CN106811444A - Monoclonal antibody and its hybridoma cell strain and the application of 1 type and 3 type DHAVs are recognized simultaneously - Google Patents
Monoclonal antibody and its hybridoma cell strain and the application of 1 type and 3 type DHAVs are recognized simultaneously Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Engineering & Computer Science (AREA)
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- Virology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
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- Microbiology (AREA)
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- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of while recognizing monoclonal antibody and its hybridoma cell strain and the application of 1 type and 3 type DHAVs, the hybridoma cell strain good stability, stably excreting monoclonal antibody is remained to by subculture in vitro separately and cryopreservation, secreted monoclonal antibody can simultaneously recognize 1 type and 3 type DHAVs, with the affinity costant of 1 type DHAV 109It is more than the order of magnitude, and not with duck enteritis virus, dhbv dna, duck tembusu virus, Goose Parvovirus, riemerella anatipestifer, E. coli isolated from ducks and Salmonella anatis occur specific reaction, so as to be that the clinical and type of grass-roots unit's not missing inspection 1 or 3 type DHAVs provide wide spectrum, high specificity, affinity detection antibody high, can be used to prepare individually detection 1 type or 3 type DHAVs and while detect the reagent of 1 type and 3 type DHAVs.
Description
Technical field
The present invention relates to a kind of monoclonal antibody, secrete the hybridoma cell strain and the monoclonal of the monoclonal antibody
The purposes of antibody.
Background technology
DHAV (DHAV) can cause duckling that acute, height lethal infectious diseases, the week old of main infection 4 occur
Following duckling, wherein the duckling death rate within 1 week old is up to 95%, principal character lesion be ill duck liver enlargement and
Bleeding.According to genome signature and antigen neutralization test, DHAV is divided into 3 genotype or serotype, i.e. duck
The type of hepatitis A virus 1 (DHAV-1), 2 types (DHAV-2) and 3 types (DHAV-3).Wherein the type of DHAV 1 and 2 types be not
Cross-protection is produced, has partial intersection neutralization reaction with 3 types.There are 1 type and 3 type duck A types in the duck culturing production of current China
The generation of hepatitis and prevalence, but find the common antibody of 1 type and 3 type DHAVs and set up and can simultaneously detect 1 type and 3
The easy method for quick of type DHAV be always academia and production in there is no the problem for solving very well.
The content of the invention
In view of this, 1 type DHAV can be recognized, can also recognize 3 type ducks it is an object of the invention to developing
The monoclonal antibody of hepatitis A virus, so as to be that the clinical and type of grass-roots unit's not missing inspection 1 or 3 type DHAVs are provided
Wide spectrum, high specificity, affinity detection antibody high.
For achieving the above object, through research, the present invention provides following technical scheme：
1. hybridoma cell strain DHAV-MAb1, deposit number is CCTCC NO：C201701.
2. the monoclonal antibody secreted by hybridoma cell strain DHAV-MAb1.
3. monoclonal antibody described in prepare individually detection 1 type or 3 type DHAVs and detect simultaneously 1 type and
Application in the reagent of 3 type DHAVs.
The beneficial effects of the present invention are：The present invention is using the 1 type DHAV that purifies as immunogene and screening
Antigen, the hybridoma cell strain for being capable of stably excreting monoclonal antibody has been filtered out by monoclonal antibody technique.Demonstrate,proved through experiment
Real, the hybridoma cell strain good stability remains to stably excreting monoclonal antibody by subculture in vitro separately and cryopreservation；
Secreted monoclonal antibody can simultaneously recognize 1 type and 3 type DHAVs, the parent with 1 type DHAV
With constant 109It is more than the order of magnitude, and not with duck enteritis virus, dhbv dna, duck tembusu virus, gosling blast
There is specific reaction in poison, riemerella anatipestifer, E. coli isolated from ducks and Salmonella anatis, so that for clinical and grass-roots unit not
The type of missing inspection 1 or 3 type DHAVs provide wide spectrum, high specificity, affinity detection antibody high, can be used to prepare list
Solely detect 1 type or 3 type DHAVs and detect the reagent of 1 type and 3 type DHAVs simultaneously.
Biomaterial preservation
Hybridoma cell strain DHAV-MAb1 is preserved in China typical culture collection center on January 18th, 2017 (referred to as
CCTCC, address：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road), deposit number is CCTCC NO：C201701.
Brief description of the drawings
Fig. 1 is the form of immune mouse spleen cell, SP2/0 murine myeloma cells and feeder cells, and wherein a is immune small
Mice spleen cell, b is the feeder cells for cultivating 24h, and c is the feeder cells for cultivating 7d, and d is SP2/0 murine myeloma cells.
Fig. 2 is the hybridoma after fusion, wherein 1 is the hybridoma after culture 5d；2 is the hybridization after culture 9d
Oncocyte.
Fig. 3 is the SDS-PAGE analyses for purifying ascites, and wherein M is albumen Marker, and 1 is hybridoma cell strain DHAV-
The purifying ascites of MAb1.
Fig. 4 is determined for the affinity constant of monoclonal antibody.
Fig. 5 is the ultraviolet spectra of colloid gold particle and gold mark monoclonal antibody.
Fig. 6 is the assembling schematic diagram of colloidal gold strip.
Fig. 7 is the how anti-coating concentration optimization of type DHAV of rabbit-anti 1 of colloidal gold strip, and 1-6 is respectively many
Anti- concentration 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, 1.2mg/mL, 1.4mg/mL and 1.6mg/mL.
Fig. 8 is the sheep anti-mouse igg coating concentration optimization of colloidal gold strip, and 1-6 is respectively sheep anti-mouse igg concentration 0.2mg/
ML, 0.6mg/mL, 1.0mg/mL, 1.4mg/mL, 1.8mg/mL and 2.0mg/mL, 7 is feminine gender.
Fig. 9 is the gold mark monoclonal antibody coating concentration optimization of colloidal gold strip, and 1-8 is respectively 1mL gold mark monoclonals
Precipitation after antibody-solutions centrifugation is anti-with 100 μ L, 150 μ L, 200 μ L, 250 μ L, 300 μ L, 350 μ L, 400 μ L and 450 μ L gold mark
Body weight suspension dilutes.
Figure 10 is the sensitivity test result that colloidal gold strip detects 1 type DHAV, and 1-9 is respectively the positive
Duck embryos allantoic fluid (0.6mg/mL) presses 1:1、1:2、1:4、1:8、1:16、1:32、1:64、1:128 and 1:256 dilutions, 10 is the moon
Property control.
Figure 11 is the sensitivity test result that colloidal gold strip detects 3 type DHAVs, and 1-7 is respectively the positive
Duck embryos allantoic fluid (0.3mg/mL) presses 1:1、1:2、1:4、1:8、1:16、1:32 and 1:64 dilutions, 8 is negative control.
Figure 12 is the specific test result that colloidal gold strip detects 1 type and 3 type DHAVs, 1-5 and 7 point
Wei not CZ160312 plants, QL plants, QL13 plants, MY02 plants, YA151006 plants and SD151227 plants of 3 type DHAV, 6 Hes
8-10 is respectively BZB plants, CD1231 plants, DY0903 plants and X plants of 1 type DHAV, and 11 is negative control.
Figure 13 is the specific test result of other common infective pathogens of colloidal gold strip detection duck.
Figure 14 is the replica test result that colloidal gold strip detects 1 type and 3 type DHAVs, 1-3 difference
For the 1st, 2, the colloidal gold strip of 3 batches, a is 1 type DHAV of detection；B is detection 3 type duck hepatitis A disease.
Figure 15 is the stability test result of colloidal gold strip, and 1-3 is respectively preservation 1,2,3 months.
Specific embodiment
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, below in conjunction with accompanying drawing to the present invention
Preferred embodiment be described in detail.
The main material used in preferred embodiment is as follows：
1st, cell, bacterial strain and strain
SP2/0 murine myeloma cells are purchased from Chinese Academy of Sciences's cell bank；1 type DHAV (BZB plants,
CD1231 plants, DY0903 plants and X plants), 3 type DHAVs (CZ160312 plants, QL plants, QL13 plants, MY02 plants,
YA151006 plants and SD151227 plants), duck enteritis virus (DPV CHv plants), dhbv dna (DHBV), duck Tan Busu
Viral (DTMUV), Goose Parvovirus (GPV), E. coli isolated from ducks (E.coli, preserving number：CVCC83003), Salmonella anatis
(SE, preserving number：CMCC50083 during), riemerella anatipestifer (RA CH-1 plants) is studied by Sichuan Agricultural University's poultry disease prevention and control
The heart is preserved and provided.
2nd, experimental animal
6-8 week old and 10-12 week old SPF BALB/c female mices test Company of Animals Ltd. purchased from Chengdu up to rich fruit；10 days
Age duck embryos are purchased from Sichuan Agricultural University farm.
3rd, reagent and material
HAT culture medium additives (50 ×), polyethylene glycol (PEG 1450), Freund's complete adjuvant and incomplete Freund's adjuvant
It is Sigma Products；Hyclone (FBS) and the culture mediums of RPMI 1640 are Gibco Products；Bovine serum albumin(BSA)
(BSA) it is BIOSHARP Products；Mouse monoclonal antibody Ig class subgroup identifications ELIAS secondary antibody suit is exempted from purchased from Beijing Bo Aolong
Epidemic disease Technology Co., Ltd.；Disposable cell bottle, Tissue Culture Plate and ELISA ELISA Plates are purchased from Corning companies of the U.S..
Resist to be preserved by Sichuan Agricultural University's poultry disease prevention and control research center more the type DHAV of rabbit-anti 1 and provide；20nm
Colloidal gold solution, glass fibre element film (MA0280, GL0194), nitrocellulose (NC) film (Sartorius CN140,
Sartorius CN95, vivid 170 and Millipore 135), sample pad (GL-b03 and GL-b04), absorption pad
(H5072), base plate (DB-6), white plastic card (A-9) and linking adhesive tape are purchased from Shanghai one Bioisystech Co., Ltd of outstanding person；Glass
Glass cellulose membrane (CB-0801, SB06, VL78) is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd..
4th, instrument and equipment
Ultra-pure water instrument (WaterPro, Labconco companies of the U.S.)；Nucleic acid-protein detector (SmartSpec 3000, it is beautiful
Bio-rad companies of state)；Gel is into phase system (GelDoc 3000, Bio-rad companies of the U.S.)；Inverted fluorescence microscope
(TE2000-U, Japanese Nikon companies)；CO2gas incubator (3951Reach-In, the U.S. Thermo Fisher
Scientific companies)；ELIASA (MODEL 680, Bio-rad companies of the U.S.).
Three-dimensional planar point film gold spraying instrument (HM3030, Shanghai Jinbiao Bio-Tech Co., Ltd.)；Case pressing machine (YK725, Shanghai
Jin Biao bio tech ltd)；The automatic cutting machine of micro computer (Shanghai Jinbiao Bio-Tech Co., Ltd.).
Embodiment 1, the acquisition of hybridoma cell strain DHAV-MAb1 and identification
First, the breeding and purifying of 1 type DHAV
By X plants of 1 type DHAV, (preserving number is CCTCC NO：C201538 virus liquid) makes of physiological saline
Appropriate dilution, with 0.22 μm of filtering with microporous membrane, the duck embryos of 10 ages in days, 0.1mL/ pieces is inoculated in by allantoic cavity；Separately take 5 piece 10
The duck embryos of age in days are inoculated with physiological saline as a control group；It is incubated under the conditions of 37 DEG C, discards dead duck embryos in inoculation 24h, often later
It shines egg 2 times, observes 3-5d；Collect that 36-72h after inoculation is dead and the obvious duck embryos allantoic fluid of lesion, -20 DEG C freeze it is standby
With.
Take the above-mentioned duck embryos allantoic fluid for freezing, after multigelation 3 times, in 4 DEG C, 6000r/min centrifugation 15min, it is heavy to discard
Form sediment；Add equal amounts of chloroform to shake 20min strongly in supernatant, in 4 DEG C, 6000r/min centrifugation 20min, take supernatant, repeat
Above-mentioned chloroform operates 3 times to remove lipid and some foreign proteins；By supernatant in 4 DEG C, 40000r/min centrifugation 2h, harvest
Precipitate and be suspended in the PBS of 0.01mol/L, pH7.4, its light at 260nm and 280nm is determined with ultraviolet specrophotometer
Density, calculates the concentration of 1 type DHAV of purifying, dispenses, and -20 DEG C save backup.
2nd, mouse immune
It is immunogene with the 1 type DHAV for purifying, the BALB/c female mices of 6-8 week old is immunized.Exempt from first
Epidemic disease (1d), mixes simultaneously in equal volume according to the dosage of every type DHAV of 100 μ g of mouse 1 with Freund's complete adjuvant
It is fully emulsified, dorsal sc multi-point injection；Second immune (14d), 1 type of same dose is processed with incomplete Freund's adjuvant
DHAV carries out dorsal sc multi-point injection；Third time is immune (28d), processes identical with incomplete Freund's adjuvant
1 type DHAV of dosage carries out dorsal sc multi-point injection.10d after third time is immune, from mouse tail vein blood sampling,
37 DEG C of standing 2h, 4 DEG C overnight, and the 10min of 5000r/min centrifugations afterwards separates serum, is resisted using indirect ELISA method detection serum
Body potency, chooses serum titer and reaches 1:104BALB/c mouse above carries out cell fusion.3d before cell fusion, mouse abdomen
Chamber injection only carries out booster immunization without the μ g/ of 1 type DHAV of adjuvant 50.
Indirect ELISA method is as follows：The 1 type DHAV carbonate of 0.05mol/L, pH 9.6 that will be purified
Buffer solution by volume 1:ELISA Plate is added after 800 dilutions, 100 μ L/ holes (187.5ng/ holes), overnight, PBS-T is washed 4 DEG C of coatings
Wash, the μ L of 0.01g/mL BSA solution 200 are added per hole, 37 DEG C of closing 2h, PBS-T washings add 100 μ L 0.01mol/ per hole
The PBS of L, pH7.4 by volume 1:The immune serum of 800 dilutions is primary antibody, and 37 DEG C are incubated 30min, and PBS-T is washed, often
Hole adds the 100 μ L PBS of 0.01mol/L, pH7.4 by volume 1:The HRP mark sheep anti-mouse iggs of 5000 dilutions, 37 DEG C incubate
1h is educated, cleaning solution washing adds TMB nitrite ions 100 μ L, 37 DEG C of colour developing 10min per hole, 2mol/L H are added per hole2SO4Solution
50 μ L terminating reactions, ELIASA reads OD with double wave long form450-OD630Value.The OD of test hole450-OD630Value is designated as P, negative
The OD of control wells450-OD630Value is designated as N, calculates P/N values, if P/N value >=2.1 are the positive, otherwise is feminine gender.
3rd, cell fusion
SP2/0 myeloma cell and immune mouse spleen cell are pressed 1:5 ratio is mixed, and is put into 50mL centrifuge tubes,
1800r/min is centrifuged 5min, and abandoning supernatant is slow between 60s-90s to be at the uniform velocity added dropwise 37 DEG C during centrifuge tube put into 37 DEG C of water-baths
The 1mL of PEG 1450 of preheating, are finished and stand 90s in 37 DEG C of water-baths, and the serum-free RPMI 1640 for adding 37 DEG C of preheatings is trained
Nutrient solution terminates fusion, and 800r/min centrifugation 5min, abandoning supernatant, cell precipitation is trained with the RPMI 1640 of 37 DEG C of preheatings
After nutrient solution washing, select nutrient solution resuspended with the HAT containing 20% hyclone, be added to 96 porocyte plates containing feeder cells
In, 37 DEG C, 5%CO are put in 100 μ L/ holes2Cultivated in incubator.
It is prepared by feeder cells：More than 10-12 week old non-immune BALB/c mouse is taken, draws neck to put to death, flowing clear water is rinsed,
5-10min is carried out disinfection in being soaked in 75% alcohol, and the mouse after sterilization is put on superclean bench, and abdominal cut skin makes
Peritonaeum is exposed, and peritonaeum is lifted with tweezers, and the nutrient solution about 5mL of 37 DEG C of preheatings is expelled in mouse peritoneal, careful massage
Mouse web portion about 3min, extracts out, repeats several times, and Extract is put in 10mL centrifuge tubes, 1500r/min centrifugations, supernatant discarded
Liquid, cell precipitation is resuspended with nutrient solution, and it is 2-3 × 10 adjust after viable count to cell number5Individual/mL, by 100 μ L/
Hole is added in 96 porocyte plates, in 37 DEG C, 5%CO2Overnight incubation in incubator, next day observation is pollution-free to be can be used.
It has been observed that immune mouse spleen cell is rounded, smaller, the form to SP2/0 myeloma cell is similar；Mouse abdomen
Chamber feeder cells are in fusiformis after incubated overnight, and the state of the more explanation feeder cells of spindle cell quantity is better, for fusion is thin
The condition that intracellular growth is provided is also better (Fig. 1).
4th, the screening of hybridoma and subclone
Hybridoma Cell Culture after fusion selects nutrient solution partly to be changed liquid after one week with HAT, and cell culture is taken after 2d
Supernatant, the screening in positive hole is carried out using foregoing indirect ELISA method.The positive hole cell for filtering out uses limiting dilution assay
3-4 subclone is carried out, positive cell rate is reached 100%.By can stably excreting monoclonal antibody hybridoma cell strain and
When Amplification Culture and freeze.
The visible clone cell groups of 3d after hybridoma cell fusion, the clone cell collection grown in most culture hole
Group's number more than two, elapses over time, and clone cell group quantity continues to increase (see Fig. 2).Positive hole cell is by 3 Asias
After clone, 5 plants of hybridoma cell strains for being capable of stably excreting monoclonal antibody are finally given.
5th, the Stability Determination of hybridoma cell strain DHAV-MAb1
The hybridoma cell strain that DHAV-MAb1 is named as in the strain of 5 strain of hybridoma is carried out into continuous Secondary Culture, point
Do not take the culture supernatant of its 1st generation, the 5th generation and the 10th generation cell, and after freezing 1 month recovery cell culture supernatant
Liquid, with SP2/0 murine myeloma cell culture supernatants as negative control, using foregoing indirect ELISA method to the hybridoma
Cell line carries out antibody-secreting Detection of Stability.
The results are shown in Table 1,1st generation, the 5th generation, the 10th generation cell culture supernatant and after freezing 1 month in recovery cell culture
The OD of clear liquid450-OD630Value no significant difference, shows that hybridoma cell strain DHAV-MAb1 has stably excreting monoclonal antibody
Ability.
The hybridoma cell strain DHAV-MAb1 of table 1 passes on and freezes rear stability measure
Hybridoma cell strain DHAV-MAb1 is preserved in China typical culture collection center on January 18th, 2017 (referred to as
CCTCC, address：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road), deposit number is CCTCC NO：C201701.
The preparation of embodiment 2, monoclonal antibody and identification
First, a large amount of preparation and purifications of monoclonal antibody
The BALB/c female mices of 10-12 week old are taken, incomplete Freund's adjuvant sensitization, 0.5mL/, abdomen after 5d are used in advance
Chamber injects hybridoma cell strain DHAV-MAb1 0.5mL/ (2.0 × 106Individual/mL), after 7-10d, when mouse web portion significantly increases
When big, mouse ascites are extracted, 37 DEG C are placed 2h, and 4 DEG C overnight, and next day is centrifuged 15min with 3000r/min, collect supernatant, -20
DEG C save backup.Ascites can be extracted every 2d 1 time, can repeatedly extract mouse ascites multiple.The ascites that will be preserved is first with pungent
Acid-ammonium sulfate precipitation method is slightly carried, then is crossed post with rProtein A/G Beads and purified, and obtains purifying ascites.
Purifying ascites is taken, the purification effect of monoclonal antibody is detected using SDS-PAGE, as a result see Fig. 3.
2nd, the hypotype identification of monoclonal antibody
Hybridoma cell strain DHAV-MAb1 culture supernatants and purifying ascites are made into doubling dilution, it is anti-according to murine monoclonal
Body Ig class subgroup identifications ELIAS secondary antibody is set with specification, and monoclonal antibody Ig hypotypes are carried out using foregoing indirect ELISA method
Identification, OD450-OD630ELIAS secondary antibody corresponding to value highest hole is the monoclonal antibody of hybridoma cell strain DHAV-MAb1 secretions
Hypotype.
2 are the results are shown in Table, the hypotype of the monoclonal antibody of hybridoma cell strain DHAV-MAb1 secretions belongs to Ig G1.
The hypotype qualification result of the monoclonal antibody of table 2
3rd, the titration of monoclonal antibody
Hybridoma cell strain DHAV-MAb1 culture supernatants and purifying ascites are made into doubling dilution, using foregoing indirect
ELISA method determines the potency of monoclonal antibody, parallel dilute with negative ascites with SP2/0 murine myeloma cells culture supernatant
Release as negative control.
Result shows that the antibody titer of hybridoma cell strain DHAV-MAb1 culture supernatants is 1:1280；Purifying
The antibody titer of ascites is 1:819200.
4th, the specificity identification of monoclonal antibody
Under 37 DEG C, 180r/min controlled conditions, to common duck pathogenic bacteria (riemerella anatipestifer, Salmonella anatis, duck
Escherichia coli) carry out Multiplying culture 24h after, ultrasonication, centrifugation, take each strain culturing supernatant coated elisa plate；Duck is caused
Virus (1 type DHAV, 3 type DHAVs, duck enteritis virus, dhbv dna, the smooth cloth of duck
Soviet Union virus, Goose Parvovirus) virus liquid coated elisa plate；With PBS as blank；With hybridoma cell strain DHAV-MAb1
Culture supernatant is primary antibody, while with SP2/0 murine myeloma cell culture supernatants as negative control, using foregoing indirect
ELISA method carries out monoclonal antibody specificity identification.
The results are shown in Table 3, the monoclonal antibody of hybridoma cell strain DHAV-MAb1 secretions not with duck enteritis virus,
Dhbv dna, duck tembusu virus, Goose Parvovirus, riemerella anatipestifer, E. coli isolated from ducks, Salmonella anatis occur special
Opposite sex reaction, but there is cross reaction with 3 type DHAVs, illustrate the list of hybridoma cell strain DHAV-MAb1 secretions
Clonal antibody can simultaneously recognize 1 type and 3 type DHAVs and have preferably specificity.
The specificity identification result of the monoclonal antibody of table 3
5th, the affinity costant of monoclonal antibody is determined
Hybridoma cell strain DHAV-MAb1 culture supernatants are made into doubling dilution, 100 μ L/ holes, 37 DEG C of incubation 2h are used
Foregoing indirect ELISA method determines OD450-OD630Value, draws antibody extension rate and OD450-OD630The relation curve of value, according to
50%OD in curve450-630nmThe corresponding antibody extension rate of value, based on formula Kaff 150000 × A/ of (L/mol) ≈ [Ag]
Affinity costant (Kaff) is calculated, A is 50%OD in formula450-OD630The corresponding antibody extension rate of value, [Ag] is AC.
Result is shown in Fig. 4, and the affinity costant of the monoclonal antibody of hybridoma cell strain DHAV-MAb1 secretions is 2.4 × 1010L/
Mol, with preferable antigen binding capacity.
The preparation of embodiment 3, colloidal gold strip and performance evaluation
First, the preparation and purification of gold mark monoclonal antibody
1st, the treatment of monoclonal antibody
The hybridoma cell strain DHAV-MAb1 of purifying is induced during ascites puts bag filter, in 0.01mol/L, pH7.4
4 DEG C of dialysed overnights in PBS, dialyzate is changed every 4h, and then 4 DEG C, 10000r/min centrifugation 30min take supernatant, determine egg
Bai Hanliang, obtains final product the monoclonal antibody for recognizing 1 type and 3 type DHAVs simultaneously, dispenses, and -20 DEG C save backup.
2nd, the preparation of gold mark monoclonal antibody
20nm colloidal gold solution 1mL are taken, 0.1mol/L K are added2CO3The μ L of solution 12, stir and evenly mix, and add identification 1 simultaneously
The μ g of monoclonal antibody 34 of type and 3 type DHAVs, stir and evenly mix, and stand 30min, add 0.1g/mL BSA molten
Liquid is stirred and evenly mixed to final concentration of 0.01g/mL, stands 15min, obtains gold mark monoclonal antibody solution, and 4 DEG C save backup.
3rd, the purifying of gold mark monoclonal antibody
Take 1mL gold mark monoclonal antibody solution, in 4 DEG C, 2000r/min centrifugation 15min, collect supernatant, in 4 DEG C,
9000r/min is centrifuged 20min, abandoning supernatant, precipitation with gold labeling antibody re-suspension liquid (by 1g BSA, 10g sucrose, 5g trehaloses,
0.05g caseins, 0.1g sodium azide and 1g PEG 20000 are dissolved with the PBS 100mL of 0.01mol/L, pH8.0 and are obtained) it is dilute
Release to original volume, in 4 DEG C, 9000r/min centrifugation 20min, abandoning supernatant, precipitation repeats aforesaid operations 1-2 times, finally with gold
Labeling antibody re-suspension liquid dilutes, 4 DEG C of sealing preserves.
The gold mark monoclonal antibody of purifying is taken, ultraviolet spectra is scanned in the range of wavelength 400-660nm.Result is shown in Fig. 5, with
Unlabelled colloid gold particle is compared, and the maximum absorption band of gold mark monoclonal antibody has moved right 4nm, illustrates monoclonal antibody
Succeed by colloid gold label.
2nd, the preparation of colloidal gold strip
Prepare the colloidal gold strip for detecting 1 type and 3 type DHAVs simultaneously using double antibody sandwich method.Test paper
Bar is made up of base plate, nitrocellulose (NC) film, sample pad, 5 parts of gold standard pad and absorption pad, wherein the nature controlling line on NC films
(C lines) is coated with sheep anti-mouse igg, and detection line (T lines) coating rabbit-anti 1 type DHAV resists more；Gold standard pad coating gold mark is single
Clonal antibody (see Fig. 6).When 1 type and/or 3 type DHAV is contained in testing sample, in chromatography process, virus
Antigen is first combined with the gold mark monoclonal antibody specificity in gold standard pad, then sick with the type duck hepatitis A of rabbit-anti 1 in detection line
The how anti-specific binding of poison, so that detection line is presented red stripes, and the gold not combined with viral antigen marks monoclonal antibody
Specifically bound with the sheep anti-mouse igg on nature controlling line, so that nature controlling line is also presented red stripes；When not contained in testing sample
Or during containing 1 type and/or 3 type DHAV less than detection limit, then detection line is not presented red stripes, only matter
Control line is presented red stripes.
The specific preparation method of colloidal gold strip is as follows：By sheep anti-mouse igg and the type duck hepatitis A of rabbit-anti 1 of purifying disease
After the PBS of how anti-0.01mol/L, the pH7.4 of use respectively of poison dilutes, formation nature controlling line and detection line, 37 on NC films are coated in respectively
DEG C be dried overnight, then by NC films with 0.01g/mL BSA solution in 37 DEG C of closings, dry, 4 DEG C of sealing preserves are standby；By what is purified
Gold mark monoclonal antibody solution is added dropwise on glass fibre element film, and 37 DEG C are dried overnight, and obtain gold standard pad；To be coated with and closed
The NC films for the treatment of, gold standard pad, sample pad, absorption pad are bonded on base plate successively after drying, that is, colloidal gold strip is obtained.
1st, the coating concentration optimization resisted the type duck hepatitis A of rabbit-anti 1 of purifying more
First slightly carried how anti-the type DHAV of rabbit-anti 1 is with octanoic acid-ammonium sulfate precipitation method, then used rProtein
A/GBeads crosses post purifying, and the type DHAV of rabbit-anti 1 for being purified resists more.The type duck A type liver of rabbit-anti 1 that will be purified
The scorching how anti-PBS of 0.01mol/L, pH7.4 of virus be diluted to concentration be respectively 0.6,0.8,1.0,1.2,1.4,1.6mg/mL,
It is coated on NC films and forms detection line, colloidal gold strip is obtained in the case where other conditions are constant, observes the aobvious of test strips
Pornographic condition, it is determined that reaching the optimal coating concentration resisted the type DHAV of rabbit-anti 1 of test strips susceptibility requirement more.As a result
See Fig. 7, when the coating concentration resisted when the type DHAV of rabbit-anti 1 is 0.8mg/mL more, the colour developing color of test strips is shallower,
Viewing test result is difficult, therefore the optimal coating concentration resisted the type DHAV of rabbit-anti 1 is 1.0mg/mL more.
2nd, the coating concentration optimization of sheep anti-mouse igg
By sheep anti-mouse igg with the PBS of 0.01mol/L, pH7.4 be diluted to concentration be respectively 0.2,0.6,1.0,1.4,1.8,
2.0mg/mL, is coated on NC films and forms nature controlling line, and colloidal gold strip, observation examination are obtained in the case where other conditions are constant
The colour developing situation of paper slip, it is determined that reaching the optimal coating concentration of the sheep anti-mouse igg of test strips susceptibility requirement.Result is shown in Fig. 8, when
When the coating concentration of sheep anti-mouse igg is 0.6mg/mL, the colour developing color of test strips is shallower, is difficult viewing test result, therefore sheep
The optimal coating concentration of anti-mouse IgG is 1.0mg/mL.
3rd, the coating concentration optimization of gold mark monoclonal antibody
Take 1mL gold mark monoclonal antibody solution, in 4 DEG C, 2000r/min centrifugation 15min, collect supernatant, in 4 DEG C,
9000r/min is centrifuged 20min, and abandoning supernatant, precipitation gold labeling antibody re-suspension liquid is diluted to original volume, in 4 DEG C, 9000r/
Min is centrifuged 20min, and abandoning supernatant, precipitation repeats aforesaid operations 1-2 times, finally respectively with 100,150,200,250,300,
350th, 400,450 μ L gold labeling antibodies re-suspension liquids dilution, the gold mark monoclonal antibody solution for being purified.By the purifying of various concentrations
Gold mark monoclonal antibody solution be added dropwise respectively formed objects glass fibre element film on form gold standard pad, in other conditions not
Colloidal gold strip is obtained in the case of change, the colour developing situation of test strips is observed, it is determined that reaching the gold of test strips susceptibility requirement
Mark the optimal coating concentration of monoclonal antibody.Result is shown in Fig. 9, and precipitation after 1mL gold mark monoclonal antibody solutions are centrifuged uses 450
When the gold labeling antibody re-suspension liquid of μ L dilutes, the colour developing color of test strips is shallower, is difficult viewing test result, therefore gold mark monoclonal
The optimal coating concentration of antibody is that the precipitation after 1mL gold mark monoclonal antibody solution centrifugations is dilute with 400 μ L gold labeling antibody re-suspension liquids
Release.
4th, the optimization of NC films
This 4 kinds of Sartorius CN140, Sartorius CN95, vivid 170 and Millipore 135 is respectively adopted
The NC films of model, colloidal gold strip is obtained in the case where other conditions are constant, observes the colour developing situation of test strips.Result is shown in
Table 4, Sartorius CN140 and Millipore 135 develops the color comparatively fast, can be developed the color in 10min, but Sartorius
CN140 colour developings rear backdrop is not clean, there is the incomplete phenomenon of chromatography, therefore preferably Millipore 135 (is that aperture is 8 μm, layers
Analysis speed is the NC films of 120-150s/4cm).
The optimum results of the NC films of table 4
5th, the optimization of glass fibre element film
The glass fibre element film preparation of this 5 kinds of models of CB 0801, SB 06, VL 78, MA0280, GL 0194 is respectively adopted
Gold standard pad, then colloidal gold strip is obtained in the case where other conditions are constant, gold mark is single on observation different glass cellulose membrane
The release conditions of clonal antibody.Result shows that the gold mark monoclonal antibody release on CB0801, SB06, VL78 and GL0194 is not
Completely, have it is more gold mark monoclonal antibody remain on film, and on CB0801 gold mark monoclonal antibody rate of release it is extremely slow, and
Gold mark monoclonal antibody release on MA0208 is complete, therefore preferably MA0208 (is thickness for 0.40 ± 0.05mm, grammes per square metre are 68
±10g/m2Polyester cellulose film).
6th, the optimization of sample pad
Two sample pads of article No. of GL-b03 and GL-b04 are respectively adopted, colloid is obtained in the case where other conditions are constant
Gold test paper strip, negative, positive duck embryos allantoic fluid is added dropwise in sample pad, observes two kinds of release conditions of sample pad.Result shows
Show, sample flowing velocity on GL-03 is slower, be unfavorable for the combination of sample and gold mark monoclonal antibody, therefore preferably GL-04
(it is thickness for 0.78 ± 0.05mm, grammes per square metre are 92 ± 10g/m2Glass fibre element film).
7th, the optimization of NC membrane closures time
In order to reduce the non-specific adsorption of NC films, after NC films completion coating is processed and is dried, 0.01g/mL BSA are used
Solution closes off 0 in 37 DEG C, 30,60,120min, dry, collaurum examination is then obtained in the case where other conditions are constant
Paper slip, observes the colour developing situation of test strips, determines the optimal off-period of NC films.Result shows, closes the examination of 0min and 30min
Paper slip traction is than more serious, and the test strips color developing effect for closing 60min and 120min is preferable, the two no significant difference, because
The optimal off-period of this NC film is 60min.
3rd, the performance evaluation of colloidal gold strip
1st, the detection method and result judgement of colloidal gold strip
Take 100 μ L samples to be added drop-wise in sample pad, colour developing result is observed after 5-10min.If the detection line of test strips and
Nature controlling line shows clearly red stripes, then judge that testing result is the positive, 1 type and/or 3 type duck hepatitis A in sample
The content of virus is higher, and detection line colour developing is deeper；If test strips only nature controlling line shows red stripes, detection is judged
Result is feminine gender；If the nature controlling line of test strips does not show red stripes, this detection failure is judged, need to re-start inspection
Survey.
2nd, the cracking treatment of duck embryos allantoic fluid or bacterial culture fluid
To added in duck embryos allantoic fluid or bacterial culture fluid sample isometric lysate (0.01mol/L, pH7.4's
PBS, X-100 containing 2%Triton), 5min is acted on, acutely vibration, 4 DEG C, 8000r/min centrifugation 5min, collecting supernatant is used for
Detection.
3rd, the sensitivity test of colloidal gold strip
After 1 type or 3 type DHAV infected duck embryo allantoic liquids are processed through cracking, viral egg is determined with BCA methods
White concentration, according to 1:1、1:2、1:4、1:8、1:16、1:32、1:64 and 1:After 128 doubling dilutions, pressed with colloidal gold strip
Foregoing detection method detected, observation colour developing result.
The testing result of 1 type DHAV infected duck embryo allantoic liquid is shown in Figure 10, the disease measured after being processed through cracking
Toxalbumin concentration is 0.6mg/mL, according to 1:Developed the color during 64 dilution shallower, according to 1:Only have nature controlling line to develop the color during 128 dilution, because
This, colloidal gold strip is 0.9 μ g to the limit of identification of 1 type DHAV.3 type DHAV infected ducks
The testing result of embryo allantoic liquid is shown in Figure 11, and the virus protein concentration measured after being processed through cracking is 0.3mg/mL, according to 1:16 is dilute
Developed the color when releasing shallower, according to 1:Only have nature controlling line to develop the color during 32 dilution, therefore, colloidal gold strip is to 3 type duck hepatitis A disease
The limit of identification of poison is 1.8 μ g.The above results show that colloidal gold strip prepared by the present invention has good detection sensitive
Degree.
4th, the specific test of colloidal gold strip
Normal duck embryos allantoic fluid, the 1 type duck through cracking treatment are detected respectively by foregoing detection method with colloidal gold strip
Hepatitis A virus infection duck embryos allantoic fluid, 3 type DHAV infected duck embryo allantoic liquids, duck enteritis virus sense
Mo Shi bars in dye duck embryos allantoic fluid, duck tembusu virus infected duck embryo allantoic liquid, Goose Parvovirus infected duck embryo allantoic liquid, pest of duck
The bacterial culture fluid of the bacterial culture fluid of bacterium, the bacterial culture fluid of E. coli isolated from ducks and Salmonella anatis, observation colour developing result.
The testing result of 1 type or 3 type DHAVs is shown in Figure 12, and colloidal gold strip is to 4 plant of 1 type duck hepatitis A
Viral (BZB plants, CD1231 plants, DY0903 plants and X plants) and 6 plant of 3 type DHAV (CZ160312 plants, QL plants, QL13
Strain, MY02 plants, YA151006 plants and SD151227 plants) testing result be positive.The inspection of other common infective pathogens of duck
Survey result and see Figure 13, colloidal gold strip is to normal duck embryos allantoic fluid, duck enteritis virus, duck tembusu virus, gosling
The testing result of pestivirus, riemerella anatipestifer, E. coli isolated from ducks and Salmonella anatis is negative.The above results show,
Colloidal gold strip prepared by the present invention has good detection specificity.
5th, the replica test of colloidal gold strip
With 3 colloidal gold strips of different batches, positive duck embryos allantoic fluid is detected simultaneously respectively by foregoing detection method
(1 type DHAV infected duck embryo allantoic liquid, 3 type DHAV infected duck embryo allantoic liquids) and negative duck embryos urine
Cyst fluid, each batch duplicate detection 3 times, observation colour developing result.
Result is shown in Figure 14, no significant difference between the testing result that 3 different batches colloidal gold strips are respectively repeated 3 times,
Between showing the colloidal gold strip of present invention preparation there is good batch and batch in detection repeatability.
6th, the stability test of colloidal gold strip
Colloidal gold strip sealing preserve is used into duck hepatitis A respectively every other month in 4 DEG C, room temperature and 37 DEG C respectively
Viral feminine gender duck embryos allantoic fluid and 1 type DHAV positive duck embryos allantoic fluid are detected by foregoing detection method, seen
Examine colour developing result.
Result is shown in Figure 15, negative, the positive of the colloidal gold strip after 4 DEG C, room temperature and 37 DEG C are preserved 1,2,3 months respectively
Detection results are preferable, no significant difference between testing result, show present invention preparation colloidal gold strip have it is good steady
It is qualitative, can at least be preserved 3 months at 4-37 DEG C.
7th, the detection of clinical sample
To 162 parts of samples of plant's censorship, after aseptic collection duck pathological material of disease, shred, be put into and fill about 500 μ L physiological saline
Sterilizing EP pipes in, multigelation 3 times acutely vibrates 1-3min, add isometric lysate (0.01mol/L, pH7.4's
PBS, X-100 containing 2%Triton), 5min is acted on, acutely vibration, 4 DEG C, 8000r/min centrifugation 5min collect supernatant, point
Do not detected with RT-PCR method with colloidal gold strip.
5 are the results are shown in Table, in the positive of 115 detected in RT-PCR, colloidal gold strip detects 111 sun
Property；In 47 negative samples that RT-PCR is detected, colloidal gold strip detects 1 positive, 46 feminine genders；Two kinds of detections
The positive coincidence rate of method is 96.5% (111/115), and negative match-rate is 97.9% (46/47).
Testing result of the colloidal gold strip of table 5 to clinical sample
Finally illustrate, above example is merely to illustrate technical scheme, do not constitute in the present invention
The limitation of appearance.Although by above-described embodiment the present invention has been done it is more detailed enumerate, those skilled in the art are still
It can so be made respectively in the form and details according to the technology contents described by Summary and embodiment part
The change of various kinds is planted, without departing from the spirit and scope of the present invention that appended claims are limited.
Claims (3)
1. hybridoma cell strain DHAV-MAb1, deposit number is CCTCC NO：C201701.
2. the monoclonal antibody secreted as the hybridoma cell strain DHAV-MAb1 described in claim 1.
3. the monoclonal antibody described in claim 2 is preparing individually detection 1 type or 3 type DHAVs and is examining simultaneously
The application surveyed in the reagent of 1 type and 3 type DHAVs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| KR20090012686A (en) * | 2007-07-31 | 2009-02-04 | 전남대학교산학협력단 | Attenuated duck hepatitis virus strains, method of separating duck hepatitis virus and preparing process using the same |
| CN103923882A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
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| KR20090012686A (en) * | 2007-07-31 | 2009-02-04 | 전남대학교산학협력단 | Attenuated duck hepatitis virus strains, method of separating duck hepatitis virus and preparing process using the same |
| CN103923882A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
| CN105368786A (en) * | 2015-05-19 | 2016-03-02 | 福建省农业科学院畜牧兽医研究所 | Hybridoma cell strain for secreting duck type-1 hepatitis A virus subtype monoclonal antibody |
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| M. LIU ET AL.: ""Goose haemorrhagic hepatitis caused by a new subtype duck hepatitis type 1 virus"", 《VETERINARY MICROBIOLOGY》 * |
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Application publication date: 20170609 |