CN114685673A - Fibroblast activation protein antibodies and methods of use - Google Patents
Fibroblast activation protein antibodies and methods of use Download PDFInfo
- Publication number
- CN114685673A CN114685673A CN202210325036.1A CN202210325036A CN114685673A CN 114685673 A CN114685673 A CN 114685673A CN 202210325036 A CN202210325036 A CN 202210325036A CN 114685673 A CN114685673 A CN 114685673A
- Authority
- CN
- China
- Prior art keywords
- activation protein
- fibroblast activation
- polypeptide
- reagent
- protein antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002950 fibroblast Anatomy 0.000 title claims abstract description 49
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 230000004913 activation Effects 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 15
- 229920001184 polypeptide Polymers 0.000 claims abstract description 45
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 45
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 45
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 38
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 38
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 38
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 claims abstract description 28
- 101000652736 Homo sapiens Transgelin Proteins 0.000 claims abstract description 28
- 102100031013 Transgelin Human genes 0.000 claims abstract description 28
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007853 buffer solution Substances 0.000 claims abstract description 21
- 239000007822 coupling agent Substances 0.000 claims abstract description 19
- 239000002904 solvent Substances 0.000 claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 15
- 102000036639 antigens Human genes 0.000 claims abstract description 15
- 108091007433 antigens Proteins 0.000 claims abstract description 15
- 210000002966 serum Anatomy 0.000 claims abstract description 13
- 239000011550 stock solution Substances 0.000 claims abstract description 13
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims abstract description 12
- 239000011543 agarose gel Substances 0.000 claims abstract description 11
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims abstract description 11
- 229940116357 potassium thiocyanate Drugs 0.000 claims abstract description 11
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 10
- 239000002671 adjuvant Substances 0.000 claims abstract description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000010171 animal model Methods 0.000 claims description 14
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 claims description 8
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 5
- 238000001042 affinity chromatography Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 238000004132 cross linking Methods 0.000 claims description 4
- 125000005842 heteroatom Chemical group 0.000 claims description 3
- 241000283073 Equus caballus Species 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 7
- 230000003213 activating effect Effects 0.000 abstract description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 206010027476 Metastases Diseases 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- 230000009401 metastasis Effects 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 210000002919 epithelial cell Anatomy 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 210000001608 connective tissue cell Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
Abstract
The invention discloses a fibroblast activation protein antibody and a using method thereof, relates to the technical field of biology, and aims at the problems of high cost, complex means and the like of the existing fibroblast activation protein antibody, the following scheme is proposed, and the scheme comprises the following materials: freund's incomplete adjuvant, BCG, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent. The fibroblast activation protein antibody has the functions of promoting and inhibiting proliferation, metastasis, immunity and the like of tumor cells, is more efficient by activating proteins in the fibroblast to form the antibody and activating the fibroblast in a mode of combining polyclonal antibody serum stock solution and a polypeptide antigen reagent, and is stable in components, low in activation means cost and capable of better forming the fibroblast activation protein antibody.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a fibroblast activation protein antibody and a using method thereof.
Background
Fibroblasts are a type of biological cells that synthesize extracellular matrix and collagen, produce structural framework (stroma is the animal) tissue, and play a critical role in wound healing, are the most common connective tissue cells in animals, and unlike epithelial cells that line the body structure, fibroblasts do not form a flat monolayer of cells, nor are they limited by polarized attachment to one side of the basement membrane, although in some cases they may promote basement membrane components (e.g., fibroblasts may secrete under the epithelium in the gut) the alpha-2 chain-carrying components of laminin, which are only absent from follicle-associated epithelial cells lacking the lining of myofibroblasts, and, as opposed to epithelial cells, fibroblasts may also migrate slowly as individual cells on the stroma, although the epithelial cells line the body structure, it is the fibroblasts and associated connective tissue that shape the "whole body" of the organism.
The fibroblast-associated activated protein is one of the specific markers of CAFs, which is not generally expressed in normal tissue cells but is expressed in more than 90% of malignant tumors derived from epithelial cells, the existing fibroblast-associated activated protein antibody has high cost and complex means, and correspondingly, the activation threshold is increased, so that the fibroblast-associated activated protein antibody is difficult to apply to medical treatment for benefiting human beings, and the technical problems are solved by the existing fibroblast-associated activated protein antibody and the use method thereof.
Disclosure of Invention
Objects of the invention
The fibroblast activation protein antibody has the functions of promoting and inhibiting proliferation, metastasis, immunity and the like of tumor cells, forms an antibody by activating protein in the fibroblast, activates the fibroblast in a mode of combining polyclonal antibody serum stock solution and a polypeptide antigen reagent more efficiently, has stable components and low activation means cost, and can better form the fibroblast activation protein antibody.
(II) technical scheme
The invention provides a fibroblast activation protein antibody, which comprises the following materials: freund's incomplete adjuvant, BCG, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent.
In a preferred embodiment of the present invention, the method further comprises the following steps:
s1: after the BCG vaccine is inactivated, injecting the inactivated BCG vaccine and Freund's incomplete adjuvant into an experimental animal body, and then extracting blood of the experimental animal and centrifuging to obtain a polyclonal antibody serum stock solution;
s2: dissolving an SMCC coupling agent in a DMF solvent, adding ovalbumin into a round-bottomed flask, adding a PBS (phosphate buffer solution), slowly dropwise adding the dissolved SMCC solution into the round-bottomed flask, stirring at room temperature for reaction, dialyzing by using the PBS buffer solution, and removing free SMCC coupling agent;
s3: pouring the dialyzed ovalbumin into a centrifuge tube, dissolving a polypeptide reagent by using a PBS buffer solution, dripping the dissolved polypeptide reagent and N-terminal amino acid into the centrifuge tube, uniformly mixing and reacting in a vertical mixer at room temperature, and after the reaction is finished, supplementing the polypeptide reagent and an SMCC coupling agent to continuously crosslink with the activated ovalbumin to obtain a polypeptide antigen reagent;
s4: preparing affinity chromatography column from the polyclonal antibody serum stock solution and polypeptide antigen reagent obtained in the above steps and Sepharose4B agarose gel, washing away unbound hetero protein, eluting with potassium thiocyanate to obtain fibroblast activation protein antibody, and adding triethanolamine for protection.
In a preferred embodiment of the present invention, the experimental animal in step S1 is a mammal.
Specifically, mice, dogs, horses, cows, apes are used, and the injection time of the above mammals is kept normal and healthy.
In a preferred embodiment of the present invention, the PBS buffer solution in step S2 is used to put ovalbumin to a final concentration of 12ng/ml to 15 ng/ml.
As a preferable scheme of the present invention, the blending reaction time of the vertical blender in the step S3 is between 3.5h and 4.5h, and the reaction temperature is normal.
In a preferred embodiment of the present invention, in step S3, an Ellman reagent is used to detect thiol in polypeptide, and a Nano spectrophotometer is used to measure the uv absorption value of the polypeptide antigen reagent.
In a preferred embodiment of the invention, the Nano spectrophotometer is supplemented with the polypeptide when the OD value is less than 0.15 and greater than 0.05, until the OD value is greater than 0.15.
Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:
the fibroblast activation protein antibody has the functions of promoting and inhibiting proliferation, metastasis, immunity and the like of tumor cells, is more efficient by activating proteins in the fibroblast to form the antibody and activating the fibroblast in a mode of combining polyclonal antibody serum stock solution and a polypeptide antigen reagent, and is stable in components, low in activation means cost and capable of better forming the fibroblast activation protein antibody.
Detailed Description
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. In the present invention, the term "pharmaceutically acceptable" means that the indicated material is not of a nature that would cause reasonable caution in a physician avoiding administration of the material to a patient, taking into account the disease or condition to be treated and the corresponding route of administration. For example, it is generally desirable that such materials be substantially sterile, and in the present invention, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting and/or stopping one or more clinical symptoms of a disease or disorder before, during and/or after injury or intervention, and in the present invention, the term "patient" or "subject" refers to an animal, including a mammal, e.g., a mouse, dog, horse, cow, ape or human, and particularly a human, being treated with a pharmaceutical composition or according to the methods described herein.
While the various publications, patents and published patent specifications cited herein have been incorporated by reference in their entirety, for the purpose of describing a preferred embodiment of the invention, the disclosure is to be clearly and completely described below in connection with the examples, it is to be understood that the examples are illustrative of only some of the examples, and not restrictive of the scope of the invention, and that all other examples, which would be within the purview of one of ordinary skill in the art based on the examples and are intended to be protected by the present invention without the exercise of inventive faculty.
The first embodiment is as follows: preparation of fibroblast activation protein antibody material: freund's incomplete adjuvant, BCG, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent;
the preparation process of the fibroblast activation protein antibody comprises the following steps:
after the BCG vaccine is inactivated, injecting the inactivated BCG vaccine into a muscle layer of an experimental animal through an injector, then injecting a Freund's incomplete adjuvant into the body of the experimental animal, after waiting for 1-3 days of immune antibody generation of the experimental animal, extracting blood of the experimental animal, putting the blood into a centrifugal tube, and centrifuging to obtain polyclonal antibody serum stock solution;
dissolving 20mg of SMCC coupling agent in 2ml of DMF solvent, adding ovalbumin into a 25ml round-bottom flask, supplementing PBS buffer solution to wash the ovalbumin to enable the final concentration of the ovalbumin to be 15mg/ml, slowly dripping the dissolved SMCC solution into the round-bottom flask, stirring at room temperature for reaction for 1h, dialyzing the SMCC solution in the round-bottom flask by using 1L of PBS buffer solution at the temperature of 4 ℃ for 5-6 h, and removing free SMCC;
pouring the dialyzed ovalbumin into a 50ml centrifuge tube, determining the volume of the dialyzed ovalbumin through the scales of the centrifuge tube, calculating the concentration of the dialyzed ovalbumin according to the amount of the ovalbumin added before reaction, transferring the ovalbumin solution into a 5ml centrifuge tube according to the concentration, dropwise adding the ovalbumin solution and N-terminal amino acid into the centrifuge tube after a polypeptide reagent is dissolved by a PBS buffer solution, detecting the sulfydryl in the polypeptide by using an Ellman reagent (if the Ellman reagent shows yellow, the coupling between the polypeptide and the ovalbumin is incomplete, if the Ellman reagent does not show yellow, the coupling between the polypeptide and the ovalbumin is complete), adding an Ellman reagent stock solution into a 96-well plate, adding the polypeptide solution, measuring the ultraviolet absorption value of the polypeptide solution by using a Nano spectrophotometer at lambda 412nm, reacting the centrifuge tube for 4 hours by a vertical mixer at room temperature, finishing the reaction, further continuously crosslinking the polypeptide reagent and the SMCC coupling agent with the activated ovalbumin, obtaining a polypeptide antigen reagent;
preparing affinity chromatography column from the polyclonal antibody serum stock solution and polypeptide antigen reagent obtained in the above steps and Sepharose4B agarose gel, washing away unbound hetero protein, eluting with potassium thiocyanate to obtain fibroblast activation protein antibody, and adding triethanolamine for protection.
Example two: based on the example 1, the preparation material of the fibroblast activation protein antibody is changed into: monoclonal antibody serum, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent;
the preparation process of the fibroblast activation protein antibody is changed into the following steps: .
Dissolving 20mg of SMCC coupling agent in 2ml of DMF solvent, adding ovalbumin into a 25ml round-bottomed flask, supplementing PBS buffer solution to wash the ovalbumin to enable the final concentration of the ovalbumin to be 15mg/ml, slowly dripping the dissolved SMCC solution into the round-bottomed flask, stirring at room temperature for reaction for 1h, dialyzing the SMCC solution in the round-bottomed flask for 5-6 h at the temperature of 4 ℃ by using 1L or so of PBS buffer solution, removing free SMCC, pouring the dialyzed ovalbumin into a 50ml centrifuge tube, determining the volume of the centrifuge tube by the scale of the centrifuge tube, calculating the concentration of the dialyzed ovalbumin according to the amount of the ovalbumin added before the reaction, transferring the ovalbumin solution into a 5ml centrifuge tube according to the concentration, adding the ovalbumin solution into the centrifuge tube after a polypeptide reagent is dissolved by the PBS buffer solution and an N-terminal amino acid drop, adding an Ellman reagent stock solution into a 96-well plate, adding polypeptide solution, measuring the ultraviolet absorption value of the solution under the condition that lambda is 412nm by using a Nano spectrophotometer, uniformly mixing the solution in a centrifugal tube by using a vertical mixer at room temperature for 4 hours, finishing the reaction, adding a polypeptide reagent and an SMCC coupling agent, continuously crosslinking the polypeptide reagent and the activated ovalbumin to obtain a polypeptide antigen reagent, preparing the polypeptide antigen reagent and Sepharose4B agarose gel into an affinity chromatography column, washing away the unbound heteroprotein, then adding monoclonal antibody serum, and eluting the monoclonal antibody by using potassium thiocyanate to obtain the fibroblast activation protein antibody.
And (3) implementation three: on the basis of the first embodiment, the operation steps of preparation are changed, and the prepared material is not changed;
preparation of fibroblast activation protein antibody material: freund's incomplete adjuvant, BCG, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent;
the preparation process of the fibroblast activation protein antibody comprises the following steps:
the method comprises the steps of inactivating BCG vaccine, injecting the inactivated BCG vaccine into a muscle layer of an experimental animal through an injector, injecting Freund's incomplete adjuvant into the experimental animal, after waiting for 1-3 days of immune antibody generated by the experimental animal, drawing blood of the experimental animal, placing the blood into a centrifuge tube, centrifuging to obtain a polyclonal antibody serum stock solution, dissolving an SMCC coupling agent in a DMF solvent, adding ovalbumin into a round-bottomed flask, adding a PBS buffer solution, slowly dripping the dissolved SMCC solution into the round-bottomed flask, stirring for reaction at room temperature, dialyzing with the PBS buffer solution, removing free SMCC coupling agent, pouring the dialyzed ovalbumin into the centrifuge tube, adding a polypeptide reagent and an N-terminal amino acid into the centrifuge tube after the polypeptide reagent is dissolved with the PBS buffer solution, dripping the N-terminal amino acid into a vertical mixer at room temperature for uniform mixing reaction, supplementing the polypeptide reagent and the SMCC coupling agent to continue crosslinking with the activated ovalbumin after the reaction is finished, and (3) obtaining a polypeptide antigen reagent, preparing an affinity chromatography column by using the polyclonal antibody serum stock solution and the polypeptide antigen reagent obtained in the steps and Sepharose4B agarose gel, washing away unbound hybrid protein, eluting with potassium thiocyanate to obtain a fibroblast activation protein antibody, and finally adding triethanolamine for protection.
Experimental example:
the fibroblast activation protein antibody obtained in each example is prepared into a reagent state, then labeled for cold storage, then 60 mice with the same size, bright fur and healthy state are selected, 20 mice are equally divided into three units (the three units are respectively labeled as A, B and C), the mice with the three units are placed in an environment suitable for the survival of the mice for 2 days for housing, the tumor cell reagent is injected into each mouse, the housing is continued for 2-5 days for waiting for the growth of the tumor cells in the mice, the mice with different degrees of tumor rejection are cultured, the fibroblast activation protein antibody reagent in the example 1 is injected into the mice in the A unit, the fibroblast activation protein antibody in the example 2 is injected into the mice in the B unit, the process is repeated, and after the mice are normally housed for one week, the survival condition and the inhibition condition of the tumor cells in each unit are observed (see tables 1 and 2) .
Table 1
| Item (survival) | 7 to 10 days | 11 to 15 days | 16-25 days | 26-40 days |
| A unit mouse | 20 pieces of | 17 are only | 16 are | 16 pieces of |
| B unit mouse | 19 are only | 17 are only | 17 are only | 15 pieces of |
| C unit mouse | 20 are all | 18 are only | 17 are only | 15 pieces of |
Table 2
| Item (suppression) | 7-10 days | 11 to 15 days | 16-25 days | 26 to 40 days |
| A unit mouse | 72.1％ | 74.7％ | 71.4％ | 73.8％ |
| B unit mouse | 70.6％ | 70.3％ | 72.4％ | 72.2％ |
| C unit mouse | 74.7％ | 70.7％ | 68.1％ | 70.6％ |
As can be seen from tables 1-2, the fibroblast activation protein antibody provided by the invention can effectively inhibit the growth of tumor cells and can improve the survival rate of mice.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. A fibroblast activation protein antibody comprising the following materials: freund's incomplete adjuvant, BCG, ovalbumin, polypeptide reagent, N-terminal amino acid, SMCC coupling agent, Sepharose4B agarose gel, potassium thiocyanate, triethanolamine, DMF solvent, PBS buffer solution and DMF solvent.
2. The fibroblast activation protein antibody of claim 1, further comprising the following method of use:
s1: after the BCG vaccine is inactivated, injecting the inactivated BCG vaccine and Freund's incomplete adjuvant into an experimental animal body, and then extracting blood of the experimental animal and centrifuging to obtain polyclonal antibody serum stock solution;
s2: dissolving an SMCC coupling agent in a DMF solvent, adding ovalbumin into a round-bottomed flask, adding a PBS (phosphate buffer solution), slowly dropwise adding the dissolved SMCC solution into the round-bottomed flask, stirring at room temperature for reaction, dialyzing by using the PBS buffer solution, and removing free SMCC coupling agent;
s3: pouring the dialyzed ovalbumin into a centrifuge tube, dissolving a polypeptide reagent by using a PBS buffer solution, dripping the polypeptide reagent and N-terminal amino acid into the centrifuge tube, uniformly mixing and reacting in a vertical mixer at room temperature, and after the reaction is finished, supplementing the polypeptide reagent and an SMCC coupling agent and continuously crosslinking with the activated ovalbumin to obtain a polypeptide antigen reagent;
s4: preparing affinity chromatography column from the polyclonal antibody serum stock solution and polypeptide antigen reagent obtained in the above steps and Sepharose4B agarose gel, washing away unbound hetero protein, eluting with potassium thiocyanate to obtain fibroblast activation protein antibody, and adding triethanolamine for protection.
3. The fibroblast activation protein antibody and the use method thereof according to claim 2, wherein the experimental animal in the step S1 is a mammal.
Preferably, the injection time of the mammal is kept in a normal and healthy state by using a mouse, a dog, a horse, a cow or a ape.
4. The fibroblast activation protein antibody and the using method thereof according to any one of claims 1 and 2, wherein the PBS buffer solution in the step S2 puts ovalbumin with a final concentration of 12ng/ml to 15 ng/ml.
5. The fibroblast activation protein antibody and the use method thereof as claimed in claim 2, wherein the mixing reaction time of the vertical mixer in the step S3 is between 3.5h and 4.5h, and the reaction temperature is normal.
6. The fibroblast activation protein antibody and the using method thereof as claimed in claim 2, wherein in step S3, Ellman' S reagent is used to detect the thiol group in the polypeptide, and the Nano spectrophotometric is used to measure the uv absorption of the polypeptide antigen reagent.
7. The fibroblast activation protein antibody and method of use of claim 6, wherein the Nano spectrophotometer is supplemented with polypeptide at OD values less than 0.15 and greater than 0.05 until OD value is greater than 0.15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210325036.1A CN114685673A (en) | 2022-03-29 | 2022-03-29 | Fibroblast activation protein antibodies and methods of use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210325036.1A CN114685673A (en) | 2022-03-29 | 2022-03-29 | Fibroblast activation protein antibodies and methods of use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114685673A true CN114685673A (en) | 2022-07-01 |
Family
ID=82140638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210325036.1A Pending CN114685673A (en) | 2022-03-29 | 2022-03-29 | Fibroblast activation protein antibodies and methods of use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114685673A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103154038A (en) * | 2010-08-13 | 2013-06-12 | 罗切格利卡特公司 | Anti-fap antibodies and methods of use |
| CN112062832A (en) * | 2020-09-24 | 2020-12-11 | 河南赛诺特生物技术有限公司 | Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit |
-
2022
- 2022-03-29 CN CN202210325036.1A patent/CN114685673A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103154038A (en) * | 2010-08-13 | 2013-06-12 | 罗切格利卡特公司 | Anti-fap antibodies and methods of use |
| CN105884898A (en) * | 2010-08-13 | 2016-08-24 | 罗切格利卡特公司 | Anti-FAP antibodies and methods of use |
| CN112062832A (en) * | 2020-09-24 | 2020-12-11 | 河南赛诺特生物技术有限公司 | Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rachakatla et al. | Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors | |
| TW414799B (en) | Compositions and methods for stimulating megakaryocyte growth and differentiation | |
| JPH03500644A (en) | Cationized antibodies transported across the blood-brain barrier | |
| JP2003159083A (en) | New neurotrophic factor | |
| JP3502125B2 (en) | Extraction and culture of transformed cells and production of antibodies against them | |
| JPH07504650A (en) | Inhibition of transforming growth factor β to prevent extracellular matrix accumulation | |
| JP2002155100A (en) | Stem cell proliferation factor | |
| NZ203042A (en) | Increasing milk production in cows by administering synthetic bovine growth hormone | |
| US11938172B2 (en) | Method for regulating and controlling GLP-1/GLP-1R and drug | |
| US6262024B1 (en) | Neuron regulatory factor for promoting neuron survival | |
| DE69133485T2 (en) | Use of Staphylococcal Enterotoxin Homologs for Cancer Therapy | |
| CN109125715A (en) | A kind of method and drug of regulation GLP-1/GLP-1R | |
| CN109111517B (en) | Modified growth differentiation factor and preparation method and application thereof | |
| CN107760661A (en) | PEG trims of medicinal kininogenase and its preparation method and application | |
| CN102666576A (en) | Peptides that bind the alpha-fetoprotein (AFP) receptor and uses thereof | |
| CN103906760A (en) | Human lactoferrin derived peptide for use as an antigen masking agent | |
| JPH03195496A (en) | Growth factor | |
| JPS62187500A (en) | Novel protein and its use | |
| US20160175365A1 (en) | Methods for treating PKU | |
| CN114685673A (en) | Fibroblast activation protein antibodies and methods of use | |
| CN104558150A (en) | Novel growth hormone releasing hormone analog peptides and application thereof in preparing medicines for treating infertility | |
| CN108586609B (en) | Monoclonal antibody for resisting porcine epidemic diarrhea virus and application | |
| Swanborg | The effect of selective modification of tryptophan, lysine and arginine residues of basic brain protein on encephalitogenic activity | |
| JP2003504379A (en) | Anti-idiotypic antibodies of fibroblast growth factor and their use as drugs | |
| Zhang et al. | Legumain correlates with neuroblastoma differentiation and can be used in prodrug design |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |