CN106520703A - Preparation method for cylindrospermopsin monoclonal antibody hybridoma cells and monoclonal antibody - Google Patents
Preparation method for cylindrospermopsin monoclonal antibody hybridoma cells and monoclonal antibody Download PDFInfo
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- CN106520703A CN106520703A CN201611026166.6A CN201611026166A CN106520703A CN 106520703 A CN106520703 A CN 106520703A CN 201611026166 A CN201611026166 A CN 201611026166A CN 106520703 A CN106520703 A CN 106520703A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
Abstract
The invention provides a preparation method for cylindrospermopsin monoclonal antibody hybridoma cells and a monoclonal antibody. The method comprises the steps of 1, preparing a KLH-CYN linker; 2, immunizing experimental animals by using the KLH-CYN linker; 3, performing potency determination on serums of the experimental animals in the step 2; and 4, performing booster immunization and cell fusion on the experimental animals with the serum titers greater than 1 : 104, and performing screening to obtain the hybridoma cells. According to the preparation method for the cylindrospermopsin monoclonal antibody hybridoma cells and the monoclonal antibody, a cylindrospermopsin antibody product is prepared for the first time, so that the blank of domestic products is filled; and in addition, a method basis is provided for further developing a method for more sensitively and effectively monitoring cylindrospermopsin pollution, so that toxic substances can be effectively pre-warned, toxic events are reduced, and water supply safety is ensured.
Description
Technical field
The present invention relates to bioengineering field, more particularly, it relates to it is thin to intend post spore Algae toxins monoclonal antibody hybridoma
Born of the same parents and the preparation method of monoclonal antibody.
Background technology
Cyanophycean toxin is that, by the secondary metabolite of some poisonous bloom blue algaes generations in poisons in freshwater, frequent species includes
Microcystin, anabena toxin and plan post spore Algae toxins etc., can produce potential liver toxicity, neurotoxicity, gene to human body
In toxicity, fetal toxicity and carcinogenecity, therefore drinking water, the residual of Algae toxins can bring serious health risk for the mankind.
Intend post spore Algae toxins (Cylindrospermopsin, CYN) main by plan post spore algae (Cylindrospermopsis
Raciborskii) produce, be one of very concerned in recent years new species in cyanophycean toxin, at present, intend post spore Algae toxins
Australia, the U.S., Germany, Brazil etc. it is multiple country eutrophication water in detect, Guangdong Province of China some
Reservoir is intended post spore Algae toxins concentration and is also up to 8.25 μ g L-1。
Intend the alkaloid (C that post spore Algae toxins are that a kind of molecular weight is 415.4315H21N5O7), S, has acute poison to mice
Property, the LD of intraperitoneal injection of mice50For 2.1mg kg-1.Research shows that CYN can liver injury, kidney, heart after entering in vivo
Etc. multiple organs, into after cell albumen can be suppressed to synthesize, damage dna has extensive cytotoxicity, may act on substantial amounts of water
Raw, semi-aquatic plant and animal and phytoplankton；In addition, CYN to be enriched with biology and can be shifted, this causes the toxin
Environmental effect extends to terrestrial organism, more has research to think that CYN is a kind of potential carcinogen, serious harm human health.
Antibody is studied as diagnosis and analytical reagent and develops a nearly century.More than one since century, antibody
Used as protein molecule most marvellous in vivo, the always study hotspot of life sciences, especially biomedical sector is the mankind
The prevention of various diseases, diagnosis and treatment are made that tremendous contribution.The development experience of antibody technique three phases, conventional antibodies
With obtaining after antigen-immunized animal, referred to as polyclonal antibody, also referred to as first generation antibody.Polyclonal antibody can be used for general anti-
Former detection and passive immunotherapy etc..Due to the complicated component of this antibody, prepare costly, only have in infection early stage
Effect, with certain toxicity, therefore is restricted its extensive application.
1975, the appearance of the monoclonal antibody that second generation antibody B lymphocyte hybridoma technology is produced, into making a living
One of epoch-making great progress of life science.Hybridoma technology depends on the B of a myeloma cell line and immune animal
Cell is blended, and the fused cell of acquisition had both had the ability of B cell secreting specificity antibody, has murine myeloma cell again
The characteristic of immortality.At present this technology is still one of core technology in biotechnology, thousands of medical science and diagnosticss' phase
The hybridoma of pass has been formed.The secretory cell of monoclonal antibody all derives from same ancester cell, and antibody has high
Homogeneity, which has the incomparable specificity of polyclonal antibody with the reactivity of antigen, therefore has caused life sciences to grind
Study carefully and clinical disease diagnosis, the significant innovation for the treatment of, the diagnosis derived as basic fundamental with monoclonal antibody, treatment technology layer
Go out not poor.Monoclonal antibody has been used for the treatment of various diseases such as organ transplantation, tumor, infectious disease, cardiovascular disease and inflammation, into
For researching and developing one of most active biotech drug.
ELISA is the abbreviation of enzyme linked immunoadsorbent assay (Enzyme-Linked Immunosorbent Assay).This
Since item technology came out from the beginning of the seventies, development is very rapid, has been widely used for biology and medical science applied many at present
Field.Immunological detection based on ELISA is widely used in the detection of cyanophycean toxin nearly ten years, and ELISA method has
Quickly, the advantages of sensitivity is high, sample pre-treatments are simple, market application foreground is wide.The two kinds of CYN examinations sold in the market
The direct competive ELISA method that agent box (Beacon and Abraxis LLC) is all based on rabbit polyclonal antibody and sets up, both inspections
Survey limit and be respectively 0.1 and 0.05ppb, detection range within 2ppb, the chemistry of the sensitivity of ELISA method apparently higher than CYN
And biological test method.
Presently commercially available import plan post spore Algae toxins ELISA kit is prohibitively expensive, and single sample testing cost reaches up to a hundred
Unit, and the range of linearity is extremely narrow.Foreign countries have the ELISA kit based on CYN antibody to sell at present, but still no single CYN
Antibody product.
The content of the invention
In view of this, the technical problem to be solved in the present invention is the defect for overcoming prior art, there is provided intend post spore algae
The preparation method of toxin monoclone antibody hybridoma, comprises the steps：
Step one, prepares KLH-CYN junctional complexs；
Step 2, with the KLH-CYN junctional complexs immunization experiment animal；
Step 3, carries out titration to the laboratory animal serum of the step 2；
Step 4, takes serum titer more than 1:104The laboratory animal carry out booster immunization, cell fusion and screen
Obtain hybridoma cell strain.
Further, the step one, KLH-CYN junctional complex preparation methoies are：KLH albumen is directly dissolved in phosphate to delay
Liquid is rushed, CYN and formaldehyde stirring reaction is subsequently added, is dialysed after the completion of reaction.
Further, comprised the steps with the KLH-CYN junctional complexs immunization experiment animal in the step 2：
Step one, takes KLH-CYN junctional complexs and laboratory animal is administered；
Step 2, gathers laboratory animal serum；
Step 3, reaches 1 using indirect elisa method real-time detection serum:104It is during the above, stand-by.
Further, described in the step 3, titration method comprises the steps：
Step one, prepares BSA-CYN junctional complexs, is dissolved in carbonate buffer solution coated elisa plate；
Step 2, takes immunization experiment animal serum and adds the ELISA Plate, and add label；
Step 3, develops the color and tests the potency of immune serum.
Further, the method for titration described in the step 3 is indirect elisa method.
Further, the laboratory animal is BALB/c mouse.
The application provides a kind of preparation method for intending post spore Algae toxins monoclonal antibody, comprises the steps：
Step one, the plan post spore Algae toxins monoclonal antibody hybridoma cell is bred；
Step 2, the plan post spore Algae toxins monoclonal antibody hybridoma cell to breeding carry out purification.
The propagation in the step one comprises the steps：
Step one, by the hybridoma with 1 × 106/ amount injection only is all through the 8-10 of liquid paraffin pretreatment
The laboratory animal abdominal cavity in age；
Step 2, extracts ascites after raising 6-7 days.
Described in the step 2, purification comprises the steps：Ascites is taken, is centrifuged, by purification column eluting.
The purification column adopts Protein G fillers.
In the application, first, BSA-CYN junctional complexs and KLH-CYN junctional complexs are prepared for；Further, KLH-CYN companies are taken
Connecing thing has carried out immunity to laboratory animal, determines immune serum potency come tracking test process using indirect elisa method, and treating excess syndrome is tested
Animal serum potency reaches 1:104Further booster immunization is carried out, and carries out cell fusion, then carried out by indirect elisa method
Screening experiment, finally obtains stably excreting and intends post spore Algae toxins monoclonal antibody hybridoma cell strain.
Further, post spore Algae toxins monoclonal antibody hybridoma cell will be intended by propagation and purification, obtains intending post spore
Algae toxins monoclonal antibody.
Presently commercially available import plan post spore Algae toxins ELISA kit is prohibitively expensive, and single sample testing cost reaches up to a hundred
Unit, and the range of linearity is extremely narrow.Foreign countries have the ELISA kit based on CYN antibody to sell at present, but still no single CYN
Antibody product.The preparation method of the plan post spore Algae toxins monoclonal antibody hybridoma cell in the application and plan post spore Algae toxins list
The preparation method of clonal antibody realizes the preparation of CYN antibody products first, has filled up the blank of home products.
Description of the drawings
It should be appreciated that the following drawings illustrate only some embodiments of the application, therefore it is not construed as to model
The restriction enclosed, for those of ordinary skill in the art, on the premise of not paying creative work, can be with according to these
Accompanying drawing obtains other related accompanying drawings.
Fig. 1 is monoclonal antibody and plan post spore Algae toxins response characteristic figure in the application.
Specific embodiment
The claim of the present invention is described in further detail with reference to the mode of specific embodiment, following
Elaborate many details in order to fully understand the present invention in description.But the present invention can be much retouching different from here
Implementing, those skilled in the art can do similar improvement in the case of without prejudice to intension of the present invention to the alternate manner stated, because
This present invention is not embodied as being limited by following public.
This application provides intending the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell, comprise the steps：
Step one, prepares KLH-CYN junctional complexs；
Step 2, with the KLH-CYN junctional complexs immunization experiment animal；
Step 3, carries out titration to the laboratory animal serum of the step 2；
Step 4, takes serum titer more than 1:104The laboratory animal carry out booster immunization, cell fusion and screen
Obtain hybridoma cell strain.
It is to be appreciated that cyanophycean toxin is the secondary metabolism produced by some poisonous bloom blue algaes in poisons in freshwater producing
Thing, can produce potential liver toxicity and Nervous toxicity to human body including Microcystin, anabena toxin and plan post spore Algae toxins etc.
Property, cause health risk, receive much concern.
It is to be appreciated that it is non-in recent years in cyanophycean toxin to intend post spore Algae toxins (Cylindrospermopsin, CYN)
Often one of new species of concern, is mainly produced by plan post spore algae (Cylindrospermopsis raciborskii).Intend post
Spore Algae toxins are detected in the eutrophication water of multiple countries such as Australia, the U.S., Germany, Brazil, China Guangdong
Some reservoirs for saving are intended post spore Algae toxins concentration and are also up to 8.25 μ g L-1。
It is to be appreciated that intend post spore Algae toxins to be, by a kind of cyanophycean toxin that the cyanophyceaes such as post spore algae produce is intended, is a kind of
Molecular weight is 415.43 alkaloid (C15H21N5O7S), have acute toxicity, the LD of intraperitoneal injection of mice to mice50For 2.1mg
kg-1.Research shows, CYN enter in vivo after can multiple organs such as liver injury, kidney, heart, albumen can be suppressed into after cell
Synthesis, damage dna have extensive cytotoxicity, may act on substantial amounts of aquatic, semi-aquatic plant and animal and phytoplankton；
In addition, CYN to be enriched with biology and can be shifted, this causes the environmental effect of the toxin to extend to terrestrial organism, more grinds
Study carefully and think that CYN is a kind of potential carcinogen, serious harm human health.Therefore researcher be devoted to always development it is more sensitive,
Effectively monitoring method, so that the appearance to noxious substance carries out effective early warning, reduces the generation of toxic events, ensures the peace that supplies water
Entirely.
Further, the step one, KLH-CYN junctional complex preparation methoies are：KLH albumen is directly dissolved in phosphate to delay
Liquid is rushed, CYN and formaldehyde stirring reaction is subsequently added, is dialysed after the completion of reaction.
It is to be appreciated that keyhole limpet hemocyanin (KLH) is the protein macromolecule with high degree of immunogenicity, as carrier
Albumen is used for immunogenic preparation, is crosslinking in hapten and other antigens, strengthens the immunogenicity of micromolecular compound.
Above-mentioned, KLH-CYN junctional complexs voluntarily can be prepared, and concretely comprise the following steps, by 1.5mg KLH albumen be directly dissolved in 200 μ
The phosphate buffer of L, is subsequently added 250 μ g CYN, adds 6 μ L of formaldehyde, and under room temperature dark condition, stirring reaction 50 is little
When, to be dialysed after the completion of coupled reaction, lyophilizing is standby.
Further, comprised the steps with the KLH-CYN junctional complexs immunization experiment animal in the step 2：
Step one, takes KLH-CYN junctional complexs and laboratory animal is administered；
Step 2, gathers laboratory animal serum；
Step 3, reaches 1 using indirect elisa method real-time detection serum:104It is during the above, stand-by.
Above-mentioned, immunization experiment animal methods are：The KLH-CYN junctional complexs for taking lyophilizing are mixed with 250 μ L adjuvants, with 100 μ g/
The amount subcutaneous abdomen multi-point injection BALB/c mouse of 500 μ L, is spaced three weeks amount subcutaneous abdomen multi-point injections with 100 μ g/500 μ L
BALB/c mouse, from the beginning of third time immunity, the second week tail vein after immunity gathers mice blood, indirect ELISA method every time
Detection serum titer reaches 1:104More than after prepare fusion, merge it is first 3 days, tail vein injection booster immunization once, antigen dose
100μg。
Further, described in the step 3, titration method comprises the steps：
Step one, prepares BSA-CYN junctional complexs, is dissolved in carbonate buffer solution coated elisa plate；
Step 2, takes immunization experiment animal serum and adds the ELISA Plate, and add label；
Step 3, develops the color and tests the potency of immune serum.
It is to be appreciated that bovine serum albumin (BSA), also known as BSA, is a kind of albumin in Ox blood serum,
It is widely used in biochemical test.
It is above-mentioned, immune serum potency is determined using indirect elisa method in the application.
Above-mentioned, BSA-CYN junctional complexs voluntarily can be prepared, and concrete grammar is：10mg BSA are dissolved in MES buffer, with
Afterwards EDC the and NHS liquid of same dissolving is slowly added in BSA solution, after mixing, room temperature is placed 5 minutes, then by 1M's
During 50 μ L of Jeffamine (polyetheramine) add above-mentioned mixed liquor, and react 3 hours.Jeffamine-BSA junctional complex PD-10
Pillar lyophilizing after purification.The cryodesiccated Jeffamine-BSA of 2mg is dissolved in into the phosphate buffer of 400 μ L, is subsequently added
550 μ g of CYN, add about 12 μ L of formaldehyde, and stirring reaction 50 hours under room temperature dark condition are carried out after the completion of coupled reaction
Dialysis, lyophilizing are standby.
Further, indirect elisa method is carried out to laboratory animal serum and determines potency, take 30 μ g BSA-CYN junctional complexs molten
Solution is coated in ELISA Plate in carbonate buffer solution, 100 μ L/ holes, and 4 DEG C overnight.Next day, (contain 0.1% using PBS
(V/V) Tween-20) board-washing three times, with 0.5% gelatin confining liquid, 200 μ L/ holes, 37 DEG C are closed 1 hour, using PBS
Board-washing three times, mice 15 days tail vein bloods after third time immunity, Mus immune serum is with containing 2% new-born calf serum 10mM PBS
Buffer dilutes, and adds 1 after adding ELISA Plate, 100 37 DEG C of μ L/ holes 1 hour, board-washing three times：104Horseradish peroxidase is diluted again
Enzyme labelling goat anti-mouse igg, 100 37 DEG C of μ L/ holes 1 hour, after board-washing, 100 μ L/ holes add TMB to develop the color, room temperature lucifuge 20min,
Plus 50 μ L/ holes 2M H2SO4Terminating reaction, survey 450nm absorption values, using before immunity mice serum as negative control, with measured value
The potency of immune serum must be judged with control value than >=2.1 for the positive.
Further, the method for titration described in the step 3 is indirect elisa method.
It is to be appreciated that ELISA is the abbreviation of enzyme linked immunoadsorbent assay.ELISA method is after immunofluorescence and puts
A kind of immunoenzyme technics grown up after penetrating immunological technique.Since technique came out from the beginning of the seventies, develop very fast
Speed, has been widely used for biology and medical science applied many fields at present.
Immunological detection based on ELISA (enzyme linked immunoadsorbent assay) is widely used in cyanophyceae nearly ten years
The detection of toxin, ELISA method have the advantages that quickly, sensitivity is high, sample pre-treatments are simple, market application foreground is wide.Mesh
The direct competive ELISA method that several CYN test kits sold on front market are all based on rabbit polyclonal antibody and set up, both
Test limit is respectively 0.1 and 0.05ppb, and, within 2ppb, the sensitivity of ELISA method is apparently higher than detection CYN for detection range
Chemistry and biological test method.
Further, the laboratory animal is BALB/c mouse.
It is to be appreciated that BALB/c is albino lab mouse, as numerous conventional subbreed, originate from house mouse
Mus musculus.From nineteen twenty, they are born so far in New York, and BALB/c mouse has been multiplied more than 200 in global research institution
In generation, it is widely used in the zoopery of immunology, physiology.
The application provides a kind of preparation method for intending post spore Algae toxins monoclonal antibody, comprises the steps：
Step one, the plan post spore Algae toxins monoclonal antibody hybridoma cell is bred；
Step 2, the plan post spore Algae toxins monoclonal antibody hybridoma cell to breeding carry out purification.
The propagation in the step one comprises the steps：
Step one, by the hybridoma with 1 × 106/ amount injection only is all through the 8-10 of liquid paraffin pretreatment
The laboratory animal abdominal cavity in age；
Step 2, extracts ascites after raising 6-7 days.
Described in the step 2, purification comprises the steps：Ascites is taken, is centrifuged, by purification column eluting.
The purification column adopts Protein G fillers.
It is above-mentioned, it is the preparation method of plan post spore Algae toxins monoclonal antibody provided herein, specially：By hybridoma
Cell is with 1 × 106The BALB/c female mices abdominal cavity of the 8-10 week old of/amount injection liquid paraffin pretreatment only, breeding observing
Ascites is extracted when mouse web portion expands after 6-7 days.Mouse ascites are gathered after 7-10 days, and centrifugation 10min collects supernatant, uses
Protein G affinity chromatographs technologies carry out purification：By Protein G fillers loaded in purification column, with equilibration buffer, treat
The ascites of purification with level pad dilute 10 times after with 1.5mL/min speed loadings, be washed till base with level pad after loading
Line, uses elution buffer antibody elution, collects antibody peak, the antibody of eluting with Tris-HCl buffer and after, that is, intended
Post spore Algae toxins monoclonal antibody.Intending post spore Algae toxins monoclonal antibody can be frozen in -20 DEG C.
Further, the plan post spore Algae toxins monoclonal antibody for preparing in the application can be glimmering using direct competitive time resolution
The response characteristic of light immunoassay examination purified monoclonal antibody and CYN, such as Fig. 1, under 0.1ng/mL concentration, CYN shows good
Competitive inhibition reaction, shows that the affinity intended post spore Algae toxins monoclonal antibody with intend post spore Algae toxins is high.
Embodiment
Prepare plan post spore Algae toxins monoclonal antibody hybridoma cell and monoclonal antibody need to be followed the steps below, specifically
For：
1st, KLH-CYN junctional complexs are prepared
1.5mg KLH albumen is directly dissolved in into the phosphate buffer of 200 μ L, 250 μ g CYN is subsequently added, is added first
6 μ L of aldehyde, under room temperature dark condition, stirring reaction 50 hours, are dialysed after the completion of coupled reaction, and lyophilizing is standby.
2nd, mouse immune
The KLH-CYN junctional complexs for taking lyophilizing are mixed with 250 μ L adjuvants, are noted with the amount subcutaneous abdomen multiple spot of 100 μ g/500 μ L
BALB/c mouse is penetrated, three weeks amount subcutaneous abdomen multi-point injection BALB/c mouse with 1100 μ g/500 μ L are spaced, is exempted from from third time
Epidemic disease starts, every time the second week tail vein collection mice blood after immunity, and indirect ELISA method detection serum titer reaches 1:104
More than after prepare fusion, merge it is first 3 days, tail vein injection booster immunization once, 100 μ g of antigen dose.
3rd, immune serum potency tracking and measuring
Immune serum potency is determined using indirect elisa method.Take 30 μ g BSA-CYN and be dissolved in carbonate buffer solution, be coated with
ELISA Plate, 100 μ L/ holes, 4 DEG C are overnight.Next day, using PBS (containing 0.1% (V/V) Tween-20) buffer board-washing three times, use
0.5% gelatin confining liquid, 200 μ L/ holes, 37 DEG C are closed 1 hour, and using PBS board-washing three times, mice is in third time immunity
15 days tail vein bloods afterwards, Mus immune serum with containing the dilution of 2% new-born calf serum 10mM PBS, addition ELISA Plate, 100
1 is added after 37 DEG C of μ L/ holes 1 hour, board-washing three times：104Horseradish peroxidase-labeled goat anti-mouse igg, 100 μ L/ are diluted again
37 DEG C of hole 1 hour, ibid after board-washing, TMB colour developings, 100 μ L/ holes, room temperature lucifuge 20 minutes, plus 50 μ L/ holes 2M H2SO4Terminate anti-
Should, 450nm absorption values are surveyed, using before immunity, mice serum, must be than >=2.1 as positive with control value with measured value used as negative control
To judge the potency of immune serum.
4th, prepare and intend post spore Algae toxins monoclonal antibody hybridoma cell
Serum titer is taken more than 1:104Mice, merge it is first 3 days, take KLH-CYN and mix with isopyknic PBS
Afterwards, booster immunization is carried out with the amount lumbar injection BALB/c mices to be fused per only 100 μ g/500 μ L.It is aseptic to take mouse spleen,
The murine myeloma cell strain SP20 of splenocyte suspension and exponential phase is made by 10：Supernatant is abandoned in 1 ratio mixing, centrifugation,
Centrifuge tube is placed in 37 DEG C of water-baths, by 50% Polyethylene Glycol of 37 DEG C of water bath heat preservations with dropper it is one after another drop of addition centrifuge tube in,
Centrifuge tube is shaken in drop, is dripped off in 1 minute, after dripping off, stand 2 minutes, added the serum-free of 37 DEG C of preheatings every 1 minute
Terminating the effect of Polyethylene Glycol, cell mixture is centrifuged 5 minutes for 1640 culture medium 1mL, 2mL, 3mL, 4mL, 5mL and 10mL,
Supernatant is abandoned, HAT culture fluid re-suspended cells are added, by cell point into 96 orifice plates, per 200 μ L of hole.After culture three days, observation of cell
Fusion situation, changes half HAT culture fluid, continuous a few days, until there is Clone formation, changes HT culture fluid culture three days, passes through
Indirect ELISA method is filtered out can be changed 1640 complete mediums, obtains final product with the hybridoma of BSA-KLH junctional complexs reaction
Intend post spore Algae toxins monoclonal antibody hybridoma cell.
5th, the hybridoma of the anti-CYN monoclonal antibodies of screening secretion
Cells and supernatant is screened using indirect elisa method, the positive colony hybridoma for selecting potency higher is carried out
Subcloning, and with limiting dilution assay continuous cloning 2-3 time, until to 100% cell positive rate, finally obtaining stably excreting
1 plant of anti-CYN cell strain of monoclonal antibody.By positive rate after cloning up to liquid nitrogen cryopreservation after 100% cell amplification culture.
6th, cell propagation and purification
By hybridoma with 1 × 106The BALB/c females of the 8-10 week old of/amount injection liquid paraffin pretreatment only are little
Ascites is extracted when mouse web portion expands after 6-7 days in Mus abdominal cavity, breeding observing.Mouse ascites are gathered after 7-10 days, are centrifuged 10 minutes
Supernatant is collected, and purification is carried out with Protein G affinity chromatographs technologies：It is by Protein G fillers loaded in purification column, slow with balance
Rush liquid balance, ascites to be purified dilute 10 times with level pad after with 1.5mL/min speed loadings, it is slow with balance after loading
Rush liquid and be washed till baseline, use elution buffer antibody elution, collect antibody peak, the antibody of eluting is neutralized with Tris-HCl buffer
Afterwards, obtain final product plan post spore Algae toxins monoclonal antibody.Intending post spore Algae toxins monoclonal antibody can be frozen in -20 DEG C.
7th, the response characteristic analysis and checking intended post spore Algae toxins monoclonal antibody and intend post spore Algae toxins
Plan post spore Algae toxins monoclonal antibody and plan is examined or check and is verified with direct competitive Timed resolved fluoroimmunoassay
The response characteristic of post spore Algae toxins.
Claims (10)
1. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended, it is characterised in that：Comprise the steps：
Step one, prepares KLH-CYN junctional complexs；
Step 2, with the KLH-CYN junctional complexs immunization experiment animal；
Step 3, carries out titration to the laboratory animal serum of the step 2；
Step 4, takes serum titer more than 1:104The laboratory animal carry out booster immunization, cell fusion and screening obtain miscellaneous
Hand over tumor cell strain.
2. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended as claimed in claim 1, it is characterised in that：
The step one, KLH-CYN junctional complex preparation methoies are：KLH albumen is directly dissolved in into phosphate buffer, is subsequently added
CYN and formaldehyde stirring reaction, are dialysed after the completion of reaction.
3. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended as claimed in claim 1, it is characterised in that：Institute
Comprised the steps with the KLH-CYN junctional complexs immunization experiment animal in stating step 2：
Step one, takes KLH-CYN junctional complexs and laboratory animal is administered；
Step 2, gathers laboratory animal serum；
Step 3, reaches 1 using indirect elisa method real-time detection serum:104It is during the above, stand-by.
4. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended as claimed in claim 1, it is characterised in that：Institute
State titration method described in step 3 to comprise the steps：
Step one, prepares BSA-CYN junctional complexs, is dissolved in carbonate buffer solution coated elisa plate；
Step 2, takes immunization experiment animal serum and adds the ELISA Plate, and add label；
Step 3, develops the color and tests the potency of immune serum.
5. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended as claimed in claim 1, it is characterised in that：Institute
The method for stating titration described in step 3 is indirect elisa method.
6. the preparation method of post spore Algae toxins monoclonal antibody hybridoma cell is intended as claimed in claim 1, it is characterised in that：Institute
Laboratory animal is stated for BALB/c mouse.
7. the preparation method of post spore Algae toxins monoclonal antibody is intended, it is characterised in that：Comprise the steps：
Step one, the plan post spore Algae toxins monoclonal antibody hybridoma cell is bred；
Step 2, the plan post spore Algae toxins monoclonal antibody hybridoma cell to breeding carry out purification.
8. the preparation method of post spore Algae toxins monoclonal antibody is intended as claimed in claim 7, it is characterised in that：The step one
In the propagation comprise the steps：
Step one, by the hybridoma with 1 × 106Reality of/amount the injection only through the 8-10 week old of liquid paraffin pretreatment
Test animal abdominal cavity；
Step 2, extracts ascites after raising 6-7 days.
9. the preparation method of post spore Algae toxins monoclonal antibody is intended as claimed in claim 7, it is characterised in that：The step 2
Described in purification comprise the steps：Ascites is taken, is centrifuged, by purification column eluting.
10. the preparation method of post spore Algae toxins monoclonal antibody is intended as claimed in claim 9, it is characterised in that：The purification
Post adopts Protein G fillers.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926218A (en) * | 2023-08-14 | 2023-10-24 | 中国科学院水生生物研究所 | Probe combination, gene chip, kit and method for detecting ascophyllum sp |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101125889A (en) * | 2007-07-18 | 2008-02-20 | 清华大学 | Microcystin monoclonal antibody and its preparation method and application |
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| CN116926218A (en) * | 2023-08-14 | 2023-10-24 | 中国科学院水生生物研究所 | Probe combination, gene chip, kit and method for detecting ascophyllum sp |
| CN116926218B (en) * | 2023-08-14 | 2024-03-15 | 中国科学院水生生物研究所 | Probe combination, gene chip, kit and method for detecting ascophyllum sp |
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