37 ℃ of lucifuges develop the color and add the stop buffer (H of 2 mol/L behind 10 min
2SO
4) termination reaction; ELIASA is measured OD
450Value.With the blank zeroing, P is the OD in each hole
450Value, N is the OD in the hole of the negative reference serum of adding
450Value.When N≤0.1, add the OD in the hole of positive reference serum
450The OD in the hole of value and the negative reference serum of adding
450The ratio (P/N)>=2.1 of value, i.e. under the situation that positive and negative contrast is set up, the detection hole of P/N>=2.1 is judged to the positive, and the detection hole of 1.5≤P/N＜2.1 is judged to suspicious, and the detection hole of P/N＜1.5 is judged to feminine gender.
5, the subclone of hybridoma
Step 4 is screened the positive hybridoma cell of the secretion monoclonal antibody specific that is obtained; (Liu Xiu is of ancient India in time to adopt limiting dilution assay; The application (first version) [M] of monoclonal antibody on agricultural. Hefei: Anhui science tech publishing house, 1994:35-36) carry out subclone.Select positive hole, adopt limiting dilution assay to carry out 2-3 time subclone continuously.At first the viable cell in the positive hole of hybridoma is carried out dyeing counting, with 10 cell/mL of DMEM perfect medium dilution written treaty, the cell suspension that dilution is good joins 96 porocyte culture plates, every hole 100 μ L, 37 ℃, 5% CO
2Cultivate in the incubator after 4-5 days, observe, mark all single clonal growth holes, get cell conditioned medium when being cultured to 8-9 days and do indirect ELISA detection (embodiment two step 4 methods) with inverted microscope; Select the male monoclonal cell to carry out subclone same more than 3 times, all cells hole supernatant detects all positively behind subclone, and the OD value that each hole is detected is more approaching, with frozen after the positive porocyte strain enlarged culturing behind the subclone repeatedly.Obtaining can the anti-hybridoma cell strain 2G4 that has a liking for kidney type IBV DS10 strain monoclonal antibody of stably excreting.
Embodiment three is anti-to have a liking for kidney type avian infectious bronchitis virus DS10 strain MONOCLONAL ANTIBODIES SPECIFIC FOR
Get the female mouse (available from Yangzhou University's Experimental Animal Center) of healthy BALB/c in 7 ~ 8 ages in week; Abdominal injection whiteruss (0.5 ml/ only) is after the week; Hybridoma cell strain 2G4 is injected mouse peritoneal with PBS damping fluid dilution back, and every injected in mice 0.2 mL contains 1～2 * 10 approximately
6Individual hybridoma., gathers mouse web portion ascites after 7-10 days when obviously expanding.With the ascites of gathering under 4000 rpm conditions centrifugal 5 minutes, discard grease and post precipitation, the gained supernatant is the ascites of preliminary purification.With the ascites packing in a small amount of preliminary purification, subsequent use in-70 ℃ of preservations.
Embodiment four is anti-to have a liking for mensuration and the subgroup identification that kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody ascites is tired
With the ascites of preliminary purification with the PBS damping fluid with 2 times of doubling dilutions; The ascites of each concentration is joined in the good enzyme plate hole, 96 hole that is coated with deactivation purifying antigen (diluting with 1:200 with carbonate buffer solution) of sealing (first ranked first row hole adding PBS damping fluid as blank); 100 μ L/ holes; 37 ℃, hatch 90 min; With PBST damping fluid washing 3 times, 5 min/ time, last clap dried; The sheep anti-mouse igg (available from Beijing gold China fir bio tech ltd) that adds 1:3500 dilution HRP mark, 100 μ L/ holes, 37 ℃ of effect 1 h, PBST washing 3 times, 5 min/ time, last clap dried; Add substrate colour developing liquid (Biosharp company, TMB-S-002), 100 μ L/ holes, 37 ℃ of lucifuges develop the color and add stop buffer (2 mol/L H behind 10 min
2SO
4) termination reaction; Measure the OD450nm value through ELIASA.With the blank zeroing, P detects the value in hole for each, and N is the OD in the hole of the negative reference serum of adding
450Value.When N≤0.1, add the OD in the hole of positive reference serum
450The ratio (P/N)>=2.1 of value and N, i.e. under the situation that positive and negative contrast is set up, the detection hole of P/N>=2.1 is judged to the positive, tires as the ascites of monoclonal antibody with the greatest dilution in positive hole.The result shows that anti-to have a liking for kidney type IBV DS10 strain monoclonal antibody ascites indirect ELISA titer be 1:102000 to hybridoma cell strain 2G4 excretory.
Adopt mouse monoclonal antibody subgroup identification test kit (Sigma company; ISO2-1KT) antagonism is had a liking for kidney type IBV DS10 strain monoclonal antibody and is carried out subgroup identification; Experimental procedure is carried out according to the test kit operation instructions, and the result shows: hybridoma cell strain 2G4 excretory is anti-to be had a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody and belongs to the IgG1 subclass.
Embodiment five anti-reactionogenicity and the specificity evaluations of having a liking for kidney type IBV DS10 strain monoclonal antibody and having a liking for kidney type IBV DS10 strain
With Western-blotting (J. Sa nurse Brooker; D.W. draw Sai Er. molecular cloning experiment guide [M]. the third edition. Beijing: Science Press; 1998,884-895.) identify anti-reactionogenicity and the specificity of having a liking for kidney type IBV DS10 strain monoclonal antibody of hybridoma cell strain 2G4 excretory.Have a liking for kidney type IBV DS10 strain allantoic fluid, negative allantoic fluid (aseptic collection is from 4 ℃ of lethal 12 age in days SPF chicken embryos) through 5% concentrate carry out the SDS-PAGE electrophoretic separation in glue and 10% separation gel after, with 2 mA/cm
2, 90 min are transferred on the pvdf membrane, with 4 ℃ of sealings of 10% BSA (available from Sigma company) solution, 2 h; Wash film 3 times with the PBST damping fluid, 5 min/ time; To have a liking for kidney type IBV DS10 strain monoclonal antibody be one anti-with anti-, is two anti-with the sheep anti-mouse igg (available from Beijing gold China fir bio tech ltd) of the HRP mark of 1:2500 dilution; Develop the color with enhancement type DAB colouring reagents box (available from Wuhan Boster Biological Technology Co., Ltd.).The result shows; This monoclonal antibody can with have a liking for kidney type IBV DS10 strain specificity and combine; About 14 KDa, located a single reaction band, and not with the reaction of negative allantoic fluid, prove that anti-to have a liking for kidney type IBV DS10 strain monoclonal antibody be to have a liking for the specific antibody of kidney type IBV DS10 strain for this.
To contain bird flu virus (Avian Influenza Virus with carbonate buffer solution; AIV) H9 hypotype, infections chicken cloacal bursa virus (Infectious Bursal Disease Virus; IBDV), chicken egg drop syndrome virus (Egg Drop Syndrome Virus; EDSV), newcastle disease virus (New Castle Disease Virus; NDV), have a liking for the SPF chick embryo allantoic liquid of kidney type IBV DS10 strain, negative allantoic fluid 1:200 dilution back and encapsulate 96 hole enzyme plates by the method for embodiment two steps 4; Anti-to have a liking for kidney type IBV DS10 strain monoclonal antibody be one anti-with hybridoma cell strain 2G4 excretory, establishes PBS damping fluid negative control simultaneously and carry out indirect ELISA experiment (referring to embodiment two step 4 methods).The result show hybridoma cell strain 2G4 excretory anti-have a liking for kidney type IBV DS10 strain monoclonal antibody only with have a liking for kidney type IBV DS10 strain allantoic fluid poison and become strong positive reaction; Be the negative reaction (see figure 1) with negative control and other chicken virus allantoic fluid, show that hybridoma cell strain 2G4 excretory that the present invention obtains is anti-and have a liking for kidney type IBV DS10 strain monoclonal antibody and have excellent specificity.
Bird flu virus (Avian Influenza Virus wherein; AIV) the H9 hypotype is available from Nanjing Tianbang Bio-industry Co., Ltd.; Infections chicken cloacal bursa virus (Infectious Bursal Disease Virus; IBDV), chicken egg drop syndrome virus (Egg Drop Syndrome Virus, EDSV) and newcastle disease virus (New Castle Disease Virus is NDV) available from China Veterinery Drug Inspection Office.
Annotate: the reagent that each embodiment uses among the present invention is following:
(1) PBS damping fluid: sodium-chlor (NaCl) 8.0 g, Repone K (KCl) 0.2 g, Sodium phosphate, dibasic (Na
2HPO
412H
2O) 2.9 g, potassium primary phosphate (KH
2PO
4) 0.2 g, be settled to 1,000 mL after adding deionized water dissolving, be 7.4,121 ℃ of sterilization 15 min with 1 M sodium hydroxide (NaOH) adjust pH, 4 ℃ of preservations.
(2) PBST damping fluid: containing mass concentration is the PBS damping fluid of 0.5% Tween-20.
(3) carbonate buffer solution: contain 0.05 mol/L Na
2CO
3With 0.05 mol/L NaHCO
3The aqueous solution, the pH value is 9.6.
(4) 1% BSA-PBST: containing mass concentration is the PBST damping fluid of 1% bovine serum albumin (BSA).
(5) 0.5% BSA-PBST: containing mass concentration is the PBST damping fluid of 0.5% bovine serum albumin (BSA).
(6) TEN damping fluid: contain the NaCl of 50 mmol/L, the Na2EDTA of 5 mmol/L in the Tris-HCL damping fluid of 50 mmol/L, pH7.4.
Claims (3)
1. a secretion resists the hybridoma cell strain 2G4 that has a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody, and its preserving number is CGMCC No.6172.
2. the said hybridoma cell strain excretory of claim 1 is anti-has a liking for kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody.
3. one kind is used to detect the test kit of having a liking for kidney type avian infectious bronchitis virus, it is characterized in that: said test kit comprises the said anti-kidney type avian infectious bronchitis virus DS10 strain monoclonal antibody of having a liking for of claim 2.
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| CN104845941A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis virus IBV-K136, monoclonal antibody cell line 3D5 prepared by using avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of avian infectious bronchitis virus IBV-K136 and monoclonal antibodies |
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| CN104845941B (en) * | 2014-11-18 | 2018-03-30 | 天津瑞普生物技术股份有限公司 | A kind of avian infectious bronchitis virus IBV K136, cell strain of monoclonal antibody 3D5, monoclonal antibody and its application using its preparation |
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