CN106381289A - Monoclonal antibody of resisting 25-hydroxyvitamin D3 as well as preparation cell lines and method of monoclonal antibody - Google Patents
Monoclonal antibody of resisting 25-hydroxyvitamin D3 as well as preparation cell lines and method of monoclonal antibody Download PDFInfo
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- CN106381289A CN106381289A CN201610999897.2A CN201610999897A CN106381289A CN 106381289 A CN106381289 A CN 106381289A CN 201610999897 A CN201610999897 A CN 201610999897A CN 106381289 A CN106381289 A CN 106381289A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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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
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention provides a monoclonal antibody of resisting 25-hydroxyvitamin D3 as well as preparation cell lines and method of the monoclonal antibody. By optimizing the preparation process of hybridoma cells and/or the screening process of monoclonal cell lines, the monoclonal cell lines of producing the monoclonal antibody of resisting the 25-hydroxyvitamin D3 and the monoclonal antibody are obtained. According to the monoclonal antibody as well as the preparation cell lines and the method of the monoclonal antibody, by optimizing the preparation step of preparing the cell lines or the antibody, the screening time is reduced, the preparation efficiency of the cell lines as of the monoclonal antibody of resisting the 25-hydroxyvitamin D3 is improved, and the production cost is lowered. In addition, the affinity of the monoclonal antibody is larger than 3.84x10<9>, and cross reaction of the monoclonal antibody and 25-hydroxyvitamin D2 is lower than 10 percent.
Description
Technical field
The present invention relates to immunological technique field, more particularly to a kind of anti-25-hydroxy-vitamin D3Monoclonal antibody and its
Prepare cell strain and method.
Background technology
Vitamin D is a kind of fatsoluble vitamin, is also regarded as a kind of hormone precursor acting on Ca,P metabolism.Mesh
At least 10 kinds of front known vitamin D, but above all vitamin D2(vitamin D2) and vitamin D3(gallbladder calcification
Alcohol).Vitamin D3It is a kind of form of biological metabolism rate highest in vitamin D.
Vitamin D3(cholecalciferol) in addition to being present in minority animal food, mainly by human body autologous skin
7-DHC is formed after ultraviolet irradiation, and 7- dehydrogenation gallbladder alcohol is then to be changed by cholesterol to generate, so someone
It is its sun vitamin.If child can fully accept more than direct sunlight skin 4-6 hour, the vitamin of itself synthesis
D3, just substantially can meet.In addition, vitamin D3May also come from animal food, such as liver class, especially by the fish liver of marine products class
The cod-liver oil of middle refinement.
Vitamin D3There is no activity, add hydroxyl in 25 carbon potentials under liver 25- hydroxylation enzyme effect, generate 25- hydroxyl dimension
Raw element D3.25-hydroxy-vitamin D3It is vitamin D3Chief active existence form in blood.Vitamin D3Lack and may result in
Virgin rachitic generation.Current research shows, vitamin D3Also related to tumor, detect human body 25-hydroxy-vitamin D3Content is outstanding
It is important.
At present, clinically to vitamin D3Detection by quantitative become conventional sense project and health check-up project.25- hydroxyl is tieed up
Raw element D3The detection by quantitative main rationalization method of inspection and immunological method.Physical and chemical inspection method adopts liquid chromatography-mass spectrography
Multiple techniques (LC-MS), due to having that expensive equipment, Sample pretreatment be complicated, the loaded down with trivial details defect such as time-consuming of process, the method should
With being restricted.Immunological method has the advantage such as sensitive, quick, special and easy, becomes now vitamin D3The master measuring
Stream method.It is critical only that of such method prepares that affinity is strong, purity is high and the high-quality antibody of low cost.
At present, Multiple Antibodies for 25-hydroxy-vitamin D have been disclosed in prior art.For example, Chinese patent
CN105352958A discloses a kind of total 25-hydroxy-vitamin D detection kit, the antibody energy of wherein said use and vitamin
D2And D3Simultaneous reactions.Chinese patent CN103857698A also discloses that one kind is directed to 25-hydroxy-vitamin D2And D3Antibody.
Additionally, Chinese patent CN101273062A discloses a kind of antibody of anti-25-hydroxy-vitamin D, wherein said antibody and 25- hydroxyl
Base vitamin D2And 25-hydroxy-vitamin D3Two kinds of 25-hydroxy-vitamin D forms all can be reacted.
As described above, most of antibody for 25-hydroxy-vitamin D can not be distinguished at present, or at least can not be very
Distinguish vitamin D well2And D3.In addition, currently for 25-hydroxy-vitamin D3Antibody in, due in preparation process introduce
Crosslinked group impact, cause gained affinity of antibody low.Additionally, in order to obtain monoclonal in existing preparation method for antibody
Antibody, needs to carry out multiple repeated screening to exclude the antibody for crosslinked group, therefore, wastes time and energy, increased antibody system
Standby cost.
Accordingly, there exist a kind of fast and convenient for setting up, low cost prepares the high monoclonal antibody of high-affinity, sensitivity
Demand.
Content of the invention
In order to solve at least part of above-mentioned technical problem, the present invention passes through to optimize hybridoma preparation process, and/or excellent
Change the screening process of monoclonal cell strain, thus obtaining producing anti-25-hydroxy-vitamin D3The cell strain of monoclonal antibody, and base
Complete the present invention in this.Specifically, the present invention includes herein below.
An aspect of of the present present invention, provides a kind of preparation method of cell strain, and wherein said cell strain produces anti-25- hydroxyl dimension
Raw element D3Monoclonal antibody, methods described includes：
A) step using immunizing antigen immune animal, wherein said immunizing antigen is by making the macromole egg as carrier
White matter with as haptenic 25-hydroxy-vitamin D3It is coupled the water-insoluble antigen being obtained via crosslinked group, and described big
Molecule protein and described 25-hydroxy-vitamin D3Coupling Mole Ratio be 1:80 to 1:100；
B) cell fusion step, makes the splenocyte of described animal and homology myeloma cell merge, and forms hybridoma
Cell；
C) using the step of screening antigen selection positive cell strain, wherein said screening antigen does not comprise described crosslinking group
Group.
In certain embodiments, described macro-molecular protein is albumin.For example, human serum albumin, Sanguis Bovis seu Bubali are pure
Albumen, albumin rabbit serum etc., preferably bovine serum albumin.
In certain embodiments, described crosslinked group is omega-dicarboxylic acids, such as ethylenedicarboxylic acid's ester bond.
In certain embodiments, described screening antigen does not comprise described crosslinked group, and does not comprise described macromole egg
White matter.
In certain embodiments, described screening antigen is macro-molecular protein and 25-hydroxy-vitamin D3Amino derives
The coupling molecule of thing.
In certain embodiments, moved using the mixture direct immunization of immunizing antigen and the gel for being isolated
Thing.Preferably use the mixture of PAGE gel and immunizing antigen.
Another aspect of the present invention, provides a kind of cell strain, and it is prepared by method of the present invention.
Another aspect of the invention, provides a kind of production anti-25-hydroxy-vitamin D3The method of monoclonal antibody, it includes
The preparation method of cell strain of the present invention is as step therein, or includes cultivating the step of cell strain of the present invention
Suddenly.
Another aspect of the present invention, provides a kind of monoclonal antibody, itself and 25-hydroxy-vitamin D3Affinity constant
3.84×109, with 25-hydroxy-vitamin D2Cross reaction be less than 10%, preferably shorter than 8%, more preferably less than 5%, or even low
In 3%.Preferably, described monoclonal antibody is obtained by preparation method of the present invention, or by above-mentioned production anti-25- hydroxyl
Base vitamin D3The method of monoclonal antibody obtains.
In certain embodiments, the immunizing antigen of present invention preparation has higher immunogenicity, can more strongly pierce
Swash specific immunocyte, make activated immune cell, propagation, differentiation, final generation corresponding antibodies and primed lymphocyte.
In certain embodiments, the immunizing antigen of the present invention overcomes the limited source of antigen or property during antigen purification
Unstable, the defect of changeableness during purification.
In certain embodiments, present invention optimizes the step preparing cell strain or antibody, decrease screening number of times, carry
High anti-25-hydroxy-vitamin D3The preparation efficiency of cell strain of monoclonal antibody, reduces production cost.
In certain embodiments, the preparation method for antibody of the present invention eliminates the crosslinking group introducing in artificial antigen's preparation
The impact of the antibody to preparation for the group.
In certain embodiments, the antibody that the present invention prepares has higher affinity, and ties up with 25- hydroxyl
Raw element D2Cross reaction low.
Brief description
Fig. 1 exemplary immunization of the present invention antigen is coupled result electrophoretogram (SDS-PAGE glue).
Fig. 2 present invention exemplary screening antigen is coupled result electrophoretogram (SDS-PAGE glue).
The exemplary 25 hydroxyl VD of Fig. 3 present invention3Affine the trying hard to of antibody (antibody 1 to 6), wherein vertical coordinate are absorbance,
Abscissa is the number of times being diluted the antibody of 1mg/ml by coubling dilution.
The exemplary 25 hydroxyl VD of Fig. 4 present invention3Affine the trying hard to of antibody (antibody 3), wherein vertical coordinate are absorbance, horizontal seat
It is designated as the number of times that the antibody of 1mg/ml is diluted by coubling dilution.
Specific embodiment
Be will be clear from by explaining the preferred embodiment of following the application, other objects of the present invention and advantage.
It should be understood that heretofore described term is only to be to describe special embodiment, it is not intended to limit this
Bright.In addition, for the numerical range in the present invention it is thus understood that it is also specifically disclosed that this scope upper and lower bound between every
Individual intermediate value.In in the intermediate value in any statement value or stated ranges and any other statement value or in described scope
Between each less scope between value be also included in the present invention.These small range of upper and lower bounds can independently include
Or exclusion in the range of.
Unless otherwise stated, all technology used herein and scientific terminology have the routine in field of the present invention
The identical meanings that technical staff is generally understood that.Although the present invention only describes preferred experimental technique, mensure or method of testing,
It is can also to use and similar or equivalent any method described herein in the experiment of the present invention, mensure or test.This explanation
The all documents mentioned in book are incorporated by reference into, in order to the disclosure and description method related to described document or experiment.With
When any document being incorporated to conflicts, it is defined by the content of this specification.
In the present invention, vocabulary of terms had both included singulative, also included plural form, unless context separately clearly refers to
Go out.Heretofore described " at least one " or " at least one " refer not only to the situation that comprises " one " or " a kind of ", heavier
The situation also comprising " multiple " or " multiple " wanted.
Heretofore described " cell strain " refers to unicellular separation and Culture or the method by screening, by unicellular propagation
The cell colony being formed.The special nature of cell strain or mark must exist during whole culture all the time.From culture algebraically
Say, the cell strain of the present invention can cultivate 40-50 generation.For the source of cell strain, it can be any mammalian cell
Or artificial treatment cell, such as fused cell.Preferably, the cell that the cell strain of the present invention is originated for mice.
Heretofore described " 25-hydroxy-vitamin D3" compound (vitamin D that refers in formula3, abbreviation
VD3) the 25th hydroxylated compound：
Known 25-hydroxy-vitamin D3Itself all can not produce immunoreation.By vitamin D metabolism component chemical activation with
And so that them is combined with carrier molecule or reporter group be not the easy thing of part.Therefore, in order that immune success rate, indispensable bar
Part is to prepare one kind such as to comprise 25-hydroxy-vitamin D3As haptenic conjugate.
Heretofore described " immunizing antigen " is also referred to as " immunogen " or " antigen " sometimes, refers to immunogenicity
Material with reactionogenicity.The immunizing antigen of the present invention refers to by making as the macro-molecular protein of carrier and as hapten
25-hydroxy-vitamin D3It is coupled the water-insoluble antigen being obtained via crosslinked group.It should be noted that what the present invention used
Immunizing antigen needs for water-insoluble antigen.It is coupled in routine techniquess means and finish rear gained antigen water-soluble antigen.
In the present invention, macro-molecular protein and 25-hydroxy-vitamin D in immunizing antigen3Mol ratio be 1:5 to 1:150,
Preferably 1:50 to 1:100, more preferably 1:80 to 1:100, for example, 1:90,1：100 etc..25-hydroxy-vitamin D in immunizing antigen3
Ratio higher, then the antigen for preparing is less susceptible to water-soluble.
In the present invention, described " crosslinked group " refers to connect macro-molecular protein and 25-hydroxy-vitamin D3Group.Excellent
Selection of land, crosslinked group is the derivant of polybasic carboxylic acid, such as polybasic carboxylic acid ester bond.Described polybasic carboxylic acid includes, but are not limited to binary
Carboxylic acid, tricarboxylic acid, quaternary carboxylic acid etc..Preferably dicarboxylic acids.From the angle of the impact reducing antagonist, described polybasic carboxylic acid
Carbon number is preferably 2-10, more preferably 3-8, further preferred 4-6, and most preferably 4.Instantiation includes succinic acid etc..
In the present invention, the position of described " crosslinked group " is preferably placed at 25-hydroxy-vitamin D33.Preferably, crosslinked
Group passes through-COOH and the 25-hydroxy-vitamin D of polybasic carboxylic acid33-OH connected by chemical reaction.In addition, it is polynary
- NH on another-COOH of carboxylic acid and macro-molecular protein2 +React and be connected with carrier.
In the present invention, described " aminoderivative " refers to, its instantiation includes, but are not limited to formula-(CH2) nNH- represents
Compound, wherein n be 2-10, more preferably 3-8, the integer of further preferred 4-6, most preferably 4.
The preparation method of cell strain of the present invention is the preparation method of hybridoma cell.Key step
Including：Animal immune, cell fusion, the screening of hybridoma and monoclonal antibody detection, the cloning of hybridoma, frozen, single
Anti- identification etc..Specific as follows：
1st, animal immune
(1) antigen preparation
The immunizing antigen of preparation monoclonal antibody, does not require very high though saying from purity, highly purified antigen makes to obtain
The chance of required monoclonal antibody increases, and can mitigate the workload of screening simultaneously.Therefore, immunizing antigen is more pure better.Logical in practice
Immunizing antigen frequently with purification carries out immunity.
However it is discovered in the present invention that, immunity is carried out using unpurified immunizing antigen there is unforeseeable technique effect.Tool
Body ground, the present invention carries out immunity using the method cutting glue immunity, it is to avoid can not form Water-In-Oil when directly with adjuvant emulsion slow
Release the predicament of emulsifiable paste, immune effect can be made to obtain very big lifting.Meanwhile, the limited source of antigen or property are overcome not
Stable, the defect of changeableness during purification.Therefore, in the immunization method of the present invention, preliminary purification is only needed to antigen, even do not purify.
And specifically, it is preferable to use and the mixture direct immunization animal containing separating gel.The gel using when immunizing antigen separates can
For PAGE gel, agar gel etc..In the present invention, described immunization method can be any using be usually used in the art
Method, for example, the method such as injecting immune, oral immunity, aerosol immunization.
(2) selection of immune animal
Mice and rat be can be selected for as immune animal according to myeloma cell used.In the present invention, preferably all of
Mouse myeloma cell line for hybridoma technology derives from BALB/c mouse, and all of rat myeloma cell is all originated
In LOU/c rat, preferably use both inbred animals as immune animal.But, need to be planted sometimes for specific purposes
Intermolecular hybrid, also can other animals immune, for example, rabbit, chicken, Canis familiaris L. or primatess etc..The ability of the general secretory antibody of intervarietal hybridization tumor
Unstable, chromosome is easily lost, it is advantageous to the hybridization between allogenic cell.
(3) determination of immune programme for children
Immunity is one of important step in monoclonal antibody preparation process, its object is to make bone-marrow-derived lymphocyte pierce in specific antigen
Swash lower differentiation, propagation, be beneficial to cell fusion and form hybrid cell, and have increased access to the machine of the hybridoma of secreting specificity antibody
Meeting.Therefore when designing immune programme for children, should be according to the property of antigen and purity, amount of antigen, immunization route, immune time and interval
Time, the application of adjuvant and animal determine to responsibility of this antigen etc..
Immunization route includes, but does not limit in vivo immunization, and such as subcutaneous injection, abdominal cavity or intravenous injection may also be employed
Foot pad, Intradermal, collunarium or eye dripping.Last booster immunization preferably employs abdominal cavity or intravenous injection, preferably the latter, because can make
Antigen acts on rapider and abundant to splenocyte.Spleen is taken within the 3rd day after booster immunization to merge the last time, the knot of many laboratorys
Fruit shows, in initial immunity and secondary immune response reaction, extracting spleen cell and myeloma cell fusion, and the shape of specific hybrid tumor
Peak is become to be respectively the 4th day and the 22nd day, the hybridoma Major Secretory IgM antibody obtaining in primary immune response, exempt from again
The hybridoma Major Secretory IgG antibody obtaining during epidemic disease response.Peak and the titre of mice serum antibody that positive hybridoma occurs
Have no obvious parallel relation, and how before serum antibody peak.Therefore, need for reaching highest hybridoma formation rate
Plasmablast as much as possible, this takes spleen to carry out merging conveniently on the 3rd day after booster immunization the last time.Existing people's report is adopted
With immunity in the spleen, the immunoreactivity to antigen for the mice can be improved, and time-consuming, typically immunity can be merged after 3 days.
The immunization method of the present invention may also include ion vitro immunization.So-called ion vitro immunization is exactly that (or lymph node is thin by splenocyte
Born of the same parents, or peripheral blood lymphocyte) take out in vitro, under certain condition with antigen co-cultivation, then enter with myeloma cell again
Row merges.
2nd, cell fusion
(1) preparation of main agents
A, cell culture medium
Used in hybridoma technology, cell culture medium mainly has two kinds of basal mediums of RPMI-1640 or DMEM, these
Culture medium is known in the art.
Not exclusively RPMI-1640 culture medium：
RPMI-1640 culture medium stock solution 96ml
100 × L.G. solution (L-Glutamic Acid) 1ml
Dual anti-solution 1ml
7.5%NaHCO3Solution 1-2ml
HEPES solution 1ml
Not exclusively DMEM culture medium：
DMEM 13.37g
Ultra-pure water or four steaming water 980ml
NaHCO33.7g
Dual anti-solution 10ml
100 × L.G. solution 10ml 1N HCl debugs pH to 7.2-7.4, filtration sterilization, 4 DEG C of preservations of subpackage.
RPMI-1640 or DMEM culture medium completely：
Not exclusively RPMI-1640 or DMEM culture medium 80ml
Calf serum 15-20ml
For myeloma cell SP2/0 and build the Hybridoma Cell Culture after strain.
HT culture medium：
RPMI-1640 or DMEM culture medium 99ml completely
HT stock solution 1ml
HAT culture medium：
RPMI-1640 or DMEM culture medium 98ml completely
HT stock solution 1ml A stock solution 1ml
B, aminopterin-induced syndrome (A) stock solution (100 ×, 4 × 10-5mol/L):
Weigh 1.76mg aminopterin-induced syndrome (Aminopterin MW 440.4), be dissolved in 90ml ultra-pure water or four and steam in water, drip
Plus 1mol/L NaOH 0.5ml neutralization, then add ultra-pure water or four and steam water to 100ml.Filtration sterilization, subpackage bottle (2ml/
Bottle), -20 DEG C of preservations.
C, hypoxanthine and thymidine (HT) stock solution (100 ×, H:10-2mol/L, T：1.6×10-3mol/
L):
Weigh 136.1mg hypoxanthine (Hypoxanthine, MW 136.1) and 38.8mg thymidine
(Thymidine, MW 242.2), plus ultra-pure water or four steam water to 100ml, putting in 45-50 DEG C of water-bath makes to be completely dissolved, cross filter
Bacterium, subpackage bottle (2ml/ bottle), -20 DEG C are frozen.With before can put 37 DEG C of hydrotropies of heating.
D, L-Glutamine (L.G.) solution (100 ×, 0.2mol/L):
Weigh 2.92g L-Glutamine (L-glutamine, MW 146.15), with 100ml incomplete culture fluid or ultrapure
Water (or four steaming water) dissolving, filtration sterilization, subpackage bottle (4-5ml/ bottle), -20 DEG C are frozen.
E, penicillin and streptomycin (dual anti-) solution (100 ×):
Take benzylpenicillin (sodium salt) 1,000,000 unit and streptomycin (sulfate) 1g, be dissolved in 100ml sterilizing ultra-pure water or four steamings
In water, subpackage bottle (4-5ml/ bottle), -20 DEG C are frozen.
F, 7.5%NaHCO3Solution:
Weigh the pure NaHCO of analysis37.5g, is dissolved in 100ml ultra-pure water or four and steams in water, filtration sterilization, subpackage bottle (4-
5ml/ bottle), cover tightly bottle stopper, 4 DEG C of preservations.
G, HEPES solution (1mol/L):
Weigh 23.83g HEPES, N-2- hydroxyethyl piperazine-N, -2- ethylsulfonic acid, MW238.3) it is dissolved in 100ml ultra-pure water
Or four steam water in, filtration sterilization, subpackage bottle (4-5ml/ bottle), 4 DEG C preservation.
H, 8-anaguanine stock solution (100 ×):
Weigh 200mg 8-anaguanine (8-azaguanine, MW 152.1), add 4mol/LNaOH 1ml, treat that it is molten
Xie Hou, adds ultra-pure water or four steaming water 99ml, filtration sterilization；Subpackage bottle, -20 DEG C frozen.It is added to by 1% concentration during use
In culture fluid (i.e. final concentration of 20ug/ml).
I, 50%PEG:
Weigh PEG 1000 or 4000 20-50g in triangular flask, cover tightly, 60-80 DEG C of water-bath is melted, and 0.6ml is sub-packed in
In penicillin bottle, cover tightly, 8 pounds of high steams 15 minutes, -20 DEG C deposit standby.Heating and melting before use, plus equivalent is incomplete
Culture medium, uses a little 7.5%NaHCO3Adjust pH to 8.0, or buy Sigma or Gibco company off-the-shelf.
(2) preparation of myeloma cells
The mode that before fusion, myeloma cell maintains, is of paramount importance to being successfully obtained hybridoma.Target is to make carefully
The time that born of the same parents are in logarithmic growth is as long as possible, certainly can not be less than 1 week before merging.When frozen cell wants 2 weeks after recovery
Between just can be in be suitable for merge state, the myeloma cell growing be only possible within least several days recover.Locate in the lab
Myeloma cell in logarithmic growth maintains in the culture medium containing 10% calf serum, and method is with 6 dress 5ml culture medium
Culture bottle, inoculates 10 times of myeloma cells being serially diluted.Quite close and do not grew one bottle moves again to cell after 1 week
Plant.The typical doubling time is 14-16 hour.
The preparation method of myeloma cell's suspension is as follows：
A, before merging 48-36 hour, by myeloma cell's amplification culture, (test for fusion typically pressing one piece of 96 orifice plate is about
The cell needing the culture of 2-3 bottle 100ml culture bottle is prepared).
B, the fusion same day, with connector bend dropping tube, cell is gently blown down from bottle wall, be collected in 50ml centrifuge tube or fusion pipe.
C, 1000r/min are centrifuged 5-10 minute, supernatant discarded.
D, addition 30ml incomplete culture medium, with method centrifuge washing once.Then Cell resuspension is incomplete in 10ml
Culture medium, mixes.
E, take myeloma cell's suspension, plus 0.4% trypan blue dye liquor make standby after viable count.During cell counting, take
Cell suspension 0.1ml adds in 0.9ml trypan blue dye liquor, mixes, is counted with blood counting chamber.Calculate the formula of cell number
For：Every milliliter of cell number=4 block plaid cell number × 105/4；Or every milliliter of cell number=5 medium square cell number × 106/
2.
(3) preparation of splenocyte
Take the BALB/c mouse of immunity, extract eyeball blood sampling, and it is right as positive during antibody test to separate serum
According to serum.Dislocated lethal mice by neck simultaneously, be soaked in 5 minutes in 75% ethanol, in dissect on platen fixing after raise a left side
Side skin of abdomen, it can be seen that spleen, changes eye scissorss tweezer, cuts off peritoneum with aseptic operation in super-clean bench, takes out spleen and is placed in
Fill in the plate of 10ml incomplete culture medium, gently washed, and surrounding connective tissue has been peelled off in carefulness.Spleen is moved into another
Fill in the plate of 10ml incomplete culture medium, with elbow tweezers or the curved needle head that is contained on 1ml syringe gently extrudes spleen
(also can extrude spleen with plunger), makes splenocyte enter the incomplete culture medium in plate.Blow and beat for several times with suction pipe, system
Become single cell suspension.In order to remove the large crumb in splenocyte suspension, 200 mesh copper mesh are can use to filter.Harvest splenocyte suspension,
1000r/min is centrifuged 5-10 minute, and cannot be used up full culture medium centrifuge washing 1-2 time, then cell is resuspended in 10ml incomplete
Culture medium mixes, and takes above-mentioned suspension, plus platform phenol indigo plant dye liquor make standby after viable count.Generally every mice can obtain 1 × 108-
2.5×108Individual splenocyte, every Rats Spleen can obtain 5 × 108-10×108Splenocyte.
(4) preparation of feeder cells
In selectivity incubation after cell fusion, because a large amount of myeloma cells and splenocyte are died off, now
The scattered hybridoma of single or minority is difficult survival mostly it is often necessary to add other living cells to be allowed to breed, this quilt
The living cells adding are referred to as feeder cells.Feeder cells promote the mechanism of other cell proliferation still not understand it is considered that they
The growth-stimulating factor of non-species specificity may be discharged, provide necessary growth conditionss for hybridoma；Be also likely to be for
Meet the newborn dependency to cell density for the hybridoma.Conventional feeder cells have thymocyte cell, normal splenocytes and
Peritoneal macrophage.Wherein with the source of Turnover of Mouse Peritoneal Macrophages and prepare more convenient, and phagocytosis is had to remove dead cell
And its effect of fragment, therefore most commonly used.Its preparation method is as follows：By the above-mentioned method adopting mouse boosting cell by mice
After lethal, body surface is sterilized and be fixing, cut tweezer with sterilization and start skin of abdomen from rear abdomen, expose peritoneum.Wipe abdomen with cotton ball soaked in alcohol
Film is sterilized.With syringe injection 10ml incomplete culture medium to abdominal cavity, note avoiding penetrating intestinal tube.The right hand fixes syringe, makes
Syringe needle is retained in intraperitoneal, and left hand holds cotton ball soaked in alcohol gently abdomen massage 1 minute, subsequently suctions out the culture fluid of injection.1000r/
Min is centrifuged 5-10 minute, abandons supernatant.First with 5ml HAT culture medium, sedimentation cell is suspended, according to cell counts, add
HAT culture medium, makes cell concentration be 2 × 105/ ml, standby.Generally for macrophage, the every hole of 96 well culture plates needs 2 × 104
Individual cell, the every hole of 24 orifice plates needs 105Individual cell.Every mice can obtain 3-5 × 106Individual cell, therefore one mice is available for two piece 96
The feeder cells of orifice plate.Also can prepare and cultivate feeder cells within 1-2 days before cell fusion, so that cultivation plate hole bottom is first spread
One layer of feeder layer.
(5) cell fusion and the selectivity of hybridoma are cultivated
The program of cell fusion report have many kinds.For example, following methods,
A, by 1 × 108Splenocyte and 2 × 107-5×107Myeloma cell SP2/0-Ag14 is mixed in a 50ml and merges
Guan Zhong, adds incomplete culture medium to 30ml, fully mixes.
B, 1000r/min are centrifuged 5-10 minute, supernatant is tried one's best and exhausts.
C, fusion pipe bottom is touched on palm, make sedimentation cell loose uniformly；Put preheating in 40 DEG C of water-baths.
D, with 1ml suction pipe at 1 minute about (Best Times be 45 seconds) plus the 50%PEG (pH 8.0) that is preheated to 40 DEG C
1ml, side edged is gently mixed.
E, with 10ml suction pipe, in 90 seconds plus 20-30ml is preheated to 37 DEG C of incomplete culture medium；20-37 DEG C stands 10 points
Clock.
F, 1000r/min 5 minutes；Supernatant discarded.
G, add 5ml HAT culture medium, gently pressure-vaccum sedimentation cell so as to suspending and mixing, then add huge containing abdominal cavity
Phagocytal HAT culture medium is to 80-100ml.
H, subpackage 96 porocyte culture plates, every hole 0.10-0.15ml；Subpackage 24 orifice plate, every hole 1.0-1.5ml；Then will
Culture plate puts 37 DEG C, 6%CO2Culture in incubator.
I, swapped out 1/2 culture medium with HAT culture medium after 5 days.
Swapped out HAT culture medium with HT culture medium after J, 7-10 days；(after the 14th day, can use common complete medium).
L, often observation Growth of Hybridoma Cell situation, suction out supernatant when its length is to bottom hole area more than 1/10 and supply antibody
Detection.
3rd, filtering hybridoma
Hybridoma can screen for 2 weeks about after fusion, that is, secretion needed for antibody hybridoma holes from numerous
Elect in hole, also commonly referred to as antibody test.The method of antibody test is a lot, generally according to the antigen studied and laboratory
Condition depending on.But as hybridoma screening antibody detection method must have quickly, prepare, simplicity, be easy to single treatment
The features such as a large amount of sample.It is, in general, that being necessary for establishing antibody detection method before merging, and overcome that may be present asking
Topic.Another major issue is " dynamics range " required for antibody detection method, that is, detection background more than the most by force with
The ratio of weak signal, depending on whether detection antigen used is pure.If hybridoma antibody is the proteantigen for purification,
100% antigen participates in reaction, and a male/female judgement system is just much of that.On the other hand, if hybridoma antibody is to be directed to
The micro proteantigen of cell surface, detecting system may need to measure small-signal, then dynamics range at least should be 10:
1, preferably 100:1.
In addition, the selection of detection method is also affected by the type of required hybridoma antibody and predetermined purposes.In conjunction with benefit
The antibody of body can be selected with the detection method based on cell-cytotoxic reaction.As need combine A albumen hybridoma antibody it is necessary to
Detection method with associated proteins A.As screening technique, including the Enzyme-linked Immunosorbent Assay examination of immunoenzyme technics, soluble antigen
Test (ELISA), the ELISA with full cellular antigens, antibody capture ELISA test, ABC-ELISA test, Dot-ELISA test,
Immunohistochemical Staining etc..
4th, the cloning of hybridoma
The positive hybridoma cell obtaining from archioporus, is probably derived from two or more hybridomies, therefore it
Secreted antibody be inhomogeneous.In order to obtain the monoclonal antibody of complete homogeneity it is necessary to carry out to hybridoma gram
Longhua.On the other hand, the initial stage of Hybridoma Cell Culture is unstable, some cell loss chromosome dyads, may lose
Produce the ability of antibody.In order to remove the cell of this part no longer secretory antibody, obtain the stable monoclonal of secretory antibody miscellaneous
Hand over oncocyte system (also known as sub-clone) it is also desirable to cloning.In addition, the hybridoma of long-term liquid nitrogen cryopreservation, its point after recovery
Secreting the function of antibody it is possible to losing, therefore also should make cloning, situation is secreted with detection antibody.Generally obtaining for pre-
Need after the hybridoma determining antigen to be carried out continuously 2-3 time cloning, sometimes also need to carry out repeatedly.So-called cloning be instigate single
Cell asexual propagation and obtain the whole incubation of this cell group.The method of cloning is a lot, such as limiting dilution assay, soft fine jade
Fat method, unicellular micromanipulation, monoclonal cell group micromanipulation and fluorescence activated cell sorter (FACS) separate
Method.
5th, the frozen and recovery of hybridoma
(1) hybridoma is frozen
During setting up hybridoma, Single cell fusion produces a lot " positive " holes sometimes, has little time to all of
Hybridoma makees further work, needs a portion cell cryopreservation to get up；On the other hand, in order to prevent laboratory
It may happen that contingency, such as have a power failure, pollution, the temperature of incubator or CO2Controller failure etc. is miscellaneous to setting up
Hand over tumor bring on a disaster, generally frozen as early as possible a part of cell as seed, in order to avoid suffering accident.Build in hybridoma
After vertical, with greater need for frozen large quantities of, in case taking at any time from now on.
The principle of cell cryopreservation be cell in the culture medium of increase serum and dimethyl sulfoxide with certain slow speed
Cooling degree (0 DEG C -20 DEG C, per minute decline 1 DEG C；- 20 DEG C -40 DEG C per minute to decline 2 DEG C), can steam in -196 DEG C of liquid nitrogen or liquid nitrogen
Preserve for a long time in gas.The cell freezing method of we laboratory is described below, through the in the past few years use to various kinds of cell, cell is multiple
Soviet Union's survival rate is all more than 80%.
(2) recovery of hybridoma
Hybridoma, myeloma cell or other cells preserve in liquid nitrogen, if during zero accident situation, can preserve the several years
To many decades.Melt cell speed during recovery fast, be allowed to run through -0 DEG C of the most easily damaged -5 DEG C, in case intracellular formation
Ice crystal causes cell death.Under normal circumstances, when frozen, cell quantity is many, the good hybridoma cell line of growth conditions and other
The recovery of cell can adopt following methods, and this is also the commonly used program of each laboratory.I.e. during recovery, take out from liquid nitrogen
Ampulla, melts in 37 DEG C of water-baths immediately, when last point ice cube soon melts, takes out, put on ice bath from water-bath.Use 5-
10ml HT culture medium dilute, 1000r/min 10 minutes, abandon supernatant, be resuspended in appropriate HT culture medium, proceed to culture bottle or
24 orifice plates, put 37 DEG C, and 7.5%CO2 cultivates.If cell viability is not high, dead cell is too many, can Jia 104-105/ ml mice abdomen
Chamber cell is cultivated.But, frozen cell not can 100% Successful Resuscitation, its reason is more, cell number when such as frozen
Amount is less or growth conditions are bad, or during recovery, condition of culture changes or method is improper it is also possible to cell is subject to antibacterial or mycoplasma dirty
Contaminate, and liquid nitrogen container keeping is not good at.When above-mentioned situation occurs, can be recovered these cells using some meanss to save the situation, as little
Form solid tumor method, inocalation method in spleen, mouse peritoneal induces ascites and solid tumor method under Corium Mus, and 96 orifice plate culture methods etc..
6th, the identification of monoclonal antibody characteristic
(1) the determination of monoclonicity
Identification and monoclonal antibody including the chromosome analysises of hybridoma, monoclonal antibody heavy chain immunoglobulin and light chain type are pure
Degree identification etc..
A, the chromosome analysises of hybridoma
Hybridoma is carried out chromosome analysises can obtain its be whether real hybridoma objective indicator it
One, the chromosome number of hybridoma should close to two kinds of parental cell Chromosome number purpose summations, normal mouse splenocyte
Chromosome number is 40, and murine myeloma cell SP2/0 is 62-68, and NS-1 is 54-64；The chromosome of myeloma cell simultaneously
The feature of two kinds of parental cells is reflected on structure, in addition to majority is for telocentric chromosome, minority mark chromosome also occurs.Separately
On the one hand, the chromosome analysises of hybridoma have certain meaning to the ability understanding hybridoma secretion monoclonal antibody, in general,
Hybridoma chromosome number is more and relatively concentrates, and its secretion capacity is then high, conversely, its secretion monoclonal antibody ability is then low.Check
The method the most frequently used Colchicine method of hybridoma chromosome, its principle be the application special destruction spindle fiber of Colchicine and
Obtain division phases cell；Use the Hypotonic treatment such as 0.075mol/L KCl solution again, make cell expansion, volume increases, dyeing
Body is loose；Fix through methanol-glacial acetic acid solution, you can observe and check.
B, the classification of anti-immunoglobulin and subgroup identification
The class of antibody and subclass are very helpful to the method determining purification.Except using special immunization method and detection
Method, the monoclonal antibody most frequently obtaining is IgM and IgG, and the hybridoma of secretion IgE is rarely found, and the hybridoma secreting IgA leads to
Often only just can obtain from gut-associated lymphoid tissue in the lymphocyte for merging.As in antibody test using Fructus Vitis viniferae
Coccus A protein reagent, then can not possibly obtain the antibody of other classes beyond IgG.The method of identification monoclonal antibody Ig class and subclass is main
There are two kinds, one kind is immunodiffusion, another kind is ELISA.
C, the identification of monoclonal antibody purity
Polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, isoelectric focusing electropho- resis (IEF) and immunoblotting analysis
Etc. (WB) method can be used in identifying the purity of monoclonal antibody.The principle of PAGE, SDS-PAGE, IEF, WB and method refer to You Guan special
Write, repeat no more here.
(2) the identification of monoclonal antibody physicochemical property
Say from Practical significance, monoclonal antibody is all physicochemical property to the sensitivity of temperature and pH change and the affinity of monoclonal antibody
The main project of identification, they can using and preserve offer important evidence for monoclonal antibody.Wherein monoclonal antibody affinity mensure is more multiple
Miscellaneous, briefly introduce below.
Antibody affinity refers to the intensity that antibody is combined with antigen or hapten, and its height is mainly divided by antibody and antigen
The size of son, the appropriate level of spatial configuration and antigenic determinant between for the antibody molecule haptophore (portion) determine.Affinity
Generally represented with averagely inherent binding constant (K).
It is highly important that monoclonal antibody affinity measures, and it can provide foundation for the correct monoclonal antibody selecting different purposes.Building
The monoclonal antibody of high affinity when founding various detection method, should be selected, to improve Sensitivity and Specificity, and reagent can be saved.And
During affinity chromatograph, should be from the moderate monoclonal antibody of affinity as immunoadsorbent, because affinity is too low being difficult to adsorb, affinity
Too high it is difficult eluting.
The affinity accurately measuring monoclonal antibody is more difficult, during the good monoclonal antibody of selection in actual applications, generally only needs to measure
The relative affinity of each monoclonal antibody and its height ordering.Conventional method has competitive ELISA, noncompetitive ELISA, indirectly
ELISA, indirect method sandwich ELISA etc., only introduce the method that competitive ELISA measures monoclonal antibody affinity costant here.Its step is：
Take the purifying antigen coated elisa plate of suitable concentration, 100 μ l/ holes, 4 DEG C overnight.After washing, add confining liquid
(0.5%BSA-PBS, pH7.2) 100 μ l/ hole, 37 DEG C 1 hour.Take certain density monoclonal antibody, the antigen with serial doubling dilution
Mixing, 4 DEG C overnight, makes reaction reach balance；Must be noted that antigen concentration used is high more than 10 times at least than antibody concentration.Will
Antigen antibody complex after balance adds in ELISA Plate hole, 100 μ l/ holes, 37 DEG C 1 hour.After washing, add acceptable diluent degree
HRP labelling anti-mouse IgG antibody, 100 μ l/ holes, 37 DEG C 1 hour.After washing, addition substrate (OPD) solution, 100 μ l/ holes,
37 DEG C are developed the color 15 minutes；2mol/L H2SO4The absorbance (A) in each hole after terminating reaction, is measured in 495nm wavelength.
Calculate the affinity costant (K) of each monoclonal antibody by following equation：A0/ (A0-A)=1+K/a0 A when wherein A0=is nonantigenic
Value；A=adopts A value during variable concentrations antigen；A0=antigen total amount；K=affinity costant.
(3) monoclonal antibody and the reactivity of corresponding antigens measure
Monoclonal antibody is decided by, with the reactivity of corresponding antigens, the epitope that it is identified, determines that the epi-position that monoclonal antibody is directed to is resisting
Position on original structure, is the key link of monoclonal antibody CHARACTERISTICS IDENTIFICATION, analyzes the difference of this kind of epi-position further meanwhile, can be correct
Evaluate specificity and the cross reactivity of monoclonal antibody, such as some antigens are to belong to different serotypes together to have, and even do not belong to together in section
Common, and other epitopes are then certain serotype or even a certain bacterial strain or strain institute is peculiar.Additionally, the reaction of monoclonal antibody
Property often assumes one or more immunity test specificitys, and this is measured when building plant, is conducive to these monoclonal antibodies proper use of.
Monoclonal antibody reactivity method for measuring is a lot, including the test of all kinds of immunoserologies, biological test and immunochemistry
Technology etc., select which kind of method according to different monoclonal antibody characteristics and test objective depending on, refer to being discussed in detail of relevant treatise.
The production of the monoclonal antibody of the present invention
After obtaining stable hybridoma cell line, you can produce monoclonal antibody in a large number as needed, for different purposes.At present
The method preparing monoclonal antibody in a large number mainly has two big systems, and one is working system in animal body；Another is extracorporeal culture-ing.In the present invention
Preferably employ extracorporeal culture-ing.
Generally speaking, hybridoma cell line is not strict wall dependent cells (anchorage dependent
Cell, ADC), therefore both can carry out monolayer cell culture, suspension culture can have been carried out again.The cell monolayer of hybridoma
Culture method is the most frequently used means of each laboratory, will add in culture bottle, with calf serum containing 10-15% by hybridoma
Culture medium culturing, cell concentration is with 1 × 106-2×106/ ml is preferred, and then collects culture supernatant, wherein monoclonal antibody content about 10-
50ug/ml.The monoclonal antibody amount of this method preparation is extremely limited, and is not suitable for the large-scale production of monoclonal antibody beyond doubt.Want in body
Prepare monoclonal antibody in a large number outward, be necessary for carrying out a large amount of (high density) culture of hybridoma.Unit volume inner cell quantity is more,
The cell survival time is longer, and the concentration of monoclonal antibody is higher, and yield is bigger.
The mass propgation of hybridoma includes suspension culture system, using the bioreactor of rolling bottle or fermentation pot type,
Including using microcarrier；Or cell fixation culture systems, including doughnut cell culture system and microencapsulated cell
Culture systems.
A. Maitland culture：Adopting spinner culture small-scale suspension culture more at present, make cell be in suspension by stirring
State；And using the bioreactor of fermentation type, the U.S., Canada, French and German Deng Ji company more than extensive suspension culture
Produce this kind of reactor, its training method can be divided into pure batch, stream plus formula, semi continuous and continuous way.
B. microcarrier culture method：Microcarrier (Microcarrier) is the load using little solid particle as cell growth
Body, in culture fluid, cell then grows into monolayer in solid particles surface to microcarrier suspension under stirring.Can be used as cell
The microcarrier of mass propgation mainly using Sepharose or glucosan, polystyrene, glass etc. as the product of substrate, wherein with
Cytodex I, Biosilon and Superbeads are preferably.Identical with suspension culture in the basic skills of microcarrier culture.Recently
Research show, this method is one of desirable route of hybridoma mass propgation.
C. doughnut cell culture system：This system by hollow-fiber bioreactor, medium container, oxygen supply and
Peristaltic pump etc. forms.For cell culture doughnut by acetate fiber, polrvinyl chloride-polypropylene composition, poly carbonic acid silicon
Make Deng material, external diameter is generally 100-500um, wall thickness 25-75um, and wall is in spongy, has many micropores above.Doughnut
Surface of internal cavity be one layer of semipermeable ultrafilter membrane, its aperture only allows nutrient substance and metabolic waste to come in and go out, and to cell and
Macromolecular substances (as monoclonal antibody etc.) have delay to act on.The hollow-fiber bioreactor using at present be divided into pillar, plate and frame and
Center perfusion type.Although this culture systems cost in large-scale production monoclonal antibody is relatively low, and it is highly purified anti-to obtain high yield
Body, because equipment price is expensive, limits its range.
D. microencapsulated cell culture systems：This system is first by hybridoma microencapsulation, then this is had semipermeable membrane
Microcapsule be placed in culture fluid and carry out suspension culture, after certain time, isolate microcapsule from culture fluid, after flushing, open capsule
Film, can obtain the monoclonal antibody of high concentration after centrifugation.
The monoclonal antibody of the present invention and its antigen 25-hydroxy-vitamin D3Affinity be 3.84 × 109.Preferably, resist
Body affinity is yyy.In addition, the monoclonal antibody of the present invention and 25-hydroxy-vitamin D2Cross reaction be less than 10%, preferably
Less than 5%.
Embodiment
1. immunogenic preparation：
25- hydroxyl VD3Carboxyl extension is coupled on BSA, is coupled (BSA:25- hydroxyl VD3) mass ratio 1:1, make 25-
Hydroxyl VD3Excessive.Coupling mode is as shown below：
After coupling finishes, precipitation is stayed in centrifugation.After precipitation uses PBS resuspended, run SDS-PAGE glue (referring to Fig. 1), cut glue nitrogen
Grinding carries out immunity.
Experimental detail：
A. weigh 25- hydroxyl VD3Carboxyl extension 2mg is dissolved with 500 μ l DMSO, adds 2mgEDC (with 100 μ l
The 0.01M EMS dissolving of pH5.6), and 2mg NHS (being dissolved with the 0.01M EMS of 100 μ l pH5.6), activation is stirred at room temperature
1h.
B. 0.1M sodium hydroxide is used to adjust the 25- hydroxyl VD of NHS activation3Afterwards, it is added dropwise to 500 μ l 0.01M PBS molten
In the BSA of solution, it is stirred at room temperature 3 hours.
C. use 0.01M PBS, change liquid three times.It is centrifuged after taking-up and must precipitate and supernatant.Precipitation is heavy with 200 μ l PBS
Outstanding.Precipitation and supernatant take out 20ul respectively and carry out SDS-PAGE race glue identification, and precipitate molecules amount is substantially than the BSA molecule not being coupled
Greatly.
E. configure a plate and do not insert comb, only separation gel and the SDS-PAGE concentrating glue, precipitation resuspended for 150 μ l is used 2
After × loading buffer is processed, loading is run in glue.After nontoxic dyeing-decolorzing, purpose band is cut glue.
F. collect after glue liquid nitrogen grinding purpose band being cut, be divided into five deciles, -20 freezen protective.
2. immunologic process：
100ug/ mice, Freund's complete adjuvant initial immunity, often minor tick uses incomplete Freund's adjuvant in 14 days later
50ug/ mice booster immunization, carries out cell fusion after strengthening three times altogether.
Detailed process：
A. just exempt from：Take two parts of ground glue (about 400 μ g albumen), add to 800 μ l with PBS after merging, add
Point 6 points after 800 μ l Freund's complete adjuvant emulsifyings, the BalB/C mice of 60 μ l/ point four 6 week old of immunity.
B. strengthen：After initial immunity, took out a glue every 14 days, complement to 800 μ l with PBS, add 800 μ l Freunds
Point 6 points immunity, every 60 μ l after Freund's incomplete adjuvant emulsifying.
C. impact：Resuspended protein precipitation 15 μ l/ only, is merged after abdominal cavity impact immunity on the 4th day.
3. screening process：
The preparation of screening antigen：As described below, 25- hydroxyl VD3Amino extension is coupled on BSA, is coupled (BSA:
25- hydroxyl VD3) mass ratio 4:1. it is coupled centrifugation after terminating to go to precipitate.Collect supernatant and do screening antigen.
Experimental detail：
A.4mg BSA is dissolved in the 0.01M EMS of 500 μ l pH5.6, adds 4mgEDC (with 200 μ l pH5.6's
0.01M EMS dissolves), and 2mg NHS (being dissolved with the 0.01M EMS of 200 μ l pH5.6), activation 1h is stirred at room temperature.
B., after using 0.1M sodium hydroxide to adjust the BSA of NHS activation, 50 μ l0.1MPBS are added.It is added dropwise over while stirring
The 25- hydroxyl VD of 500 μ l DMSO dissolvings3Aminoderivative, is stirred at room temperature 3 hours.
C. use 0.01M PBS, change liquid three times.It is centrifuged after taking-up and must precipitate and supernatant.Precipitation is heavy with 200 μ l PBS
Outstanding.Precipitation and supernatant take out 5 μ l respectively and carry out SDS-PAGE race glue (referring to Fig. 2) identification, the only considerably less albumen of precipitation.On
Albumin is than the BSA albumen slightly disperse not being coupled it was demonstrated that being coupled.
D. supernatant is used as screening antigen.
When E. screening, with BSA-25- hydroxyl VD3Antigen and the BSA antigen not being coupled are coated elisa plate simultaneously and are sieved
Choosing, after the positive colony to BSA reaction for the exclusion, obtains 6 plants and BSA-25- hydroxyl VD3Antigen-reactive, responseless with BSA
Monoclonal cell strain.As produce anti-25- hydroxyl VD3The cell strain of antibody.
4. antibody purification is finally identified：
The 6 plants of positive colonies filtering out beat ascites, are purified into antibody and carry out ELISA and finally identify, with 25- hydroxyl VD3(contracting
Write 25VD3) competitive reaction, there are obvious Competitive assays, wherein No. 3 cell strain suppression are the most obvious.Use VD simultaneously3Reaction, no appoints
What competitive inhibitory effect.It is directed to 25- hydroxyl VD with showing 6 plants of cell high specials3, with VD3No cross reaction, and No. 3 cell strains
Most preferably.
Table 1, the affinity analysis of the antibody of 6 plants of positive colony generations
Note：Compare as Roche Products Vitamin D total.
Antibody dilution refers to the multiple being diluted the antibody of 1mg/ml by coubling dilution, wherein carries out for the first time
The antibody dilution of doubling dilution is 200.
5. the antibody purification of the present invention and vitamin D2Cross reaction
Experimental procedure：
BSA-25 hydroxyl VD3Screening antigen 4ug/ml, 100ul/ hole adds elisa plate, and 4 DEG C of degree are coated overnight.
TBST washs 3 times
2%BSA adds elisa plate, 200ul/ hole, 37 DEG C of degree closing 2h.
TBST washs 3 times
Configuration 25 hydroxyl VD are added respectively in elisa plate3Standard substance, 25 hydroxyl VD2Standard substance, and VD3Standard
Product.50ul/ hole.
The Anti-25VD having configured 0.2ug/ml is added respectively in elisa plate3- 3 antibody and control antibodies, 50ul/
Hole, mixes.
After 37 DEG C of degree reaction 45min, TBST washs 5 times
Every hole adds 100ul sheep anti-mouse igg-HRP (1：20000).
After 37 DEG C of degree reaction 45min, TBST washs 5 times
Every hole adds 50ul substrate A, 50ul substrate B, mixes
37 DEG C of degree reaction 5min
Every hole adds terminate liquid 50ul, OD450Read absorbance.
Crossing-over rate calculation：Antibody is in 25 hydroxyl VD3Standard substance 1/2 standard substance are absorbance when 0, corresponding mark
Quasi- product concentration or immediate concentration, with 25 hydroxyl VD2Reach the concentration proportion corresponding to same absorbance and be crossing-over rate.
Anti-25VD shown in following table3- 3 immediate absorbances reaching 1/2 × 3.056 are 1.079, corresponding standard concentration
For 50ng/ml, reach the 25 hydroxyl VD that same absorbance needs2Concentration be more than 1000ng/ml, therefore, 50ng/ml be more than
1000ng ratio is less than 5%.
Table 2 cross reaction result
Note：Compare as Roche Products Vitamin D total.
Table 2 shows：The 3rd strain antibody obtained by the present invention, to 25 hydroxyl VD3In reaction, specificity is high, with 25 hydroxyls
VD2Within intersecting at 5%, and and VD3No any cross reaction.Under equal conditions, control antibodies and 25 hydroxyl VD2Crossing-over rate
More than more than 70%, and and VD3Also there is certain intersection.
6. colloidal gold strip preparation：
With BSA-25- hydroxyl VD3Screening antigen 5mg/ml draws on NC film, after 10ug/ml antibody labeling nanogold particle
It is layered in gold pad, with the 25- hydroxyl VD having configured3Standard substance react, and with the increase of standard concentration, T line is also more shallow, or even
Disappear (disappear line).Wherein No. 3 antibody exhibits are optimal.
Conclusion：Prepare high-affinity, high special 25- hydroxyl VD3Antibody can be used for ELISA kit and gold colloidal
The production of test kit.
The biomaterial that the optimal technical scheme of reference the application describes the application in detail removes cell washing device, so
And, it should be noted that in the case of without departing from spirit herein, those skilled in the art can be in above disclosure
On the basis of make any transformation, modification and variation.The application includes above-mentioned specific embodiments and its any equivalents.
Claims (10)
1. a kind of preparation method of cell strain, wherein said cell strain produces anti-25-hydroxy-vitamin D3Monoclonal antibody, described
Method includes：
A) step using immunizing antigen immune animal, wherein said immunizing antigen is by making the macro-molecular protein as carrier
With as haptenic 25-hydroxy-vitamin D3It is coupled the water-insoluble antigen being obtained via crosslinked group, and described macromole
Protein and described 25-hydroxy-vitamin D3Coupling Mole Ratio be 1:80 to 1:100；
B) cell fusion step, makes the splenocyte of described animal and homology myeloma cell merge, and forms hybridoma；
C) using the step of screening antigen selection positive cell strain, wherein said screening antigen does not comprise described crosslinked group.
2. preparation method according to claim 1, wherein said macro-molecular protein is serum albumin.
3. preparation method according to claim 1 and 2, wherein said crosslinked group is polybasic carboxylic acid ester bond.
4. preparation method according to claim 1 and 2, wherein said screening antigen does not comprise polybasic carboxylic acid ester bond.
5. preparation method according to claim 1 and 2, wherein said screening antigen is macro-molecular protein and 25- hydroxyl
Vitamin D3The coupling molecule of aminoderivative.
6. use described immunizing antigen in preparation method according to claim 1 and 2, wherein step a) and be used for its point
From gel mixture direct immunization animal.
7. a kind of cell strain, the preparation method that it passes through according to any one of claim 1-6 obtains.
8. the anti-25-hydroxy-vitamin D of a kind of production3The method of monoclonal antibody, it is included according to any one of claim 1-6 institute
The preparation method stated is as step therein, or includes the step cultivating cell strain according to claim 7.
9. a kind of monoclonal antibody, itself and 25-hydroxy-vitamin D3Affinity be more than 3.84 × 109, and itself and 25- hydroxyl dimension
Raw element D2Cross reaction be less than 10%.
10. a kind of colloidal gold strip, it comprises monoclonal antibody according to claim 9.
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| CN109134657A (en) * | 2018-09-29 | 2019-01-04 | 宁波奥丞生物科技有限公司 | A kind of preparation method for antibody of 25-hydroxyvitamin D3 |
| WO2022022532A1 (en) * | 2020-07-28 | 2022-02-03 | 东莞市朋志生物科技有限公司 | Antibody capable of specifically binding to 25-hydroxyvitamin d, use thereof and diagnostic kit |
| CN113391076A (en) * | 2021-07-20 | 2021-09-14 | 深圳健丰源科技有限公司 | Immunodetection method of 25-hydroxy vitamin D and application thereof |
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