CN109400706A - Anti- Larimichthys crocea IgM monoclonal antibody and preparation method thereof - Google Patents
Anti- Larimichthys crocea IgM monoclonal antibody and preparation method thereof Download PDFInfo
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Abstract
Anti- Larimichthys crocea IgM monoclonal antibody and preparation method thereof.Anti- Larimichthys crocea IgM monoclonal antibody is hybridoma cell strain IgM-9G7.Prepare Larimichthys crocea IgM antigen；Prepare hybridoma cell strain IgM-9G7；Prepare the monoclonal antibody of anti-Larimichthys crocea IgM.Larimichthys crocea IgM monoclonal antibody is to be generated by hybridoma cell strain IgM-9G7, have specific antibody to Larimichthys crocea IgM.The immunoglobulin of cell secretion can efficiently specifically bind Larimichthys crocea IgM, and highest potency reaches 2 × 104.Animal is immunized in the Larimichthys crocea IgM of purifying, takes it to generate the splenocyte of anti-Larimichthys crocea IgM monoclonal antibody and merges the hybridoma cell strain for obtaining and capableing of secreting specificity antibody with myeloma cell, which can secrete the monoclonal antibody of anti-Larimichthys crocea IgM.This antibody specificity is high, high sensitivity, can be used for structural analysis, the detection of immune response level of Larimichthys crocea IgM etc..
Description
Technical field
The present invention relates to monoclonal antibodies and preparation method thereof, more particularly, to a kind of anti-Larimichthys crocea (Larimichthys
Crocea) the monoclonal antibody and preparation method thereof of IgM.
Background technique
China is the first in the world aquaculture big country.With the continuous multiplicity expanded and cultivate type of aquaculture scale
Change, the farming disease harms prevention and treatment problem becomes increasingly conspicuous, and becomes the research emphasis of aquatic products researcher.From the immunocompetence of fish itself
It sets out to study its mechanism for resisting affect, not only facilitates evolution laws and the spy of understanding vertebrate immune system
Point, and peomote the development and development of vaccines for fish.Fish are the biologies for occurring immunoglobulin earliest, although with the food in one's mouth
The immune system of newborn animal has been formed including nonspecific immune reaction, cellular immunity, body fluid compared to also more original
The fairly perfect immune response system such as immune.
Immunoglobulin (Immunoglobulin, Ig) is molecule crucial in fish specific immunity.IgM is B lymph
Cell surface molecule mark only has found a kind of Larimichthys crocea immunoglobulin of IgM in rheum officinale fish serum at present, and heavy chain is about
74kD, light chain about 25kD (building of [1] Han Yifan vibrio alginolyticus outer membrane protein DNA recombinant expression carrier and rabbit-anti Larimichthys crocea
IgM serum prepares [D]；Chinese Marine University, 2006).By the anti-Larimichthys crocea IgM monoclonal antibody of high quality, can carry out big
Yellow croaker IgM+Bone-marrow-derived lymphocyte sorting, will provide help for the function of Larimichthys crocea bone-marrow-derived lymphocyte and related immunological Mechanism Study.
In addition, after infection of marine fishes cause of disease or vaccine inoculation, the specific antibody for the antigen that fish body generates can also pass through the list
Clonal antibody carries out specific antibody titres detection ([2] ZHANG Y A, SALINAS I, LI J, et al.IgT, a
primitive immunoglobulin class specialized in mucosal immunity[J].Nature
Immunology,2010,11(9):827).Therefore, using anti-Larimichthys crocea IgM monoclonal antibody, by detecting rheum officinale fish serum
In the specific IgM potency, the diagnosis of Larimichthys crocea disease and the evaluation of vaccine using effect can be carried out, to Larimichthys crocea disease
Research and prevention and treatment have most important theories and realistic meaning.
Summary of the invention
It is an object of the invention in order to solve using monoclonal antibody research Larimichthys crocea immunoglobulin structure and function,
Monoclonal antibody is used for Pseudosciaena crocea microorganism detection, fish disease diagnosis and prevention and treatment etc. is assisted, anti-Larimichthys crocea IgM Dan Ke is provided
Grand antibody and preparation method thereof.
The anti-Larimichthys crocea IgM monoclonal antibody is hybridoma cell strain IgM-9G7, and depositary institution is Chinese Typical Representative culture
Object collection, preservation address are Wuhan Wuhan University, and collection deposit number is CCTCC NO:C2018104, preservation day
Phase is on May 15th, 2018.
The preparation method of the Larimichthys crocea IgM monoclonal antibody the following steps are included:
1) Larimichthys crocea IgM antigen is prepared；
In step 1), the specific method of the preparation Larimichthys crocea IgM antigen can are as follows: anaesthetizes Larimichthys crocea, carries out tail portion
Blood is taken, is solidified after blood sampling, is placed, after serum is sufficiently precipitated, serum and PBS are diluted, made after filtering by centrifuging and taking upper serum
The IgM antigen in rheum officinale fish serum is obtained with albumin A affinity column and molecular sieve, which includes heavy chain and light chain, institute
Stating heavy chain is 76kD, and the light chain is 25kD；The anesthesia of MS-222 anesthetic can be used in the Larimichthys crocea anesthesia；The tail portion takes blood
2mL injector for medical purpose can be used；The solidification can solidify 1h at room temperature；The placement can be in 4 DEG C of placement 12h；The centrifugation
Condition can be 3000g, 5min；The PBS proportional diluted that can be 8 by serum and pH value by serum and PBS dilution；The mistake
0.45 μm of membrane filtration can be used in filter, and the filter membrane and albumin A affinity column, molecular sieve are tool commonly used in the trade；It is obtained
IgM antigen in rheum officinale fish serum is the Larimichthys crocea serum IgM of high-purity.
2) hybridoma cell strain IgM-9G7 is prepared；
In step 2), the specific method for preparing hybridoma cell strain IgM-9G7 can are as follows: by the Larimichthys crocea of purifying
IgM is mixed with Freund's adjuvant, and Balb/C mouse is immunized as antigen, with myeloma cell and generates anti-Larimichthys crocea IgM antibody
Mouse boosting cell fusion, must secrete the hybridoma cell strain IgM-9G7 of anti-Larimichthys crocea IgM antibody.
3) monoclonal antibody of anti-Larimichthys crocea IgM a kind of is prepared.
In step 3), a kind of specific method of the monoclonal antibody for preparing anti-Larimichthys crocea IgM can are as follows: by hybridoma
Cell strain IgM-9G7 is combined using two methods of limiting dilution assay and ELISA, and it is anti-to filter out the anti-Larimichthys crocea IgM monoclonal of secretion
The hybridoma cell strain IgM-9G7 of body, and then obtain monoclonal antibody.
Larimichthys crocea IgM monoclonal antibody of the present invention, be generated by hybridoma cell strain IgM-9G7, to Larimichthys crocea
IgM has the antibody of specificity.The immunoglobulin of cell secretion can efficiently specifically bind Larimichthys crocea IgM, most efficiently
Valence reaches 2 × 104。
Limiting dilution assay of the present invention and ELISA are respectively used to improve the monoclonal antibody purity generated and detection antibody
Potency is method commonly used in the trade.
In conclusion the present invention is that animal is immunized in the Larimichthys crocea IgM that will be purified, it is taken to generate anti-Larimichthys crocea IgM monoclonal
The splenocyte (being derived from the animal being immunized through antigen) of antibody, which merges acquisition with myeloma cell, is capable of the miscellaneous of secreting specificity antibody
Tumor cell strain is handed over, which can secrete the monoclonal antibody of anti-Larimichthys crocea IgM.This antibody specificity is high, and high sensitivity can be used
In the structural analysis of Larimichthys crocea IgM, the detection of immune response level, etiological diagnosis and further immunological investigation etc., have wide
Wealthy application prospect.
Detailed description of the invention
Fig. 1 is the specificity that Western-blot detects anti-Larimichthys crocea IgM monoclonal antibody.In Fig. 1, M: standard molecule
Amount；Swimming lane 1: negative control sample；Swimming lane 2: Larimichthys crocea blood serum sample.Anti- Larimichthys crocea IgM 9G7 monoclonal antibody can specificity
Identify Larimichthys crocea IgM heavy chain, specificity is good.
Fig. 2 is the lymphocyte of the anti-Larimichthys crocea IgM monoclonal antibody label of flow cytomery.In Fig. 2, A: nothing is schemed
Close the head-kidney Lymphocyte samples of homotype primary antibody contrasting marking；Scheme the head-kidney Lymphocyte samples of B:9G7 labeling of monoclonal antibody.
9G7 monoclonal antibody can be with the IgM in specific marker Larimichthys crocea head-kidney lymphocyte+Bone-marrow-derived lymphocyte.
Fig. 3 is immunofluorescence label Larimichthys crocea lymphocyte.In Fig. 3, the nucleus of dark color instruction DAPI label, light color
Indicate the IgM of 9G7 monoclonal antibody specificity label.
Specific embodiment
Following embodiment will the present invention is further illustrated in conjunction with attached drawing.
One, the preparation of Larimichthys crocea IgM monoclonal antibody
(1) prepare Larimichthys crocea specific antigen: Larimichthys crocea is anaesthetized using MS-222 anesthetic, uses 2mL injector for medical purpose
It collects, in being stored at room temperature 1h, then at 4 DEG C of standing 10h, serum is sufficiently precipitated, and 3000g/min centrifugation takes its supernatant, is used
PBS (PH8.0) proportional diluted, loading is eluted, automatic sampler is received to albumin A affinity column with PBS after 0.45 μm of membrane filtration
Collect eluent, and with the protein concentration of ultraviolet specrophotometer tracking and measuring eluent.The albumen eluted at first is foreign protein, to
After foreign protein is cleaned, albumen is eluted using glycine-HCI buffer (pH3.0), elution is purer Larimichthys crocea IgM egg
It is white.After the IgM collecting protein, continues loading into HiPrep 16/60SepHacryl S-100HR molecular sieve, use PBS
(pH8.0) it elutes, collects the front half section of first protein peak, as high purity rhubarb fish IgM；The ferritin heavy chain molecular weight is
74kD or so, light chain molecule amount are 25kD or so.
(2) prepare a kind of hybridoma cell strain: using Larimichthys crocea IgM protein immunization mouse, with SP2/0 myeloma cell with
The Balb/C mouse boosting cell that anti-Larimichthys crocea haemocyanin IgM antibody is generated after immune merges to obtain hybridoma cell strain.
(3) prepare a kind of monoclonal antibody of anti-Larimichthys crocea immunoglobulin: being generated with the hybridoma cell strain should
Monoclonal antibody, the monoclonal antibody have specificity to the Larimichthys crocea Immunoglobulin IgM.
Mouse immune and step (3) generate the mouse immune of the antibody in step (2) specifically:
5 female Balb/C mouse are immunized with the Larimichthys crocea specific antigen, separately take 5 to do negative control.Exempt from every time
Epidemic disease is with 100 μ g/ Larimichthys crocea IgM and Freund's adjuvant mixed immunity mouse.
Immune programme is respectively as follows: 0d, 15d using fundamental immunity and secondary booster immunization, fundamental immunity time twice, reinforces
Immune: 21d, booster immunization is primary again for the preceding 3 days abdominal cavities of fusion and tail vein approach.By immunizing antigen normal saline dilution,
The antigen injection that fundamental immunity Freund's complete adjuvant emulsifies is subcutaneous (multiple spot) or intraperitoneal in the nape of the neck of mouse, and second
It is subcutaneous (multiple spot) or intraperitoneal in the nape of the neck of mouse that secondary fundamental immunity takes antigen injection that incomplete Freund's adjuvant has emulsified,
Later booster immunization is the same as above-mentioned second of fundamental immunity.
Step (3) prepares hybridoma cell strain specifically:
1. prepared by myeloma cell (SP 2/0)
The good myeloma cell of growth conditions is selected, supernatant is abandoned, is washed one time, is reused using incomplete culture medium
10mL incomplete culture medium gently blows down it.
2. the preparation of feeder cells
A normal mouse is put to death using cervical dislocation, its body surface is sterilized and fixed, cuts off leg using eye scissors
Portion's skin, exposure peritonaeum.Using the HAT culture medium of 5~10mL of 10mL syringe injection into abdominal cavity, featheriness for several times, draws back abdomen
Liquid in chamber gathers the liquid.
3. the preparation of mouse spleen lymphocyte
Cervical dislocation puts to death immune mouse, and spleen is taken out in sterile abdominal cavity of cutting off after disinfection.It is washed using basal medium, with
It is transferred into the plate of DMEM afterwards, its cell is fully ground with 70 μm of strainers by syringe tail portion, it is thin that spleen is made
Born of the same parents' suspension.
4. cell fusion
A. the cell suspension in step 1 and 3 is mixed in the centrifuge tube of 50mL, 1200rpm is centrifuged 10min, by supernatant
Exhaustion；
B. by 1mL preheat PEG be added slowly in fusion pipe in 50s or so, on one side plus while the mixing of Qinging；
C. it is slowly added into the DMEM culture medium of 1mL incubation in the 1st minute, the DMEM of 2mL was allegretto added at the 2nd minute
Culture medium then gradually accelerates the DMEM culture medium that 8mL is added in ground on the 3rd minute；
D. it is centrifuged 10min in 37 DEG C of standings 10min, 1000rpm, abandons supernatant；
E. the HAT culture medium that 5mL is incubated is added, gently hangs cell, adds suitable peritoneal macrophage, mend HAT extremely
50mL or so；
F. above-mentioned cell is sub-packed in 96 porocyte culture plates, is placed in 37 DEG C, 5%CO2Incubator in cultivate；
G.5d half culture medium is replaced with HAT culture medium afterwards；
H. when hybridoma cell strain IgM-9G7 supernatant turns yellow or cell dispersion to bottom hole 10% or more when, take a small amount of thin
Born of the same parents carry out antibody test.
The method for establishing hybridoma cell line is given below:
Raising splenocyte (method is same as above) is prepared in advance, and hybridoma cell strain IgM-9G7 is lightly blown out of culture hole
Under, living cells is counted with blood cell counting plate: cell is diluted to 5,10,50 carefully with DMEM complete medium respectively
The cell suspension of above three concentration is separately added into 96 well culture plates for having prepared feeder cells by born of the same parents/mL, 100 μ L/
Hole, makes corresponding hole averagely contain 0.5,1 and 5 cell respectively, and culture can be changed the liquid once, be examined from 5~6d on the 4th day
The growing state of each hole inner cell, and record.When 7~9d cell clone covers with culture hole 1/3~1/2 after clone
Go the detection for carrying out antibody specificity that antibody effect may be selected such as the cell hole of the detection specific antibody positive with indirect competitive
Valence height is in single clonal growth, the good cell of upgrowth situation, continues to clone again.The cell in positive hole is moved into 24 holes
Culture plate, and fresh culture is added in corresponding foramen primum, to prevent the two pollution or cell death simultaneously.To in 24 orifice plates
Cell well-grown when, can go in 100mL Tissue Culture Flask and cultivate, and freeze in time.
The method that hybridoma cell strain IgM-9G7 freezes is given below:
It taking and grows vigorous, the good hybridoma cell strain IgM-9G7 of form, cell suspension is made, 200rpm is centrifuged 5min,
Supernatant is removed, adds frozen stock solution (+1 part of dimethyl sulfoxide of 9 parts of fetal calf serums), makes final cell density 5 × 106A/mL is thin by 1mL
Born of the same parents' suspension tightens blind nut loaded in 2mL cryopreservation tube, is then packed into cryopreservation tube in the capsule for filling cotton, be put in -80 DEG C it is ultralow
After staying overnight (8~12h) in temperature refrigerator, then immerse long-term preservation in liquid nitrogen.It is full that present invention obtains appearances, perfectly round bright, point
Split vigorous hybridoma.
The method preparation ascites induced in animal body is given below:
1~2 week before being inoculated with hybridoma, first not to Balb/C mouse peritoneal injection 0.5mL atoleine or Freund
Freund's complete adjuvant；Successful hybridoma 1000rpm centrifugation will be cloned, supernatant is abandoned, cell is suspended with 1640 basic culture solutions, and
Cell number is adjusted to 1 × 106A/mL, above every mouse peritoneal injection above-mentioned hybridoma suspension prepared of 0.5mL connect
About 10d after kind hybridoma, it is seen that mouse web portion obviously expands, and is disappeared at this time with the tincture of iodine, cotton ball soaked in alcohol to mouse part skin
After poison, ascites is extracted with 5mL syringe, by ascites in 4 DEG C, 1000rpm is centrifuged 10min, takes supernatant, dispenses -20 DEG C of postposition guarantors
It deposits.
The purifying of ascites: taking the ascites 3mL of above-mentioned preparation, and 0.06mol/L, the acetate buffer solution 6mL of pH4.8 is added, and mixes
99 μ l octanoic acids are added after even while stirring at room temperature, persistently stirs 30min, is subsequently placed in 4 DEG C of static 2h or more.By solution 4
DEG C, 1000rpm is centrifuged 30min, takes supernatant, adjusts pH value to 7.4 with the NaOH of 2mol/L, adds while stirring into supernatant solution
Enter isometric saturation sulfuric acid by solution, after 30min, 4 DEG C of static 2h or more.10000rpm is centrifuged 30min, and precipitating is taken to be dissolved in
In the PBS of 5mL 0.01M, pH 7.4, it is placed in the PBS of 0.01M and dialyses after mixing, 4 DEG C of dialysis 2d, every 8h change a not good liquor, 2d
The solution in bag filter is taken out afterwards, after collection after ultraviolet specrophotometer measures its protein content, is frozen by 5mg/mL packing
In -80 DEG C.
Two, the anti-Larimichthys crocea IgM specific antibody verifying of the present invention
(1) Western-Blot:
1) sample buffer of the equal proportion containing dodecyl sodium sulfate is added in the rheum officinale fish serum of collection, is boiled in boiling water
10min；
2) 1) processed sample is added in loading hole, 5 μ l of sample-adding product in every hole, under constant-pressure conditions, when starting is used
Low-voltage (90V), after sample after concentration glue partial concentration is into a line, high current (120V), electrophoresis to bromophenol blue is indicated
Agent can stop electrophoresis when reaching bottom margin, take out gel；
3) one piece of clip is with the pvdf membrane (0.22 μm of aperture) of running gel same size with electrotransfer buffer (electrotransfer
Buffer: 25mmol/L Tris-Base, 192mmol/L glycine, 20% methanol, pH 8.3) wetting, it is solidifying after being placed on electrophoresis
On glue.It is supported with the filter paper of wetting, the filter paper that second piece soaks is attached to the another side of gel film；By above-mentioned placement order, make
Blob of viscose and pvdf membrane and filter paper form a set of sandwich " sandwich "；
4) " gel sandwich " merging is filled in the electrophoresis tank of electrotransfer buffer, by pvdf membrane towards anode；Electrophoresis
Constant current 250mA, energization 1h；
5) transfer finishes, and takes out pvdf membrane.Pvdf membrane is washed into 10min with PBS, then sets 5% skimmed milk solution
Closing 1h in (PBS matches), 37 DEG C；
6) it is washed 3 times with TBST, each 5min；
7) pvdf membrane is placed in 5% skimmed milk solution containing 10% Hybridoma Cell Culture supernatant, 37 DEG C are slowly shaken
Dynamic 1h；
8) 6. with step；
9) goat anti-mouse ig antibody of horseradish peroxidase-labeled is added (with 5% skimmed milk solution, 1 ︰ in pvdf membrane
5000 dilutions) in, 37 DEG C slowly shake 1h；
10) 6. with step；
11) pvdf membrane is slightly rinsed with deionized water water, in darkroom by ECL kit A liquid and B liquid it is isometric
It mixes well.
12) ECL mixing liquid is covered on pvdf membrane to the one side for having adsorbed albumen, at room temperature (25 DEG C) reaction 1min,
Reflection liquid is discarded, filter paper patch angle is blotted.
13) pvdf membrane film is placed among two layers of preservative film in tabletting box in darkroom, wherein being adsorbed with the one of protein
Up, it is entirely placed in tabletting box, and places the egative film of same size on it, tabletting 1min is (according to actual effect
Adjust duration).
14) film of exposure is put into the 3min that develops in developer solution, soaks film after clear water rinses and removes developer solution
The not 1min in fixing solution.
As a result: the Larimichthys crocea IgM heavy chain that monoclonal antibody of the present invention and molecular weight are 76kD generates specific reaction, shows single item
Band, and negative control is shown without band.(Fig. 1)
(2) flow cytomery fluorescence:
The head-kidney tissue for taking healthy Larimichthys crocea is placed on 70 μm of mesh screens, and L-15 culture medium of the 2mL containing heparin sodium is added, makes
It is carefully ground with syringe tail end, gained, that is, head-kidney leucocyte.
1. the leucocyte slightly mentioned about 1 × 107It is a, the hybridoma culture supernatant weight of 5% Normal Goat Serum is contained with 500 μ L
It is outstanding, 4 DEG C of incubation 1h；
2. PBS 400g, 4 DEG C centrifuge washing 3 times, each 5min；
3. the sheep anti-Mouse Ig (1 ︰ 500 dilution), 4 DEG C of incubation 30min of FITC label is added；
4. PBS 400g, 4 DEG C centrifuge washing 3 times, each 5min；
5. PBS is resuspended, cell suspension is after 70 μm of screen filtrations, flow cytometer detection.
The results show that compared with negative control, labeling of monoclonal antibody a group has the cell (30% or so) of fluorescence,
Monoclonal antibody prepared by the present invention can be with the bone-marrow-derived lymphocyte (Fig. 2) in specific recognition Larimichthys crocea head-kidney leucocyte.
(3) immunofluorescence experiment:
The head-kidney tissue for taking healthy Larimichthys crocea is placed on 70 μm of mesh screens, and L-15 culture medium of the 2mL containing heparin sodium is added, makes
It is carefully ground with syringe tail end, gained, that is, head-kidney leucocyte.
1. the leucocyte slightly mentioned about 2 × 105It is layered in 35mm Glass bottom culture dish, creep plate 3h；
2. abandoning culture medium, PBS is washed once；
3. being washed one time with the fixed 10min of 4% paraformaldehyde, PBS；
4. 5% Normal Goat Serum closes 30min；
5. the hybridoma culture supernatant with 500 μ L containing 5% Normal Goat Serum is resuspended, 4 DEG C of incubation 1h；
6. PBS is washed 3 times, each 5min；
7. the sheep anti-Mouse Ig (1 ︰ 500 is with the dilution of 5% Normal Goat Serum) of FITC label, 4 DEG C of incubation 30min are added；
8. PBS is washed 3 times, each 5min；
9. dyeing 2min with 2 μ g/mL DAPI；
10. PBS is washed 3 times, each 5min, carries out laser co-focusing and take pictures
The results show that positive cell is 3 μm of diameter or so of round cell, nucleus is larger, cell week make a circle have it is green
Color fluorescence.Negative cells are the cell of not no green fluorescence, including the T lymphocyte close with positive cell form, volume compared with
Big macrophage etc. (Fig. 3).
Claims (10)
1. anti-Larimichthys crocea IgM monoclonal antibody, it is characterised in that be hybridoma cell strain IgM-9G7, depositary institution is Chinese allusion quotation
Type culture collection, collection deposit number are CCTCC NO:C2018104, and the deposit date is Mays 15 in 2018
Day.
2. the preparation method of Larimichthys crocea IgM monoclonal antibody as described in claim 1, it is characterised in that the following steps are included:
1) Larimichthys crocea IgM antigen is prepared；
2) hybridoma cell strain IgM-9G7 is prepared；
3) monoclonal antibody of anti-Larimichthys crocea IgM a kind of is prepared.
3. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 2, it is characterised in that described in step 1)
Prepare Larimichthys crocea IgM antigen method particularly includes: Larimichthys crocea is anaesthetized, tail portion is carried out and takes blood, solidify after blood sampling, place, to blood
After clear sufficiently precipitation, serum and PBS are diluted, are obtained after filtering using albumin A affinity column and molecular sieve by centrifuging and taking upper serum
IgM antigen in rheum officinale fish serum, which includes heavy chain and light chain.
4. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 3, it is characterised in that the Larimichthys crocea anesthesia is adopted
It is anaesthetized with MS-222 anesthetic；The tail portion takes blood using 2mL injector for medical purpose.
5. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 3, it is characterised in that the solidification is in room temperature
Lower solidification 1h；The placement is in 4 DEG C of placement 12h；The condition of the centrifugation is 3000rpm, 5min.
6. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 3, it is characterised in that described by serum and PBS
Dilution is the PBS proportional diluted for being 8 by serum and pH value.
7. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 3, it is characterised in that the filtering uses 0.45
μm membrane filtration, the filter membrane and albumin A affinity column, molecular sieve are tool commonly used in the trade.
8. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 3, it is characterised in that obtained Larimichthys crocea blood
IgM antigen in clear is the Larimichthys crocea serum IgM of high-purity；The heavy chain is 76kD, and the light chain is 25kD.
9. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 2, it is characterised in that described in step 2)
Prepare hybridoma cell strain IgM-9G7's method particularly includes: mix the Larimichthys crocea IgM of purifying with Freund's adjuvant, as antigen
Immune Balb/C mouse, is merged with the mouse boosting cell for generating anti-Larimichthys crocea IgM antibody with myeloma cell, must secrete anti-rheum officinale
The hybridoma cell strain IgM-9G7 of fish IgM antibody.
10. the preparation method of Larimichthys crocea IgM monoclonal antibody as claimed in claim 2, it is characterised in that described in step 3)
Prepare the monoclonal antibody of anti-Larimichthys crocea IgM a kind of method particularly includes: hybridoma cell strain IgM-9G7 is used into limiting dilution
Two methods of method and ELISA combine, and filter out the hybridoma cell strain IgM-9G7 for secreting anti-Larimichthys crocea IgM monoclonal antibody, into
And obtain monoclonal antibody.
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