CN102399287A - Method for preparing turbot immunoglobulin monoclonal antibody - Google Patents
Method for preparing turbot immunoglobulin monoclonal antibody Download PDFInfo
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- CN102399287A CN102399287A CN2011103735773A CN201110373577A CN102399287A CN 102399287 A CN102399287 A CN 102399287A CN 2011103735773 A CN2011103735773 A CN 2011103735773A CN 201110373577 A CN201110373577 A CN 201110373577A CN 102399287 A CN102399287 A CN 102399287A
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Abstract
The invention relates to a method for preparing a turbot immunoglobulin monoclonal antibody, which comprises the steps of separation and purification of a turbot serum immunoglobulin and preparation and screening of hybridoma cells and is characterized in that in the separation and purification step, the turbot serum immunoglobulin is subjected to crude extraction by a salting-out method to obtain crude extraction liquid and then the crude extraction liquid is subjected to treatment by a Sephadex-200 gel column after being dialyzed to obtain the turbot serum immunoglobulin. In the invention, a simple, convenient and rapid method for purifying the turbot immunoglobulin is established and the high-purity immunoglobulin can be obtained. The obtained immunoglobulin is used for preparing the monoclonal antibody of the turbot immunoglobulin. An indirect enzyme linked immunosorbent assay method which is established by the prepared monoclonal antibody can be used for detecting specific immunoglobulins of various pathogens so that the aim of early diagnosis on the pathogens is fulfilled. Moreover, the immune effect of various vaccines can be evaluated and data support is provided for establishing a correct immunizing schedule.
Description
Technical field
The invention belongs to fish molecular immunology technical field, be specifically related to turbot Tegeline MONOCLONAL ANTIBODIES SPECIFIC FOR method.
Background technology
Fish are minimum etc. the vertebratess with cellular immunization and immunity system, and Tegeline is the main medium of immune response reaction.After the fish body receives the foreign matter stimulation (like disease, vaccine etc.), can produce corresponding Tegeline in its body.In view of the above, through detection to specific immunoglobulin, not only can the early diagnosis disease, disease is made early warning, also can detect the responsing reaction of internal antibody behind the animal inoculation pvaccination vaccine in real time, formulate for effective immune programme for children and give security.
Turbot (Scophthalmus maximus) is the famous and precious fingerling of sea farming that China introduces, and because of advantages such as its growth are fast, Fresh & Tender in Texture, has become the main kind of northern China industrialized culture.Yet along with it cultures the rapid expansion of scale, the continuous increase of cultivation density, the disease problem is outstanding day by day.Under increasingly high environmental protection and food health safe requirement, serve as that the novel disease control means of main representative obtain broad development with vaccine etc.Therefore, significant in disease early detection and developing vaccines application to the research of turbot Tegeline and antibody thereof.
At present, the research report of relevant turbot Tegeline and antibody thereof is very limited.The purification process of relevant turbot Tegeline is only seen one piece of report (Wei Jianteng etc., 2008), and its purification process is loaded down with trivial details relatively, and the purpose of its purifying is preparation polyclonal antibody (resisting) more, but not monoclonal antibody (monoclonal antibody).And that monoclonal antibody preparation requires immunoglobulin purity is higher, and monoclonal antibody can catch the Tegeline of trace more accurately, improves the sensitivity and the accuracy that detect, promotes turbot disease control level with this, has important theory and realistic meaning.
Summary of the invention
The purpose of this invention is to provide a kind of turbot Tegeline MONOCLONAL ANTIBODIES SPECIFIC FOR method, improve, obtain the higher Tegeline of purity, and then prepare its monoclonal antibody to present turbot immunoglobulin purification method; And monoclonal antibody applied to the cause of disease early diagnosis of turbot and the research of immunne response rule.
MONOCLONAL ANTIBODIES SPECIFIC FOR method of the present invention; Include the separation and purification of turbot serum immune globulin and preparation, the screening step of hybridoma; It is characterized in that; Described separation and purification is to adopt salting-out process that the turbot serum immune globulin is slightly put forward the acquisition coarse body fluid, again the Sephadex-200 gel column is crossed in crude extract dialysis back and obtains the turbot serum immune globulin.
The specification of above-mentioned Sephadex-200 gel column is 80cm * 1.5cm.
The preparation process of above-mentioned hybridoma is to obtain hybridoma behind the turbot serum immune globulin immune balb/c mice with purifying.
The present invention set up a kind of simply, turbot immunoglobulin purification method easily, can obtain highly purified Tegeline; The monoclonal antibody that the Tegeline that obtains is used to prepare the turbot Tegeline; The monoclonal antibody of preparing is set up indirect enzyme-linked immunosorbent adsorption experiment method; Can detect the specific immunoglobulin (being antibody) of multiple cause of disease, reach the purpose of cause of disease early diagnosis.And can assess multiple immune effect of vaccine, formulating for correct immune programme for children provides the data support.
Description of drawings
Fig. 1: turbot Tegeline SDS-PAGE and Western-blot figure.
Wherein position shown in the arrow is the heavy chain (upper end) and the light chain (lower end) of Tegeline, M-marker,
1, turbot Tegeline SDS-PAGE; 2, turbot Tegeline western-blot analyzes
Fig. 2: indirect enzyme-linked immunosorbent assay detects healthy turbot, suffer from the sick turbot of Vibrio anguillarum and through the turbot serological specificity antibody horizontal of Vibrio anguillarum vaccine immunity.
Embodiment
One, the separation and purification of turbot serum immune globulin
Normal turbot tail vein blood, after room temperature was placed 1h, 4 ℃ were spent the night, next day centrifugal (5000r/min, 30min, 4 ℃) separation of serum, packing and to be stored in-70 ℃ of refrigerators for use.
Salting-out process is slightly carried the turbot serum immune globulin; With turbot serum with after equal-volume 0.01mol/L (pH=7.4) phosphate buffered saline buffer (PBS) dilution, 4 ℃ of dialysis in 0.01mol/L (pH=5.4) phosphoric acid buffer (PB), centrifugal (3000r/min; 30min; 4 ℃) behind the collecting precipitation, resolution of precipitate continued in 0.1mol/L (pH=5.4) PB is dialysed as stated above, be dissolved among 0.1mol/L (pH=8.6) PB behind the recentrifuge collecting precipitation and with it; Cross Sephadex-200 gel column (80cm * 1.5cm) be further purified; With 0.1mol/L (pH=8.6) PB is elution buffer, and the high sample of purity is collected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) check.The result shows, uses this method and analyzes the Tegeline that obtains 2 bands are arranged, and molecular weight is respectively 76kD and 27kD, represents the heavy chain and the light chain (Fig. 1) of Tegeline, and the result is consistent with bibliographical information; And easy, the easy handling of this purification process.
Two, turbot Tegeline MONOCLONAL ANTIBODIES SPECIFIC FOR
With the turbot serum IgM immune balb/c mice of purifying, the method for application cell engineering is produced the hybridoma that merges.Through the immunology detection screening method, filter out the hybridoma of the monoclonal antibody of secretion turbot Tegeline, the practical implementation process is following:
1. immune mouse
1) fundamental immunity: with after the turbot Tegeline of purifying and the emulsification of isopyknic Freund's complete adjuvant mixing as antigen, immunizing dose is 0.2ml, subcutaneous injection BALB/C mice in 6 age in week;
2) booster immunization: fundamental immunity is after 3 weeks, use with after the turbot Tegeline of purifying and the emulsification of isopyknic Freund's incomplete adjuvant mixing as antigen, the booster immunization mouse, immunizing dose and method are the same;
3) secondary booster immunization: at interval after 3 weeks once more booster immunization once, method is the same;
4) merge ultra exempting from of first three day: as antigen, abdominal cavity and tail vein are injected mouse respectively and are surpassed and exempt from the turbot Tegeline of purifying, and immunizing dose respectively is 0.1ml.
2. feeder cell preparation
1) takes off cervical vertebra and put to death normal BALB/C mice; Cut off belly fur (avoiding breaking peritonaeum) under the aseptic condition gently; With being expelled in the mouse peritoneum behind the syringe absorption 5ml RPMI-1640 substratum (containing 10% NBCS and 1%HT) that is equipped with No. 16 syringe needles; The simultaneously light mouse peritoneal of pressing is then with in the substratum inhalation syringe and shift out;
2) peritoneal fluid of sucking-off is transferred in the 24 porocyte culture plates, put into CO2gas incubator and cultivate, for use.
3. cytogamy
1) take off cervical vertebra and put to death immune mouse, aseptic taking-up spleen is crossed behind 100 eye mesh screens with GNK (containing glucose, NaCl, KCl, phenol red and phosphoric acid salt) solution washing 2 times, is dissolved in 10mlGNK solution then, piping and druming formation single cell suspension;
2) get 10
5The individual NS0 myeloma cell who is in logarithmic phase (being so kind as to give) by animal health research institute of Britain country, the centrifugal 10min of 1000rpm, remove supernatant after, resuspended with 40mlGNK solution;
3) splenocyte suspension and myeloma cell's suspension are mixed after, the centrifugal 10min of 1000rpm absorbs supernatant fully, flicks at the bottom of the centrifuge tube, breaks up cell, puts into 37 ℃ of water-baths; Add 37 ℃ of polyglycol solution 1ml that preheating is good, in 1min, drip, in 37 ℃ of water-baths, slowly rotate 90s then;
4) continue to add the GNK solution 15ml that has been preheating to 37 ℃ (beginning slowly drips, afterwards pick up speed) gradually, continue slowly to drip GNK solution then to 40ml, and in 37 ℃ of water-baths, leave standstill 5min;
5) behind the centrifugal 10min of 1000rpm, sedimentary cell with after the good RPMI-1640 substratum of preheating (containing 10% NBCS and the 1%HAT) dilution, is added to 96 holes and contains in the Tissue Culture Plate of feeder cell;
6) culture plate being put into 37 ℃, gas concentration lwevel is that 5% incubator is cultivated, and observation of cell growing state under the inverted microscope probably changes liquid after 5-7 days, and sucking-off 100ul nutrient solution is changed the nutrient solution that equivalent contains HAT.
4. screening
About 10 days, the observation of cell growth conditions is good and begin to detect screening, the positive hybridoma cell of screening is transferred in the 24 porocyte culture plates carried out enlarged culturing after the cytogamy.The detection screening method is following:
1) directly the ELISA method is screened positive hybridoma cell: with 0.05mol/L (pH=9.6) carbonate solution dilution turbot Tegeline to 2 μ g/mL; Getting 50 μ L diluents adds in the enzyme plate hole; Putting 4 ℃ of refrigerator overnight encapsulates; Next day is with 0.01mol/L (pH=7.4) PBST (0.05%Tween-20) washing 3 times, 3min at every turn; In 37 ℃ of sealing 1h, sealing finishes the back and washs with method with PBST with 5% porcine blood serum; Add the Hybridoma Cell Culture supernatant, every hole adds 50 μ L, and negative control is a cell culture medium, and positive control is an immune serum, behind 37 ℃ of reaction 15min, washs with method with PBST; Every then hole adds 50 μ L sheep anti-mouse igg-HRP (dilution in 1: 1000), in 37 ℃ of reaction 30min after scouring; Every hole adds 2mol/L sulphuric acid soln 50 μ L termination reactions after adding 50 μ L colour developing liquid TMB (TMB) the colour developing 5min of new preparation; The result judges with automatic ELIASA and reads OD450 value (P/N >=2.1 o'clock be judged to be the positive).
2) indirect elisa method screening positive hybridoma cell: dilute bovine serum albumin (BSA) to 2 μ g/mL with 0.05mol/L (pH=9.6) carbonate solution; Getting 50 μ L diluents adds in the enzyme plate hole; Putting 4 ℃ of refrigerator overnight encapsulates; Next day is with 0.01mol/L (pH=7.4) PBST (0.05%Tween-20) washing 3 times, 3min at every turn; In 37 ℃ of sealing 1h, sealing finishes the back and washs with method with PBST with 5% porcine blood serum; As first antibody (dilution in 1: 1000), every hole adds 50 μ L, behind 37 ℃ of reaction 15min, washs with method with PBST with the anti-BSA serum of turbot; Add the Hybridoma Cell Culture supernatant then, every hole adds 50 μ L, and negative control is a cell culture medium, and positive control is an immune serum, behind 37 ℃ of reaction 15min, washs with method with PBST; Every then hole adds 50 μ L sheep anti-mouse igg-HRP (dilution in 1: 1000), in 37 ℃ of reaction 30min after scouring; Every hole adds 2mol/L sulphuric acid soln 50 μ L termination reactions after adding 50 μ L colour developing liquid TMB (TMB) the colour developing 5min of new preparation; The result judges with automatic ELIASA and reads OD450 value (P/N >=2.1 o'clock be judged to be the positive).
5. clone
Screening obtains 16 positive hybridoma cells, chooses 4 and clones and check, and all the other are frozen.The clone, the method for inspection is following:
1) adopt limiting dilution assay that the positive hybridoma cell that filters out is cloned: with the feeder cell that add 100ul in 96 each hole of porocyte culture plate; Count the cell in the positive hybridoma cell hole that to clone with blood counting chamber then, dilute with 10 times of gradients, take out 100 hybridomas with nutrient solution; Behind the hybridoma mixing that takes out, be added drop-wise in 96 orifice plates that are paved with feeder cell, every hole adds 100ul, and a hybridoma is contained in average every hole; Putting into CO2gas incubator then cultivates.During this time, routine observation cell growth condition and record, and detect the supernatant of each culture hole through above-mentioned ELISA method, the hybridoma in gained positive colony hole as stated above again the clone once, to guarantee to form mono-clonal.
2) the Western-bloting method checks the mono-clonal positive hybridoma cell that is obtained: SDS-PAGE to separate the turbot Tegeline of being purified; Running gel is transferred on the nitrocellulose filter (aperture 0.22um), and transfer printing finishes nitrocellulose filter is sealed 1h with skim-milk, places the monoclonal hybridoma culture supernatant of acquisition slowly to shake 1h nitrocellulose filter after the PBST washing; After the method washing; Place sheep anti-mouse igg-HRP (dilution in 1: 1000) to shake 1h slowly nitrocellulose filter, after the method washing, nitrocellulose filter is put into HRP-DAB substrate colour developing liquid; To color clear till; After then nitrocellulose filter being rinsed well with deionized water, place drying between filter paper, preserve the dark place.The result shows that this monoclonal antibody and molecular weight are the heavy chain generation specific reaction of the turbot Tegeline of 76KD, are shown as puce band (Fig. 1).
6. frozen
The hybridoma hole vigorous with the growth of dropper piping and druming mixing, that form is good; It is transferred to the centrifugal 7min of 1200rpm in the centrifuge tube; Remove supernatant; Add frozen storing liquid (calf serum of sterilization, RPMI-1640 substratum and DMSO 99.8MIN., its volume ratio are 45%: 45%: 10%), making the whole density of cell is 10
6Individual/ml, transfer to then in the frozen pipe.Frozen pipe is put into capsule, put into-20 ℃ of refrigerator 1h after, transfer to-80 ℃ of refrigerator overnight, immerse again that liquid nitrogen is medium-term and long-term to be preserved.
Three, the application of the turbot Tegeline monoclonal antibody of the present invention's development
Use the monoclonal antibody of the present invention's development and set up indirect enzyme-linked immunosorbent adsorption experiment method, can detect the specific immunoglobulin (being antibody) of multiple cause of disease, reach the purpose of cause of disease early diagnosis; Simultaneously, can assess multiple immune effect of vaccine, formulating for correct immune programme for children provides the data support.
1. get the serum of three kinds of turbot: healthy turbot serum, the turbot serum of suffering from the sick turbot (mainly show as belly and expand, ascites is arranged) of Vibrio anguillarum, crossing through the Vibrio anguillarum vaccine immunity.
2. use the monoclonal antibody of the present invention's development and set up indirect ELISA method: with the Vibrio anguillarum to 10 of 0.05mol/L (pH=9.6) carbonate solution dilution Superlysoform deactivation
7Cfu/mL gets 50 μ L diluents and adds in the enzyme plate hole, puts 4 ℃ of refrigerator overnight and encapsulates, and next day is with 0.01mol/L (pH=7.4) PBST (0.05%Tween-20) washing 3 times, 3min at every turn; In 37 ℃ of sealing 1h, sealing finishes the back and washs with method with PBST with 5% porcine blood serum; Every hole adds turbot serum (2 doubling dilution) 50 μ L, behind 37 ℃ of reaction 30min, washs with method with PBST; As first antibody, every hole adds 50 μ L to add the turbot Tegeline monoclonal antibody (dilution in 1: 5000) of the present invention development then, and negative control is PBS, behind 37 ℃ of reaction 30min, washs with method with PBST; Every then hole adds 50 μ L sheep anti-mouse igg-HRP (dilution in 1: 1000) as SA, in 37 ℃ of reaction 30min after scouring; Every hole adds 2mol/L sulphuric acid soln 50 μ L termination reactions after adding 50 μ L colour developing liquid TMB (TMB) the colour developing 5min of new preparation; The result judges with automatic ELIASA and reads OD450 value (P/N>=2.1 o'clock be judged to be the positive).
3. result:
1) respectively organizes turbot serum from 10*2
2-10*2
8According to detecting its specific antibody level behind 2 doubling dilutions, the result finds that immune group, ill group of turbot antibody horizontal all are higher than healthy group (Fig. 2).This shows that turbot infects Vibrio anguillarum after being ill, can produce the specific antibody to Vibrio anguillarum in the body.Infect the early stage of Vibrio anguillarum in turbot; Can pass through the detection specificity production of antibodies; Judge whether turbot infects the Vibrio anguillarum disease (by that analogy; Can check other bacteriosises, virus disease, parasitic diseases etc.), the monoclonal antibody that makes the present invention prepare can detect the specific immunoglobulin (being antibody) of multiple cause of disease, reaches the purpose of cause of disease early diagnosis.
2) turbot behind the Vibrio anguillarum vaccine immunity; Its serological specificity antibody horizontal obviously raises; Show that this vaccine brought into play certain immune effect (by that analogy; Can check the specific antibody behind other vaccine immunities), thus the monoclonal antibody that the present invention is prepared can assess multiple immune effect of vaccine, formulating for correct immune programme for children provides the data support.
Claims (3)
1. turbot Tegeline MONOCLONAL ANTIBODIES SPECIFIC FOR method; Include the separation and purification of turbot serum immune globulin and preparation, the screening step of hybridoma; It is characterized in that; Described separation and purification is to adopt salting-out process that the turbot serum immune globulin is slightly carried the acquisition crude extract, again the Sephadex-200 gel column is crossed in crude extract dialysis back and obtains the turbot serum immune globulin.
2. preparation method as claimed in claim 1, the specification that it is characterized in that described Sephadex-200 gel column is 80cm * 1.5cm.
3. preparation method as claimed in claim 1, the preparation process that it is characterized in that said hybridoma obtain hybridoma behind the turbot serum immune globulin immune balb/c mice with purifying.
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| CN201210466421.4A CN103266087B (en) | 2011-11-22 | 2012-11-18 | A kind of hybridoma preparing preparing turbot immunoglobulin monoclonal antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266087A (en) * | 2011-11-22 | 2013-08-28 | 中国水产科学研究院黄海水产研究所 | Hybridoma for preparation of scophthalmus maximus immunoglobulin monoclonal antibody |
| CN104448000A (en) * | 2014-12-18 | 2015-03-25 | 中国海洋大学 | Monoclonal antibody of anti-turbot serum immunoglobulin M as wll as preparation method and application of monoclonal antibody |
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| CN101955534A (en) * | 2009-07-20 | 2011-01-26 | 浙江省淡水水产研究所 | Anti-trionyx sinensis immune globulin monoclonal antibody and application thereof |
| CN102399287A (en) * | 2011-11-22 | 2012-04-04 | 中国水产科学研究院黄海水产研究所 | Method for preparing turbot immunoglobulin monoclonal antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266087A (en) * | 2011-11-22 | 2013-08-28 | 中国水产科学研究院黄海水产研究所 | Hybridoma for preparation of scophthalmus maximus immunoglobulin monoclonal antibody |
| CN103266087B (en) * | 2011-11-22 | 2015-12-23 | 中国水产科学研究院黄海水产研究所 | A kind of hybridoma preparing preparing turbot immunoglobulin monoclonal antibody |
| CN104448000A (en) * | 2014-12-18 | 2015-03-25 | 中国海洋大学 | Monoclonal antibody of anti-turbot serum immunoglobulin M as wll as preparation method and application of monoclonal antibody |
| CN104448000B (en) * | 2014-12-18 | 2018-04-13 | 中国海洋大学 | Monoclonal antibody of anti-turbot serum immune globulin M and preparation method and application |
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Application publication date: 20120404 |