CN104099300B - Hybridoma cell strain and its application of anti-B-IgG IgG monoclonal antibody can be secreted - Google Patents
Hybridoma cell strain and its application of anti-B-IgG IgG monoclonal antibody can be secreted Download PDFInfo
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Abstract
The present invention relates to a kind of screening technique of the hybridoma cell strain secreting anti-B-IgG IgG monoclonal antibody for B-IgG IgG detection and hybridoma cell strain, the monoclonal antibody of its secretion and its application in B-IgG IgG detection field.This hybridoma, is deposited in Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University China typical culture collection center, and preserving number is CCTCC No：2013183.The anti-B-IgG IgG monoclonal antibody of above-mentioned hybridoma secretion has the advantages that high specificity, affinity be big, potency is high, can be widely applied to detectable or the testing equipment field of B-IgG IgG.The rapid detection card of the B-IgG IgG being set up based on immunochromatography principle can be applicable to, in the IgG detection of B-IgG in the sample such as colostrum and its product, bovine blood, there is significant advantage at aspects such as specificity, susceptiveness and detection efficiencies than common detection methods.
Description
Technical field
The present invention relates to B-IgG IgG field of immunodetection, especially relate to one kind and can secrete anti-cattle immunity ball
The hybridoma cell strain 9C6 of protein I gG monoclonal antibody and its application.
Background technology
B-IgG system on physics and chemistry properties and classification is named to other mammals and its similar, main include
IgG, IgA and IgM.IgG is divided into two subclass of IgG1 and IgG2, and wherein IgG1 is main component.IgG molecule is by 4 peptide chains
Composition, 2 long-chains are referred to as heavy chains (heavy chain, H), are made up of about 440 amino acid residues, molecular weight about 50～
70kD；Article 2, short chain is referred to as light chain (light chain, L), is made up of about 220 aminoacid, molecular weight about 22.5kD.Article 4,
Peptide chain is connected together by interchain disulfide bond (- S S-).B-IgG is widely distributed in the body fluid of cattle, in colostrum, blood
In liquid, content is higher.In many bioactive substances of colostrum, immunoglobulin G（IgG）It is most important one kind.Cattle colostrumss
The content of middle IgG is restricted by many factors such as age of milk cattle, lactation period and feeding environment.Research shows, immunoglobulin
There is antibacterial, adjust the multiple biological activities such as immunity, protection intestinal mucosa.In the exploitation of colostral milk products, the content of IgG is direct
Determine quality and the price of product.Therefore research and develop anti-B-IgG IgG monoclonal antibody, set up quick detection cattle
The method of IgG has great importance.
The detection method of B-IgG IgG mainly includes agar diffusion method at present（SRD）, liquid chromatography（HPLC）
And euzymelinked immunosorbent assay (ELISA)（ELISA）Deng.Wherein agar diffusion method is easy and simple to handle, and equipment is simple, the relatively low visual result of cost of determination.
But due to diffusion diameter measurement error, lead to result difference larger, accuracy and sensitivity are relatively low, simultaneously high labor intensive, consumption
Duration.High-efficient liquid phase chromatogram technique analysis are accurately, reproducible, however it is necessary that complex instrument equipment, sample pretreatment process is loaded down with trivial details,
Time-consuming for detection process, experimental situation is had high demands it is difficult to realize quick detection.Euzymelinked immunosorbent assay (ELISA) sensitivity is high, high specificity,
Tens even hundreds of samples can be measured simultaneously, apply universal in research abroad, but this method complex operation, and time-consuming,
Costly.Immunochromatographic method has high specificity, sensitivity is high, sample pre-treatments are simple, the low advantage of testing cost, is suitable to existing
Field batch detection, develops very fast in recent years.In the produce reality of colostrum processing and quality testing and the feeding management of milch cow calf,
In the urgent need to setting up the rapid free epidemiology detection method of the B-IgG IgG for colostrum, blood and urine.These are quick
Method is required for specific recognition B-IgG IgG（Including IgG1 and IgG2）Monoclonal antibody.Therefore, using hybridization
Anti- B-IgG IgG is secreted in the preparation of tumor technology（Including IgG1 and IgG2）Monoclonal antibody, and study on this basis
Exploitation B-IgG IgG rapid free epidemiology detection technique, significant.
Content of the invention
Problem to be solved by this invention is can to secrete anti-B-IgG IgG monoclonal using hybridoma technology selection-breeding
The hybridoma cell strain of antibody and the monoclonal antibody being produced using it set up immunological detection method.
The invention provides hybridoma cell strain 9C6, this cell strain is preserved in Chinese Typical Representative on November 28th, 2013
Culture collection (CCTCC), preservation address is, Chinese, Wuhan, Wuhan University, and deposit number is CCTCC
NO.C2013183, Classification And Nomenclature is mouse hybridoma cell 9C6.
Anti- B-IgG IgG monoclonal antibody, it is thin for the hybridoma of CCTCC NO.C2013183 by deposit number
Born of the same parents strain 9C6 secretion produces.This anti-B-IgG IgG monoclonal antibody can identify Immunoglobulin IgG1 and IgG2.
Application in B-IgG IgG mensure for the anti-B-IgG IgG monoclonal antibody.
The beneficial effects of the present invention is:
(1) the hybridoma cell strain 9C6 that the present invention provides can be used for preparing high-titer anti-B-IgG IgG Dan Ke
Grand antibody, the potency that anti-B-IgG IgG mouse ascites antibody ELISA immuning adsorpting analysis (ELISA) method records up to
5.12×105（Antigen coat concentration 2 μ g/mL）.
(2) the anti-B-IgG IgG monoclonal antibody sensitivity that the present invention provides is high, specificity is good, with Lac Bovis seu Bubali cheese
Albumen, milk albumin, Lac Bovis seu Bubali lactoferrin, bovine serum albumin, the cross reacting rate of ovalbumin are respectively less than 1%, not with mountain
Sheep IgG, sheep IgG, rabbit igg and cattle IgM reaction.
(3) the anti-B-IgG IgG monoclonal antibody that the present invention provides can be applicable to measure cattle colostrumss and its product,
B-IgG IgG content in the samples such as blood.
(4) the gold-marking immunity chromatography that the present invention provides can be applicable in the sample such as cattle colostrumss and its product, bovine blood
The field quick detection of B-IgG IgG content and the quality of production control.
Brief description
Fig. 1 is the front view of the B-IgG IgG immunochromatographydetection detection card of the embodiment of the present invention 3 preparation.
Fig. 2 is the sectional drawing of the B-IgG IgG immunochromatographydetection detection card of the embodiment of the present invention 3 preparation.
The knot of the B-IgG IgG immuno-chromatographic test paper strip detection sample that Fig. 3 provides for the application embodiment of the present invention 3
Fruit process decision chart.
Specific embodiment
Embodiment 1:The screening of hybridoma cell strain 9C6
1. animal immune
Choose 6 week old female BAl BIc/c mice 4, use B-IgG IgG（Produced by Sigma-Aldrich company,
Article No. I5506）Immunity 3 times, 100 μ g/ first, equivalent complete Freund's adjuvant, fully emulsified, lumbar injection；Carry out after 2 weeks
Secondary immunity, 100 μ g/, equivalent incomplete Freund's adjuvant, fully emulsified, lumbar injection；Carry out third time immunity, behaviour after 2 weeks
Make with second immunity.Measure serum titer, select serum titer soprano in first 3 days booster immunizations of fusion, 100 μ g/ only, no
Plus adjuvant, lumbar injection.
2. cell fusion
After booster immunization 3 days, take mouse boosting cell according to a conventional method, merge with murine myeloma cell SP2/0.Using
Fusion agent made by Polyethylene Glycol (PEG, molecular weight is 1450), carries out the culture of fused cell according to a conventional method.
3. the screening of cell strain and clone
The 8-14 days after cell fusion, cell colony grows to the 1/4-1/2 size of bottom hole area, and culture fluid turns yellow, and carries out
Antibody test.Antibody test is carried out to the culture hole having Growth of Hybridoma Cell using improved sandwich ELISA method.Using having
Limit dilution method is cloned, and is detected using same method within 8-12 days about after clone, after such repeated cloning 2-3 time,
Obtain hybridoma cell strain 9C6.
Wherein, improved Sandwich ELISA detects concretely comprising the following steps of hybridoma supernatant antibody：
（1）It is coated：With being coated liquid by goat-anti cattle IgG antibody（Sigma-Aldrich company, B1645）1000 times of dilution.?
100 μ L are added, 4 DEG C overnight in each reacting hole of ELISA Plate（Or 37 DEG C of incubation 2 ~ 3h）.Next day, discard in the hole solution, each hole adds
PBS-T lavation buffer solution 220 μ L, vibrates 1 minute, gets rid of liquid in clear opening, add PBS-T lavation buffer solution, so washs 3
Secondary.
（2）Closing：Plus 200 μ L confining liquid in above-mentioned coated reacting hole, put 37 DEG C incubation 1h.It is washed out 3 times.
（3）Sample-adding：Plus hybridoma cell strain culture fluid supernatant 100 μ L is in reacting hole, put 37 DEG C of incubation 1h.It is washed out
3 times.(negative control is done with negative culture fluid supernatant simultaneously, positive control is done with positive serum, it is blank right to be done with PBS
According to).
（4）Plus ELIAS secondary antibody：The sheep anti mouse enzyme labelled antibody of new dilution is added in each reacting hole（Sigma-Aldrich company,
A3673）100μL.37 DEG C of incubation 40min, wash 5 times.
（5）Plus substrate solution colour developing：The tmb substrate solution 100 μ L just having prepared, 37 DEG C of dark place temperature are added in each reacting hole
Educate 10min.
（6）Terminating reaction：Terminate liquid 50 μ L, reading immediately is added in each reacting hole.
（7）Microplate reader reading：Each hole absorbance is read under 450nm.
The described buffer that is coated is 1.59g sodium carbonate, 2.93g sodium bicarbonate, plus distilled water to 1000mL, regulation pH value
To 9.6.
Described PBS is 8g sodium chloride, 2.9g disodium hydrogen phosphate, 0.2g potassium chloride, 0.2g di(2-ethylhexyl)phosphate
Hydrogen potassium, plus distilled water is settled to 1000mL gained.
Described PBS-T lavation buffer solution is 0.5mL Tween-20, plus PBS 1000mL gained.
Described confining liquid is 5.349g ammonium chloride, plus distilled water 1000mL gained.
Described substrate solution is A, B liquid TMB nitrite ion（Jinan Thailand sky and biotechnology company produce）.
Described terminate liquid is 2M aqueous sulfuric acid.
Embodiment 2:Anti- B-IgG IgG monoclonal antibody prepare purification, hypotype and CHARACTERISTICS IDENTIFICATION
The anti-B-IgG IgG monoclonal antibody hybridoma strain 9C6 injection that embodiment 1 is obtained is used in advance
The BALB/c mouse that freund 's incomplete adjuvant was processed, collects the ascites of this mice, after centrifugal degreasing, using Protein A parent
With purification process antibody purification（Concrete by specification operation）.Immediately 0.01M pH7.2 phosphate buffer is dialysed after purification,
Adjustment protein concentration to 10mg/mL, -70 DEG C of refrigerator freezings, standby；
Identify the anti-B-IgG IgG monoclonal antibody of hybridoma cell strain 9C6 secretion with hypotype identification kit
Hypotype be IgG1.
The potency of mouse hydroperitoneum antibody of RGDV recording 9C6 with conventional indirect elisa method is up to 5.12 × 105（Antigen coat concentration 2
μg/mL）, can react with cattle IgG1 and IgG2, with bovine casein, milk albumin, Lac Bovis seu Bubali lactoferrin, bovine serum albumin
In vain, the cross reacting rate of ovalbumin is respectively less than 1%, does not react with goat IgG, sheep IgG, rabbit igg and cattle IgM.
The preparation of embodiment 3 immunochromatographydetection detection card or test strips and application
The anti-B-IgG IgG monoclonal antibody that embodiment 2 is obtained is used for preparing B-IgG IgG immunity
Chromatography detection card, preparation method comprises the following steps:
（1）The preparation of antibody
Secrete the anti-B-IgG IgG monoclonal antibody producing as traget antibody from described cell strain, select
With the anti-cattle polyclonal antibody in the sources such as rabbit, cattle, horse, donkey as detection line（T line）Coated antibody, sheep anti mouse polyclonal antibody is made
For nature controlling line（C line）Coated antibody.
（2）The preparation of detection film
By T line coated antibody with C line coated antibody is coated liquid with T line respectively and C line is coated liquid and is configured to 0.5-2.0mg/
The two is sprayed at nitrocellulose filter bar with the interval of 0.4-1cm using Bio-dot quantitation spray equipment by the concentration of mL respectively
On, discharge rate is 1 μ g/cm, obtains T line and C line, 30-60 minute is dried under the conditions of being placed in 37 DEG C, is then immersed in soaking in confining liquid
After 10 minutes, 3-6 hour is dried under the conditions of 30 DEG C, adds desiccant to seal up for safekeeping, standby.
It is 0.01M pH7.2-7.4 phosphate buffer that described T line is coated liquid, 0.14M sodium chloride, plus 5g/L sucrose,
0.22 μm of membrane filtration is degerming, 4 DEG C of preservations, uses in 2 weeks.
It is 0.01M pH7.2-7.4 phosphate buffer that described C line is coated liquid, plus 5g/L sucrose, 0.22 μm of membrane filtration
Degerming, 4 DEG C of preservations, use in 2 weeks.
Described confining liquid is 0.01M pH7.2 PBS, plus 10g/L sucrose, 10g/L isinglass, 0.22 μm of filter
Membrane filtration is degerming, 4 DEG C of preservations, uses in 1 week.
（3）The preparation of sample pad
Glass fibre or polyester sample pad are cut out growth 300mm, wide 25mm strip, puts into leaching in sample pad pretreatment fluid
Bubble, after 1 hour, takes out, and is dried 6 hours under the conditions of 37 DEG C, adds desiccant to seal up for safekeeping, standby.Described sample pad pretreatment fluid
0.01M pH7.2 phosphate buffer containing 1-10g/L Polyvinylpyrrolidone and the tween 20 of 0.1-2.0g/L.
（4）The preparation of gold standard pad
A, the preparation of the monoclonal antibody of nano gold mark
Before labelling by monoclonal antibody in the quadruple distillation water containing 0.05g/L sodium chloride dialysed overnight.Take nanometer gold
Solution（Grain diameter is 20-40nm）, adjust pH value to 8.2 with 0.2 M potassium carbonate, under conditions of magnetic agitation, receive by every milliliter
Meter Jin Zhong adds the amount of 8-10 μ g antibody, is added dropwise over the monoclonal antibody of purification, stirs 15 min.Add oralbumin molten
Liquid, to final concentration 5g/L, continues stirring 10 min.500 × g is centrifuged 15 min, removes precipitation, takes supernatant, and 12 000 × g is centrifuged
30 min.Suction out supernatant, the re-suspension liquid of the colloidal gold probe original volume of precipitation is redissolved and is centrifuged, suction out supernatant, it is heavy to retain
Form sediment, add the gold of original volume 1/10 to mark and preserve liquid, 4 DEG C of preservations, standby.
Described re-suspension liquid is the 0.01M pH7.2 PB buffer of 0.2g/L sodium azide containing 5g/L oralbumin,
Cross 0.22 μm of filter membrane, 4 DEG C of preservations.
It is that 0.2g/L folds containing 5g/L oralbumin, 100g/L sucrose, 25g/L trehalose that described labelling preserves liquid
The 0.01M pH7.2 PB buffer of nitrogen sodium, crosses 0.22 μm of filter membrane, 4 DEG C of preservations.
B, the preparation of gold standard pad
By glass fibre cutting growth 300mm, 5mm width slice, with Bio-dot metal spraying device resisting nano gold mark
B-IgG IgG monoclonal antibody is laterally sprayed with the discharge rate of 1 μ g/cm, then vacuum lyophilization 6h-24h, adds dry
Drying prescription, sealing preserve.
(5) preparation of absorption pad
Growth 300mm, the specification of wide 30mm are cut out in absorbent paper, obtains final product adsorptive pads;
(6) assembling of test strips:PVC backboard a face corresponding site successively paste detection film, gold standard pad, sample pad and
Adsorptive pads, adjacent sections are in junction overlap 1mm.
(7) the cutting of test strips：Cut into the wide little bar of 3mm with Bio-dot cutting cutter by assembling test strips, obtain final product cattle
Immunoglobulin IgG immuno-chromatographic test paper strip.
(8) assembling of detection card, single part of test strips of well cutting of the present invention are placed in the draw-in groove of plastic bottom card,
Cover lid, compression, see Fig. 1 and Fig. 2.Add desiccant, sealing preserve.
（9）Sample Buffer formula of liquid：0.05M Tris, 0.3M sodium chloride, 0.2g/L sodium azide, 0.005M EDTA,
pH7.2-7.4.
The application of above-mentioned B-IgG IgG immuno-chromatographic test paper strip:
Take sample 1mL, be subsequently adding 24mL sample buffer mix homogeneously, take 200 μ L to add detection card sample-adding in the hole, quiet
Result is read, if detection line shows redness, nature controlling line line shows redness, then be judged to positive findingses, show testing sample after putting 15 minutes
In B-IgG IgG content be more than or equal to 12mg/mL, see 1 in Fig. 3；If detection line does not develop the color, nature controlling line line shows
Redness, then be judged to negative findings, shows that the content of the B-IgG IgG in testing sample is less than 12mg/mL, sees in Fig. 3
2；If nature controlling line line does not develop the color, it is judged to invalid detection, see 3 in Fig. 3.
Claims (4)
1. a kind of hybridoma cell strain 9C6 is it is characterised in that anti-B-IgG IgG monoclonal antibody, its preservation can be secreted
In China typical culture collection center, deposit number is CCTCC NO.C2013183.
2. a kind of anti-B-IgG IgG monoclonal antibody, hypotype is IgG1, and it is CCTCC by deposit number
The hybridoma cell strain 9C6 secretion of NO.C2013183 produces the mice abdomen it is characterised in that being recorded with conventional indirect elisa method
The potency of water antibody is up to 5.12 × 105, can react with cattle IgG1 and IgG2, with bovine casein, milk albumin, Cow Milk
Ferritin, bovine serum albumin, the cross reacting rate of oralbumin are respectively less than 1%, not with goat IgG, sheep IgG, rabbit igg
And cattle IgM reaction.
3. a kind of B-IgG IgG detection card or test strips preparation method it is characterised in that：Comprise the following steps：
（1）The preparation of antibody
From the anti-B-IgG IgG monoclonal antibody described in claim 2 as traget antibody, from rabbit, cattle, horse,
, as detection line coated antibody, sheep anti mouse polyclonal antibody is as nature controlling line coated antibody for the anti-cattle polyclonal antibody in donkey source；
（2）The preparation of detection film
By detection line coated antibody with nature controlling line coated antibody is coated liquid with detection line respectively and nature controlling line is coated liquid and is configured to
The two is sprayed at nitric acid with the interval of 0.4-1cm using Bio-dot quantitation spray equipment by the concentration of 0.5-2.0mg/mL respectively
On cellulose membrane bar, discharge rate is 1 μ g/cm, obtains detection line and nature controlling line, 30-60 minute is dried, then under the conditions of being placed in 37 DEG C
After soaking 10 minutes in immersion confining liquid, 3-6 hour is dried under the conditions of 30 DEG C, adds desiccant to seal up for safekeeping, standby；
（3）The preparation of sample pad
Glass fibre or polyester sample pad are cut out growth 300mm, wide 25mm strip, puts into and in sample pad pretreatment fluid, soak 1
After hour, take out, be dried 6 hours under the conditions of 37 DEG C, add desiccant to seal up for safekeeping, standby；
（4）The preparation of gold standard pad
With Bio-dot metal spraying device by the anti-B-IgG IgG monoclonal antibody of nano gold mark with the discharge rate of 1 μ g/cm
It is sprayed on glass fibre, then vacuum lyophilization 6h-24h, add desiccant, sealing preserve；
（5）The preparation of absorption pad
Growth 300mm, the specification of wide 30mm are cut out in absorbent paper, obtains final product adsorptive pads；
（6）The assembling of test strips:Paste detection film, gold standard pad, sample pad and water suction in a face corresponding site of PVC backboard successively
Pad, adjacent sections are in junction overlap 1mm；
（7）The cutting of test strips：Cut into the wide little bar of 3mm with Bio-dot cutting cutter by assembling test strips；
（8）The assembling of detection card, by step（7）The little bar of reagent paper cutting is placed in the draw-in groove of plastic bottom card, covers lid, pressure
Tightly；Add desiccant, sealing preserve；
（9）Sample Buffer formula of liquid：0.05M Tris, 0.3M sodium chloride, 0.2g/L sodium azide, 0.005M EDTA, pH7.2-
7.4.
4. the detection card of B-IgG IgG according to claim 3 or the preparation method of test strips, its feature exists
In：The（4）The preparation of the gold standard pad of step comprises the following steps：
Before labelling by monoclonal antibody in the quadruple distillation water containing 0.05g/L sodium chloride dialysed overnight；The grain diameter is taken to be
20-40nm nano-Au solution, adjusts pH value to 8.2 with 0.2 M potassium carbonate, under conditions of magnetic agitation, by every milliliter of nanometer gold
Add the amount of 8-10 μ g antibody, be added dropwise over the monoclonal antibody of purification, stir 15 min；Add oralbumin solution to end
Concentration 5g/L, continues stirring 10 min；500 × g is centrifuged 15 min, removes precipitation, takes supernatant, and 12 000 × g is centrifuged 30 min,
Suction out supernatant, the re-suspension liquid of the colloidal gold probe original volume of precipitation is redissolved and is centrifuged, suction out supernatant, retain precipitation, add
The gold mark of nano-Au solution original volume 1/10 preserves liquid, and 4 DEG C preserve, standby；
Described re-suspension liquid is containing 5g/L oralbumin, the 0.01M pH7.2 PB buffer of 0.2g/L sodium azide, mistake
0.22 μm of filter membrane, 4 DEG C of preservations；
It is containing 5g/L oralbumin, 100g/L sucrose, 25g/L trehalose, 0.2g/L sodium azide that described gold mark preserves liquid
0.01M pH7.2 PB buffer, cross 0.22 μm of filter membrane, 4 DEG C preservation.
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| CN105785013A (en) * | 2016-04-21 | 2016-07-20 | 卢连伟 | Colloidal gold immunochromatographic test strip for aided detection of pancreatic cancer and preparation method of golloidal gold immunochromatographic test strip |
| CN107389940A (en) * | 2017-08-23 | 2017-11-24 | 潍坊市康华生物技术有限公司 | A kind of vaginitis multi-link detection reagent kit and preparation method thereof |
| CN107748263A (en) * | 2017-08-31 | 2018-03-02 | 北京臻惠康生物科技有限公司 | The new application and kit of a kind of plasminogen |
| CN109507421B (en) * | 2018-12-07 | 2022-02-01 | 广西大学 | Anti-buffalo IgG monoclonal antibody cell strain and preparation method and application thereof |
| CN109507420B (en) * | 2018-12-07 | 2022-02-01 | 广西大学 | Rapid detection method of buffalo fasciola gigantica antibody |
| CN111896750A (en) * | 2020-08-07 | 2020-11-06 | 杭州都林生物科技有限公司 | Bovine immunoglobulin (IgG) double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method |
| CN114264819A (en) * | 2021-12-08 | 2022-04-01 | 上海理工大学 | Quantum dot nanosphere immunochromatography test strip for rapidly detecting new coronavirus |
| CN114894911B (en) * | 2022-03-18 | 2023-10-24 | 辽宁成大生物股份有限公司 | Method for controlling quality of bovine serum products |
| CN114805589B (en) * | 2022-06-14 | 2024-03-26 | 郑州伊美诺生物技术有限公司 | Monoclonal antibody capable of simultaneously recognizing cow, goat and sheep antibodies |
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