CN110283250A - A kind of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies vaccine and preparation method thereof - Google Patents
A kind of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies vaccine and preparation method thereof Download PDFInfo
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- CN110283250A CN110283250A CN201910655349.1A CN201910655349A CN110283250A CN 110283250 A CN110283250 A CN 110283250A CN 201910655349 A CN201910655349 A CN 201910655349A CN 110283250 A CN110283250 A CN 110283250A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
Abstract
The present invention provides a kind of preparation methods of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, belong to bioengineering and vaccine preparation technology field.The method of the invention includes the following steps: that (1) by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, obtains growth hormone release inhibiting hormone immunogen complex；(2) primary immunization is carried out to animal with the growth hormone release inhibiting hormone immunogen complex, obtains somatostatin antibody；(3) secondary immunity is carried out to the same or different animal in step (2) with the somatostatin antibody, obtains growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.Growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies can be prepared using method provided by the invention.The growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies that the present invention is prepared are used as vaccine immunity animal, and animal can be made to generate somatostatin antibody, are immunized and neutralize intracorporal growth hormone release inhibiting hormone, to promote the release of growth hormone, and then promote growth of animal.
Description
Technical field
The invention belongs to bioengineering and vaccine preparation technology field, and in particular to a kind of growth hormone release inhibiting hormone antiidiotype Dan Ke
Grand antibody vaccine and preparation method thereof.
Background technique
Growth of animal relies primarily on hypothalamicpituitary axis and is adjusted, and hypothalamus generates two kinds of important hormones, makes a living respectively
Long hormone releasing hormone (GHRH) and growth hormone release inhibiting hormone (somatostatin, SS), both hormones are with blood circulation to pituitary, altogether
With the growth for adjusting animal.Wherein pig GRH promotes the release of pituitary growth hormone, and then promotes animal raw
It is long.And growth hormone release inhibiting hormone (also known as somatostatin) then inhibits pituitary growth hormone to discharge, and then inhibits animal
Growth.
According to growth of animal principle of adjustment and control, people have begun discussion by increasing Exogenous growth hormone or promoting animal
Growth hormone is secreted to reach promotion growth of animal, increases the meat, milk, eggs yield of cultivated animals.The prior art is main
It include: that (1) with the growth hormone of DNA recombinant expression adds to cultivated animals.Though this method can effectively facilitate growth of animal,
The effect for needing large dosage of multiple dosing that could obtain has at high cost, it is difficult to the shortcomings that large-scale application.In addition, recombination
The growth hormone of expression has colour line, can only work to this kind of animal, use scope is limited.(2) it is anti-to prepare growth hormone
Idiotype antibody replaces growth hormone to add to cultivated animals with it.This method can also promote growth of animal, it also requires big agent
The effect that amount multiple dosing can just play.(3) DNA recombinant expression growth hormone release inhibiting hormone is used as vaccine immunity animal, produces animal
Raw anti-somatostatin antibody, is immunized and neutralizes tumor growth chalone, tumor growth chalone level is reduced, to promote growth hormone
Secretion and release.This method can promote growth of animal, but the growth hormone release inhibiting hormone molecular weight recombinantly expressed is small, and stability is poor, enter
Decomposition failure is easy in animal body.In addition, the growth hormone release inhibiting hormone of recombinant expression is there is also colour line, use scope is limited.(4) it makes
Standby somatostatin antibody, adds to cultivated animals, carries out passive immunity to animal with somatostatin antibody, directly in neutralization animal body
Growth hormone release inhibiting hormone reduces Somatostatin, promotes growth hormone secretion and release.This method can promote growth of animal, but
The effect for needing large dosage of multiple dosing that could generate, it is time-consuming and laborious at high cost.
Summary of the invention
In view of technical problem present in background technique, the object of the present invention is to provide a kind of growth hormone release inhibiting hormone antiidiotypes
Monoclonal antibody vaccine and preparation method thereof reduces dosage, reduces cost under the preceding topic for promoting cultivated animals growth；
Break colour line and expands application range；Expand route of administration；Medication is more simple and easy to do.
The present invention provides a kind of preparation methods of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, include the following steps:
(1) by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, growth hormone release inhibiting hormone immunogen complex is obtained；
(2) primary immunization is carried out to animal with the growth hormone release inhibiting hormone immunogen complex, obtains somatostatin antibody；
(3) secondary immunity is carried out to the same or different animal in step (2) with the somatostatin antibody, is given birth to
Long chalone anti-idiotype monoclonal antibodies.
Preferably, step (1) described high molecular weight protein includes any in bovine serum albumin, hemocyanin and ovalbumin
It is a kind of.
Preferably, step (2) primary immunization or the method for step (3) described secondary immunity include subcutaneous injection, flesh
One of meat injection and immersion are a variety of.
Preferably, step (2) or step (3) described animal include one of mouse, rabbit, chicken, pig and fish or a variety of.
Preferably, further include following steps after step (3) described secondary immunity:
The splenocyte of animal after secondary immunity is merged with myeloma cell, obtains hybridoma by I；
II cultivates the hybridoma, obtains growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.
The present invention provides a kind of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, the growth hormone release inhibiting hormone anti-idiotype monoclonal
Antibody is prepared by above-mentioned preparation method.
The present invention also provides the growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies that above-mentioned preparation method is prepared to prepare
Promote the application in the drug of growth of animal.
Preferably, the drug further includes growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies medically acceptable carrier.
Preferably, the drug can be used as vaccine, so that animal is generated somatostatin antibody by immune.
The utility model has the advantages that the present invention provides a kind of preparation methods of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, including such as
Lower step: (1) by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, growth hormone release inhibiting hormone immunogen complex is obtained；(2) with the life
Long chalone immunogen complex carries out primary immunization to animal, obtains somatostatin antibody；(3) with the somatostatin antibody pair
The same or different animal in step (2) carries out secondary immunity, obtains growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.Utilize this
Growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies can be prepared in the method that invention provides.The growth hormone release inhibiting hormone that the present invention is prepared
Anti-idiotype monoclonal antibodies are used as vaccine immunity animal, and animal can be made to generate somatostatin antibody, are immunized and neutralize intracorporal life
Long chalone to promote the release of growth hormone, and then promotes growth of animal.
The growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies being prepared using the present invention, are immunized animal, including such as
Lower advantage: 1. immunizing dose is small, it is only necessary to and it is immune once to can be obtained remarkable result, it is at low cost, high-efficient；2. the vaccine is without kind
Race's boundary, a kind of vaccine can be used for many animals, play the effect of achieving many things at one stroke；3. the vaccine immunity is by way of multiplicity, injection, leaching
The effect that bubble (being used for fish immunity) etc. can get, it is easy, time saving, laborsaving；4. the vaccine is to the weight gain adjusting of animal
A kind of temporary, benign hormone immunity adjustment process is adjusted, hormone in vivo at any time to passage by animal itself feedback
Level can be gradually recovered normally；5. the vaccine does not injure animal, the meat, egg, milk that animal generation is immunized will not to the mankind
Generate food safety risk.
Biological deposits information
The hybridoma cell strain 2C11 (growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies) that the present invention obtains, in 2019 4
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 3rd, and preservation number of registering on the books is
CGMCCNo 17488。
Specific embodiment
The present invention provides a kind of preparation methods of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, include the following steps:
(1) by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, growth hormone release inhibiting hormone immunogen complex is obtained；
(2) primary immunization is carried out to animal with the growth hormone release inhibiting hormone immunogen complex, obtains somatostatin antibody；
(3) secondary immunity is carried out to the same or different animal in step (2) with the somatostatin antibody, is given birth to
Long chalone anti-idiotype monoclonal antibodies.
The present invention first by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, obtains growth hormone release inhibiting hormone immunogen complex.Due to
Growth hormone release inhibiting hormone is small-molecular peptides substance, and immunogenicity is small, and the present invention is first coupled on high molecular weight protein carrier, and SS is made
Immunogen complex.The present invention is to the growth hormone release inhibiting hormone (also known as somatostatin, somatostatin, SS)
Source is not particularly limited, this field commercial product.In more specifically embodiment of the invention, the growth hormone release inhibiting hormone is
Polypeptide comprising 14 amino acid residues is bought from sigma company, the U.S..In the present invention, the high molecular weight protein carrier is excellent
Choosing includes any one in bovine serum albumin, hemocyanin and ovalbumin, more preferably bovine serum albumin.The present invention is to institute
The specific method for stating coupling is not particularly limited.In more specifically embodiment of the invention, the pH value of the coupling is preferably
6.5~7.0, temperature is preferably 10~25 DEG C, and the time is preferably 2~3h.
After obtaining growth hormone release inhibiting hormone immunogen complex, the present invention carries out animal with the growth hormone release inhibiting hormone immunogen complex
Primary immunization obtains somatostatin antibody.Then again with the somatostatin antibody to same or different dynamic in primary immunization
The animal of species carries out secondary immunity, obtains growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.In the present invention, in being immunized twice
The type of animal used is preferably different.The type of animal used preferably includes mouse, rabbit, chicken, pig each independently in being immunized twice
It more preferably include one of mouse, rabbit and fish or a variety of with one of fish or a variety of.In the present invention, described once to exempt from
Epidemic disease or the method for secondary immunity preferably include one of subcutaneous injection, intramuscular injection and immersion or a variety of.The subcutaneous injection
It is mainly used for the immune of mouse, rabbit, chicken or pig with intramuscular injection；Described impregnate is mainly used for the immune of fish.The present invention is to the skin
Lower injection or intramuscular injection have not a particular requirement operation precision, as long as immunogenic substances can be injected into animal body,
Those of ordinary skill in the art are achievable using regular injection instrument.The present invention does not limit the method for the immersion especially
It is fixed.
After secondary immunity, the present invention preferably merges the splenocyte of the animal after secondary immunity with myeloma cell, obtains
Hybridoma.The present invention preferably filters out growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies and expresses good hybridoma, so
The hybridoma is cultivated afterwards, to quickly produce growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.The present invention is to described
The screening of cell fusion and hybridoma and cultural method are not particularly limited, this field routine operation.
The present invention provides a kind of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, the growth hormone release inhibiting hormone anti-idiotype monoclonal
Antibody is prepared by above-mentioned preparation method.
The present invention also provides above-mentioned growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies to prepare the drug for promoting growth of animal
In application.In the present invention, mass content of the growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies in the drug is preferred
It is 0.1~99.9%.The drug further preferably includes growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies medically acceptable load
Body.The growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies that the present invention is prepared are used as vaccine immunity animal, animal can be made to generate
Somatostatin antibody is immunized and neutralizes intracorporal growth hormone release inhibiting hormone, to promote the release of growth hormone, and then promotes growth of animal.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
(1) preparation of growth hormone release inhibiting hormone immunogen complex
Growth hormone release inhibiting hormone standard items are bought from sigma company, the U.S., are the growth hormone release inhibiting hormone of 14 amino acid.Due to growth hormone release inhibiting hormone
It is small-molecular peptides substance, immunogenicity is small, to prepare antibody, is coupled on the protein carrier of macromolecular, be made first
SS immunogen complex.Its coupling method is: 10mg bovine serum albumin(BSA) is dissolved into lmL PBS (pH:6.5~7.0), it will
1mg growth hormone release inhibiting hormone is dissolved into 1mL distilled water, and the two mixing is shaken up, 0.25% glutaraldehyde of 1mL is then added dropwise while stirring
Solution continues 5~10min of stirring after dripping, then the reaction was continued 2~3 hours being stored at room temperature, that is, it is multiple that SS immunogene is made
Close object.Packing is set -20 DEG C of refrigerators and is saved for use.
(2) prepared by somatostatin antibody
1. being immunized:
It is mixed with isometric SS immunogen complex and incomplete Freund's adjuvant, it is small that immune 8 week old is subcutaneously injected after stirring evenly
Mouse, every 5~10 μ g growth hormone release inhibiting hormone of per injection are immunized 3~4 times altogether, are spaced 7~10 days.
It is mixed with isometric SS immunogen complex and incomplete Freund's adjuvant, immune two monthly ages man is subcutaneously injected after stirring evenly
Rabbit, every 20~40 μ g growth hormone release inhibiting hormone of per injection.It is immunized 3~4 times, is spaced 7~10 days altogether.
2. antibody titer detects: second immune 3~5 days latter, acquires the blood of mouse and rabbit, centrifuging and taking blood respectively
Clearly.With ELISA method detection somatostatin antibody potency: if potency reaches 10-4More than, bloodletting in 5~7 days is received after third time is immune
Collect serum；If the potency of somatostatin antibody is not achieved 10-4, then make to eliminate processing, the object that moves is immunized again.
3. antibody is collected and saved: the potency of SS antibody reaches 10-4Mouse and rabbit, 5~7 days i.e. after third time is immune
Serum antibody can be harvested, takes serum after centrifugation.- 20 DEG C of refrigerators save after packing.
(3) prepared by growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies
1. animal is immunized and is immunized: 8 week old Balb/c mouse 8, by the anti-SS antibody of mouse and isometric incomplete Freund
After agent mixes, immune mouse is injected intraperitoneally, 40~50 μ g of SS amount of antibody is immunized in every mouse every time, is immunized 3~4 times altogether, interval
One week.
2. SS antiidiotype polyclonal antibody detects: selecting 1~2 mouse to cut tail at random after immune for the second time and take blood centrifuging and taking
Serum, with ELISA method detection SS anti-idiotype and potency, if potency is 10-4More than, it is within 3rd day after third time is immune
It can extracting spleen cell progress cell fusion.
3. cell fusion
I, the preparation of immune spleen cell: taking the high spleen for exempting from mouse, be put into the plate for filling the endless full nutrient solution of 5mL,
It is placed on the stainless (steel) wire of 200 mesh.Spleen is ground with plunger, and gently washes steel mesh with full nutrient solution endless in plate,
Make splenocyte all pass through mesh to be expressed in solution.Splenocyte suspension is transferred in 50mL centrifuge tube, endless full nutrient solution is added
To 30mL, it is centrifuged 5min through 1000rpm after mixing, is discarded supernatant.It cannots be used up after full nutrient solution mixes sedimentation cell and is centrifuged again
It washed once, then cell is overhang to 10mL, is mixed, is counted with cell counting board.Total cell number is adjusted to 1 × 108。
The preparation of II, sp2/0 myeloma cell's suspension
Sp2/0 myeloma cell is subjected to amplification cultivation.The myeloma cell of 4 bottles of amplification cultivations is collected into 50mL centrifugation
In pipe.1000rpm is centrifuged 5min, abandons supernatant.The addition endless full nutrient solution of 30mL, then centrifuge washing are primary, then hang cell
It hangs down to 10mL, mixes and count, total cell number is adjusted to 2~3 × 107。
Cell fusion: III draws 1 × 10 respectively8A splenocyte suspension and 2~3 × 107Myeloma cell's suspension is added
In 50mL centrifuge tube, endless full nutrient solution 30mL is added, is sufficiently shaken up.1000rpm is centrifuged 7min, abandons supernatant.50% is added to melt
Mixture polyethylene glycol 0.7mL.Add the endless full nutrient solution of 25mL, PEG is made to dilute and lose rush and melt effect.800rpm is centrifuged 7min,
It discards supernatant.20mLHAT culture solution is added, gently pressure-vaccum sedimentation cell, makes it be resuspended and mix.Cell suspension addition is covered with
In 96 well culture plate apertures of feeder cells, every hole 0.1mL contains 5%CO at 37 DEG C2Incubator in cultivate.
IV, antibody positive wells detect (ELISA method): being coated with rabbit-anti SS antibody, added with the culture solution in clonal growth hole
100 μ L of supernatant.Enzyme 100 μ L of mark dynamics.Add 100 μ L of substrate solution, room temperature is protected from light 10~20min of colour developing.Exempt from enzyme-linked
OD value is surveyed under epidemic disease detector 492nm wavelength, is greater than or equal to 2.1 times of control wells OD value to gaging hole OD value, then be can determine that as the positive
Hole.Growth has positive colony in hole.
V, cloning (with limiting dilution assay): positive colony hole is marked, and is blown and beaten hybridoma in hole with sample injector
It is counted after uniformly.Then it is diluted step by step with limiting dilution assay, is then added to 96 orifice plates and continues to cultivate.Observe clonal growth.Note
Monoclonal hole is recorded, and carries out positive detection in time.The cell in positive hole is gone into 24 orifice plate cultures, after being proliferated to certain amount again
It is transferred in cell bottle and cultivates.
VI, hybridoma freeze: the hybridoma of logarithmic growth is collected into centrifuge tube, 1000rpm centrifugation
10min abandons supernatant, and the mixing of lmL frozen stock solution is added and pours into cryopreservation tube, it is long-term to be transferred to liquid nitrogen container overnight in -70 DEG C of low temperature refrigerators
It saves.
VII, the preparation of ascitic type monoclonal antibody: the hybridoma of amplification cultivation is blown and beaten, 1000rpm centrifugation
It is spare to collect supernatant by 10min.Add serum-free medium into sedimentation cell, adjustment cell concentration is 1 × 106/ mL, every
Balb/C mouse peritoneal injection 0.5mL become larger after 7~14 days to mouse web portion, draw neck dislocation put to death mouse, draw ascites from
The heart, collection supernatant are ascitic type monoclonal antibody, and packing -20 DEG C of refrigerators of postposition save.
(4) identification of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies
1. bioactivity: examining two plants of energy stably excreting growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies with indirect elisa method
Bioactivity program and result are as follows:
Method: rabbit-anti growth hormone release inhibiting hormone is resisted with coating buffer (0.05M, the carbonate that pH value is 9.6 are coated with buffer)
Body carries out 1:1000 dilution, is added separately in ELISA Plate aperture, every 100 μ L of hole sets 37 DEG C of 0.5~1h of incubation.It goes raw in aperture
Long chalone antibody, washing buffer are washed three times, each 3min.Anti-idiotype monoclonal antibodies ascites is carried out with antibody diluent
10 times are serially diluted, and are diluted to 10 grades always.Different dilution antiidiotype monoclonal antibodies are sequentially added in ELISA Plate aperture, every hole
100 μ L, 37 DEG C of incubation 30min.Enzyme mark sheep anti-mouse igg antibody 30min.Add color developing agent (containing 0.045% hydrogen peroxide liquid and 10
5.0 phosphoric acid of pH~citrate buffer solution of~20mg o-phenylenediamine) every hole 100uL, 15~20min of colour developing is protected from light in room temperature.
Add terminate liquid, every 50 μ L of hole.At enzyme mark detector 492nm wavelength, each hole OD value, blank control zeroing, experimental port OD value are surveyed
It is positive reaction more than or equal to 2.1 times of control wells.
As a result such as table 1.Table 1 is shown: the potency of two plants of SS anti-idiotype monoclonal antibodies ascites is 10-4~10-5。
1 SS anti-idiotype monoclonal antibodies bioactivity result table of table
2. specific detection: detect the specificity of two plants of anti-idiotype monoclonal antibodies of the invention with ElISA method, method and
As a result as follows:
Method: with coating buffer (0.05M, the carbonate that pH value is 9.6 are coated with buffer) rabbit-anti is grown pressed down respectively
Plain antibody, rabbit-anti gastrin antibody, rabbit-anti growth hormone antibody, rabbit-anti insulin antibody carry out 1:1000 dilution, are added separately to
In ELISA Plate aperture, every kind plus two repeating holes, every 100 μ L of hole, 37 DEG C are incubated for 0.5~1 hour.SS of the present invention is added to resist only type list
In clonal antibody to the above ELISA Plate aperture, every hole 100uL sets 37 DEG C of incubations 30min, enzyme mark dynamics 30min,
Add chromogenic reagent.With enzyme mark detector test OD value.
As a result such as table 2.Table 2 is shown: two plants of SS anti-idiotype monoclonal antibodies are only immunoreacted with rabbit-anti SS antibody,
Other peptide matters antibody of getting along well are immunoreacted, and have stronger specificity.
2 SS anti-idiotype monoclonal antibodies specific detection result of table
3. Concentration Testing detects the concentration of SS anti-idiotype monoclonal antibodies ascites of the present invention, method with Enzyme-Linked Immunosorbent Assay
It is as follows with result:
Method: carrying out 2 times for mouse IgG standard items and antiidiotype odd contradictive hydroperitoneum of the present invention and be serially diluted, will be different dilute
The standard items for degree of releasing are sequentially added in reaction plate aperture, every kind of 12 holes, every hole 100 μ L, 37 DEG C of reaction 1h.Add rabbit anti-mouse IgG
Antibody.Enzyme mark goat anti-rabbit igg antibody, adds chromogenic reagent.Detect OD value.
As a result such as table 3.Table 3 is shown: SS antiidiotype odd contradictive hydroperitoneum concentration of the present invention is 4mg/mL.
3 SS anti-idiotype monoclonal antibodies Concentration Testing result of table
| Different dilution OD values | 3.274 | 2.311 | 2.192 | 2.0985 | 1.537 | 1.159 |
| Concentration (ug/ml) | 3050.102 | 3937.25 | 3524.316 | 5482.416 | 3803.51 | 4266.272 |
4. effect detects
Method: it is subcutaneously injected respectively with various dose SS anti-idiotype monoclonal antibodies, intramuscular injection, immersion (to fish) are exempted from
Epidemic disease different animals weigh to animal with immune the latter moon when being immunized, and calculate immune group animal and increase than control group average weight
The percentage added.
As a result such as table 4.Table 4 show: receive immune animal, after one month average weight than control-animal increase by 20% with
On.
4 SS antiidiotype monoclonal antibody effect testing result table of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, which comprises the steps of:
(1) by growth hormone release inhibiting hormone and high molecular weight protein carrier conjugation, growth hormone release inhibiting hormone immunogen complex is obtained；
(2) primary immunization is carried out to animal with the growth hormone release inhibiting hormone immunogen complex, obtains somatostatin antibody；
(3) secondary immunity is carried out to the same or different animal in step (2) with the somatostatin antibody, obtains growth suppression
Plain anti-idiotype monoclonal antibodies.
2. preparation method according to claim 1, which is characterized in that step (1) described high molecular weight protein includes cow's serum
Any one in albumen, hemocyanin and ovalbumin.
3. preparation method according to claim 1, which is characterized in that step (2) primary immunization or step (3) are described
The method of secondary immunity includes one of subcutaneous injection, intramuscular injection and immersion or a variety of.
4. preparation method according to claim 1, which is characterized in that step (2) or step (3) described animal include mouse,
One of rabbit, chicken, pig and fish are a variety of.
5. preparation method described in any one according to claim 1~4, which is characterized in that step (3) described secondary immunity
Afterwards, further include following steps:
The splenocyte of animal after secondary immunity is merged with myeloma cell, obtains hybridoma by I；
II cultivates the hybridoma, obtains growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies.
6. a kind of growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies, the growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies are by claim
Preparation method described in 1~5 any one is prepared.
7. growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies that preparation method described in Claims 1 to 5 any one is prepared or
Application of the growth hormone release inhibiting hormone anti-idiotype monoclonal antibodies as claimed in claim 6 in the drug that preparation promotes growth of animal.
8. application according to claim 7, which is characterized in that the drug further includes growth hormone release inhibiting hormone anti-idiotype monoclonal
Antibody medically acceptable carrier.
9. application according to claim 7, which is characterized in that the drug can be used as vaccine, produce animal by immune
Raw somatostatin antibody.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734496A (en) * | 2019-11-19 | 2020-01-31 | 西安咸辅生物科技有限责任公司 | Preparation method of human somatostatin anti-idiotype yolk antibodies |
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