CN105112398A - Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody - Google Patents
Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody Download PDFInfo
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Abstract
The invention disclose preparation of hybridoma cell, a monoclonal antibody secreted by the hybridoma cell and application of the monoclonal antibody and belongs to the field of microbial engineering. During screening of fusion cells prepared with the hybridoma cell, an antigen conjugated with magnetic beads and capable of specifically combining with a nonspecific antibody generated in cell culture is utilized; therefore the inhibitory effect of the nonspecific antibody upon positive reactions during the screening of the fusion cells is eliminated, and preparation efficiency is effectively improved. The invention further discloses H-FABP (Heart fatty acid-binding protein) resistant hybridoma cell strain, a monoclonal antibody produced by the strain, and application of the monoclonal antibody. The strain is capable of stably and effectively secreting high-specific H-FABP resistant monoclonal antibodies; the antibodies are of high potency and are applicable to various branch fields of in vitro diagnosis, especially immunodiagnosis.
Description
Technical field
The present invention relates to the hybridoma preparation method of bioengineering field, monoclonal antibody and Application Areas, be specifically related to the preparation method of the hybridoma of anti-H-FABP monoclonal antibody and monoclonal antibody and application.
Background technology
The ultimate principle of hybridoma cell technology is the principal character simultaneously keeping both by merging two kinds of cells.These two kinds of cells are mouse spleen lymphocyte through antigen immune and murine myeloma cell respectively.The principal character of splenic lymphocyte is its antibody-secreting function and can grows in Selective agar medium, and murine myeloma cell then can divide without limit, propagation, i.e. so-called immortality under culture conditions.Under the effect of Selective agar medium, the hybrid cell of splenocyte and myeloma cell fusion is only had just to have the ability of continuous proliferation, form the cell possessing antibody-secreting function simultaneously and keep cell immortality two kinds of features, this hybrid cell is called hybridoma.
The preparation method of hybridoma generally includes animal immune and prepares splenocyte, the preparation of myeloma cell, the preparation of feeder cell, cytogamy, the screening of hybridoma and these steps of hybridoma cell clone.
Common cellular immune processes utilizes recombinant protein immune mouse, produce immune spleen cell, in this course, the aminoacid sequence of some recombinant protein has multiple epitope, panimmunity splenocyte can be produced, comprise the immunocyte of secreting specificity antibody and non-specific antibody, form hybridoma after these cells and myeloma cell fusion, 96 orifice plates enter the filtering hybridoma stage after cultivating.
Conventional screening method utilizes indirect elisa method to filter out positive hole in 96 orifice plates, and its principle utilizes the antibody produced in antigen and 96 orifice plates to produce specific immune response, then wait two to resist by colour developing to identify positive hole, complete screening process.But above-mentioned there is the recombinant protein immune mouse of multiple epitope after, the antibody that Hybridoma Cell Culture produces comprises specificity and non-specific antibody, when utilizing indirect elisa method to screen, non-specific antibody wherein also can occur to show positive immune response, this will cause only having non-specific antibody at some and do not have in the hole of specific antibody, the selection result also has positive false positive interference, and the accuracy of impact screening, hampers the efficiency of whole flow process.
The solubility plasmosin of cardic fatty acid binding protein (H-FABP) to be molecular weight be l4 ~ 15kd, mainly be distributed in cardiac muscular tissue, account for 4% ~ 8% of myocardial cell's soluble proteins, in skeletal muscle, uriniferous tubules, cerebral tissue, mammary gland, placenta tissue, also have a small amount of distribution.H-FABP forms acidic protein by 132 amino-acid residues, and its iso-electric point (PI) is 5, and its assignment of genes gene mapping is in No. 1 karyomit(e).
Under normal circumstances, longer chain fatty acid is the main energy sources of myocardial cell.H-FABP combines with intramyocardial longer chain fatty acid as the carrier proteins of lipid acid, it is transported from cytoplasmic membrane to plastosome, thus enters oxygenolysis in energy metabolism system, generates Triphosaden, for myocardial contraction provides energy.In addition, also participate in by-passing signal conduction: as passed through the transposition of lipid acid signal to peroxisome Proliferator-activated receptor indirect adjustments and controls genetic expression, and be considered to when myocardial ischemia causes local longer chain fatty acid to assemble, H-FABP has provide protection to cardiac muscle.
Under physiological condition, little containing H-FABP or content in blood plasma and urine, the H-FABP of lower concentration can be detected in normal human blood.Sex, age and diel rhythm all can cause the change of H-FABP content if male sex's muscle ratios is higher than women, and therefore male blood plasma H-FABP concentration is higher than women; In blood, H-FABP removes primarily of kidney, and blood plasma H-FABP concentration raised with the age.
H-FABP has very strong tissue specificity, is quickly released into blood after myocardial damage, and fall ill at acute myocardial infarction (AMI) starts to raise for 1 hour, and quick peaking, can as the biochemical marker of the myocardial damage early detection such as AMI.
At present, the clinical detection of blood plasma H-FABP mainly contains the methods such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunoturbidimetry.RIA detecting step is loaded down with trivial details, and there is radiocontamination, is not suitable for clinical application; ELISA complex operation, is not suitable for the detection of single sample; Immunoturbidimetry is by clinical full-automatic immunoturbidimetry analyser, and level of automation is high, but requires high to plant and instrument, professional person is needed to operate, therefore these methods are in simple, fast application model, all there is the drawback of can not ignore, and are especially not suitable for emergency treatment and spot inspection.Colloidal gold immunochromatographimethod technology belongs to instant detection (point-of-care-testing, POCT) mature technology in field, the qualitative detection realizing index that can be quick, sensitive, simple and direct, and this technology is just gradually to detection by quantitative development, be particularly suitable for the emergency treatment inspection of monocyte sample.H-FABP monoclonal antibody (monoclonal-antibody, mAb) be the starting material of most critical in diagnostic kit, no matter in which kind of immunologic detection method, all have use, its avidity and specificity directly determine the accuracy of H-FABP mono-detection kit clinical application result.Commercialization mAb is successfully prepared abroad, and domestic this mAb that there is no can be used successfully to clinical diagnosis.
Summary of the invention
The object of this invention is to provide a kind of preparation method of hybridoma, especially provide a kind of paramagnetic particle method to screen hybridoma, after getting rid of immunity, produce the method for the right screening interference of non-specific antibody.
Another object of the present invention is the hybridoma cell strain FABP4A8 and hybridoma cell strain FABP4C2 that utilize above-mentioned preparation method to prepare to secrete anti-H-FABP monoclonal antibody, the China typical culture collection center (CCTCC) being positioned at Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province is preserved on June 26th, 2015, deposit number is respectively CCTCCNO:C2015111 and CCTCCNO:C2015112, and prepares its monoclonal antibody.
Another object of the present invention is to provide the application of above-mentioned anti-H-FABP monoclonal antibody.
Concrete scheme is as follows:
A kind of preparation method of hybridoma, comprise animal immune, cytogamy, filtering hybridoma, hybridoma cell clone, it is characterized in that in filtering hybridoma process, get rid of the interference of non-specific antibody, select object cell and carry out cell clone, concrete steps are as follows: by non-specific antibody specific combination antigen coupled bead, adding 96 orifice plates after dilution is placed in above magnet, adsorption antigen ~ magnetic bead is bottom plate, add cell conditioned medium, non-specific antibody and antigen ~ magnetic bead are combined in bottom plate, get rid of its interference, determine positive hole again, carry out object cell clone and obtain hybridoma.
In animal immune process, immunogen has specific antigen epitope and heterogenetic antigen epi-position, produces the cell of secreting specificity antibody and the cell of secretion non-specific antibody after immunity.
Described non-specific antibody interference is due to when detecting positive hole, non-specific antibody and specific antibody all can produce positive reaction, only exist in the hole of non-specific antibody there is not specific antibody, detected result also judges into positive hole, make the mistake, disturb the judgement in normal positive hole.
Utilize hybridoma prepared by aforesaid method, for secreting the hybridoma of anti-H-FABP monoclonal antibody, called after hybridoma cell strain FABP4A8 and hybridoma cell strain FABP4C2 respectively, deposit number is CCTCCNO:C2015111 and CCTCCNO:C2015112.
When preparing this cell, with H-FABP recombinant protein for immunogen, aminoacid sequence table is SEQINNO:1, there is specific antigen epitope SEQINNO:2 and SEQINNO:3 and heterogenetic antigen epi-position SEQINNO:4, the cell interference cell screening of secretion non-specific antibody is generated after heterogenetic antigen epitopic immune, utilize described cell screening method exclusive PCR, complete screening and cell clone obtain object cell.
Described hybridoma cell strain secretes the monoclonal antibody of anti-H-FABP, called after 4A8 and 4C2, and monoclonal antibody hypotype is IgG1.
An application for monoclonal antibody as above, utilizes antibody 4A8 and/or 4C2 prepared to detect respectively and capture antibody, the double antibodies sandwich detection method of Criterion, detects the H-FABP in natural sera.
The application of a kind of monoclonal antibody as above in immunodiagnosis detection kit, comprise in chemiluminescence immunoassay kit, enzyme-linked immunologic detecting kit, colloidal gold immunoassay kit, fluorescence immunoassay kit, antibody all adopts described anti-H-FABP monoclonal antibody.
Beneficial effect
The interference that the present invention adopts magnetic bead coupled antigen eliminating non-specific antibody to select positive hole sizer in filtering hybridoma process, is different from traditional screening mode, simplifies screening process, improve process efficiency.Simultaneously the present invention is by the antibody screening of ripe, mass-producing and application platform; monoclonal antibody is prepared in independent research; for diagnostic reagent provides critical biomaterial, belong to domestic initiation, breach the heavy dependence situation of material inlet that FABP diagnostic reagent field faces, reagent entry port.Hybridoma cell strain Absorbable organic halogens, efficient secretion monoclonal antibody, through continuous passage culture in vitro, frozen, still well-grown after recovery.The antibody titer that the present invention prepares is all at 1:10
5above, after qualification, its specificity is high, can be applied in myocardial infarction early detection and diagnostic preparation and test kit and go.
Accompanying drawing explanation
Fig. 1 is for using hybridoma screening method of the present invention to containing specific antibody, non-specific antibody mixed system effect schematic diagram
Fig. 2 is for using hybridoma screening method of the present invention to bright and beautiful Chinese non-specific antibody system effect schematic diagram
Fig. 3 is that ELISA identifies that monoclonal antibody 4A8 detects H-FABP recombinant protein.The extension rate of antibody is respectively 1:10
3, 1:10
4, 1:10
5, 1:10
6, 1:10
7.
Fig. 4 is that ELISA identifies that monoclonal antibody 4C2 detects H-FABP recombinant protein.The extension rate of antibody is respectively 1:10
3, 1:10
4, 1:10
5, 1:10
6, 1:10
7.
Fig. 5 is monoclonal antibody 4A8 hypotype qualification result.
Fig. 6 is monoclonal antibody 4C2 hypotype qualification result.
Wherein, in Fig. 1,2, each mark representative is as follows: 1 is specific antibody secretory cell, and 2 is non-specific antibody secretory cell, and 3 is the antigen of the coupled bead of specific binding non-specific antibody, and 4 is specific antibody, and 5 is non-specific antibody.
Embodiment
For above and other object of the present invention, feature and advantage can be become apparent, lift preferred embodiment below, and coordinate accompanying drawing, be described in detail below.
Embodiment 1: prepare immune spleen cell
With the H-FABP recombinant protein of purifying for antigen, recombinant protein aminoacid sequence is as shown in SEQIDNO:1, this aminoacid sequence has and comprises specific antigen epitope SEQINNO:2 and SEQINNO:3 and heterogenetic antigen epi-position SEQINNO:4, ordinary method immunity BALB/c mouse in 6 week age, fundamental immunity subcutaneous injection of antigens 200 μ g first, uses Freund's complete adjuvant; Carried out primary immune response every 2 weeks, totally three times, 100 μ g//times, use Freund's incomplete adjuvant, last fundamental immunity is after 2 weeks, and docking detects anti-H-FABP antibody titers in mice serum, and serum antibody titer is reached 1:10
4the restructuring FABP antigen immune of 50 μ g/ml, as the source of immune spleen cell, is inoculated into mouse peritoneal by above mouse, and gets mouse spleen acquisition splenocyte after 3 days.
Embodiment 2: the preparation of hybridoma
(1) preparation of feeder cell
Using BALB/c mouse peritoneal macrophage as feeder cell, its preparation method is: get BALB/c mouse, draws neck to put to death mouse, with 75% alcohol body surface sterilization, cut tweezer with sterilization and start skin of abdomen from rear abdomen, expose peritonaeum, with 75% cotton ball soaked in alcohol wiping peritonaeum sterilization.With injector to inject 10mlDMEM nutrient solution to abdominal cavity, repeatedly rinse, reclaim washing fluid, centrifugal 10 minutes of 1000r/min, abandons supernatant, obtains feeder cell.By the resuspended precipitation of DMEM nutrient solution containing 15% calf serum, adjustment cell concn is 2 × 10
5individual/ml.Above-mentioned cell suspension is added 96 orifice plates, and every hole 0.1ml, puts 37 DEG C, 5%CO
2incubator in overnight incubation, next day is for subsequent use.
(2) preparation of immune spleen cell
Get the BALB/c mouse after immunity, to be dislocated lethal mouse by neck, to be soaked in 75% alcohol 5 minutes, take out spleen under aseptic condition, be placed in plate, DMEM nutrient solution cleans 1 time.Spleen being moved into another fills in the plate of 10mlDMEM nutrient solution, splenocyte is made to enter nutrient solution, after spleen is ground to form cell suspension, make cell dispersal even for several times with suction pipe piping and druming, filter, results splenocyte suspension, centrifugal 10 minutes of 1000r/min, washs 2 times with DMEM medium centrifugal, then that cell is resuspended, counting, for cytogamy.
(3) enlarged culturing of myeloma cell
In first 36 ~ 48 hours of fusion, by myeloma cell's enlarged culturing, merge the same day, with connector bend dropping tube, cell is blown down gently from bottle wall, be collected in 50ml centrifuge tube, centrifugal 10 minutes of 1000r/min, supernatant discarded, add the incomplete substratum of 30ml, with method centrifuge washing once, then by Cell resuspension in the incomplete substratum of 10ml, mixing, get myeloma cell's suspension, counting, for cytogamy.
(4) immune spleen cell and myeloma cell fusion
Myeloma cell is mixed in 1: 5 ratio with immune spleen cell, 1 time is washed, 1000r/min with DMEM nutrient solution, centrifugal 10min in 50ml centrifuge tube, abandon supernatant, with the careful sucking-off residual liquid of dropper, palm touches at the bottom of fusion pipe, make sedimentation cell evenly loose, put preheating in 40 DEG C of water-baths, in 45 seconds, add 50%PEG (PH8.0) 1ml being preheated to 40 DEG C, then slowly drip DMEM nutrient solution to 10ml, effect 10min.1000r/min, centrifugal 5min, supernatant discarded, adds 5mlHAT substratum, gently pressure-vaccum sedimentation cell, makes it suspend and mixes, and then adds HAT substratum to 80ml, and packing 96 porocyte culture plate, every hole 0.1ml, then puts 37 DEG C by culture plate, 5%CO
2cultivate in incubator, to swap out 1/2 substratum with HAT substratum after 24 hours, after 4 days, change liquid completely, within two weeks, to swap out HAT substratum with HT substratum afterwards, then after maintaining two weeks, use RPMI1640 nutrient solution instead.
The compound method of HT substratum: 100 times of concentrated HT liquid (1mL) and 100 times of concentrated glutamine (1mL) are diluted into DMEM substratum.
The compound method of HAT substratum: 100 times of concentrated HT liquid (1mL), 100 times of concentrated A liquid (1mL) and 100 times of concentrated glutamine (1mL) are diluted into DMEM substratum (97mL).
The compound method of 100 times of concentrated HT liquid: take 136.1mg xanthoglobulin, 38.8mg thymidine, be successively dissolved in 100ml distilled water, filtration sterilization, packing, in-20 DEG C of preservations.
The compound method of 100 times of concentrated A liquid: take 1.76mg aminopterin-induced syndrome, adds 80ml distilled water and adds 1mol/L sodium hydroxide solution to dissolving completely, then adds 1mol/L hydrochloric acid by pH modulation 7.0, then 100mL is settled to, filtration sterilization, packing, in-20 DEG C of preservations.
The compound method of 100 times of concentrated glutamine: take 2.92gL-glutamine and be dissolved in 100mL distilled water, filtration sterilization, packing, in-20 DEG C of preservations.
(5) screening of hybridoma
Hybridoma cell fusion is after two weeks, observe the growing state of hybridoma, when clone grows to 1/3 ~ 1/2 of hole floorage, get supernatant, first get rid of the interference of non-specific antibody, then detect with indirect ELISA method: by H-FABP recombinant antigen coated elisa plate, antigen concentration is 3 μ g/ml, hatch 1h at 37 DEG C, PBST washes 2 times, pats dry; Then use the PBS (tween content is 0.05%) containing 1% gelatin to close 1h at 37 DEG C, every hole 150 μ l, PBST washes 3 times, pats dry, and-20 DEG C save backup.During detection, cell conditioned medium is added in hand-hole, every hole 100 μ l, and establish feminine gender and positive control, hatch 1h at 37 DEG C, PBST washes 5 times, add the anti-100 μ l of sheep anti mouse two of mark HRP, hatch 30min for 37 DEG C, PBST washes 5 times, add nitrite ion, stop after 10min, put into microplate reader reading, OD value carries out subclone higher than the positive hole of more than 2.0.
The method of above-mentioned eliminating non-specific antibody interference is: can with the antigen coupled bead of non-specific antibody specific combination, after antigenic solution be diluted to 1 μ g/ml add in 96 orifice plates of culturing cell, cell plate are placed in above magnet, 37 DEG C of effect 30min make the magnetic bead having adsorbed antigen be fixed on bottom cell plate, add cell conditioned medium, the non-specific antibody contained is combined with antigen ~ magnetic bead, get rid of its interference effect, detect through indirect ELISA again, determine positive hole, the clone carrying out positive porocyte obtains hybridoma.
Composition graphs 1 and Fig. 2 make an explanation to screening method of the present invention, as shown in Figure 1, wherein a, b are the hybridoma indirect ELISA screening schematic diagram not using screening method of the present invention, cell (2) wherein containing the cell (1) and non-specific antibody (5) that produce specific antibody (4) in b, being extracted out by supernatant in b carries out in indirect ELISA process, because specific antibody (4) and non-specific antibody (5) all produce positive reaction, can only judge that there is the positive in this hole, can not determine that this hole necessarily exists specific cell strain; C, d are the experimental group utilizing screening method of the present invention, wherein contain specific antibody (4) and non-specific antibody (5) in c, first the antigen (3) connecting magnetic bead is added wherein, (3) specificly can combine with the non-specific antibody in system and attracted bottom reacting hole by magnetic bead, when sucking-off supernatant carries out ELISA detection, this non-specific antibody can not be sucked out, thus ensure if there is positive reaction then in this hole, certainly there is the object cell producing specific antibody (4).
For further explanation, as shown in Figure 2, a, b, c, d are the system only containing non-specific antibody, and a, b do not use the present invention to screen system, occur positive reaction, so just cause the misjudgment selected for positive hole sizer when doing indirect ELISA and detecting; And for example shown in c, d, the present invention is used to screen system, due to add can with the antigen (3) of the connection magnetic bead of non-specific antibody (5) specific binding, non-specific antibody (5) is adsorbed on bottom hole by it, positive reaction would not be there is when sucking-off supernatant does indirect ELISA, effectively avoid the erroneous judgement that positive hole sizer is selected, improve screening efficiency.
(6) cloning of hybridoma and frozen
A) clone of hybridoma
1. getting (1) small mouse peritoneal macrophage is feeder cell.
2. prepare hybridoma suspension to be cloned, be diluted to every milliliter with the HT substratum containing 20% serum and contain the different extent of dilution of 3 kinds, 5,10 and 20 cells.
3. 5 × 10 are added by every milliliter
4~ 1 × 10
5the ratio of cell, adds peritoneal macrophage respectively in above-mentioned hybridoma suspension.
4. often kind of hybridoma packing 96 orifice plate one piece, every hole amount is 100 μ l.
5. 37 DEG C, 5%CO2 cultivates 6 days, occurs macroscopic clone and detectable antibody; Observe under inverted microscope, mark the hole only having single clonal growth, get supernatant and do antibody test.
6. the cell expansion getting the positive hole of antibody test is cultivated, and frozen.
B) hybridoma is frozen
Cells frozen storing liquid: 20% calf serum+70%DMEM nutrient solution+10% dimethyl sulfoxide (DMSO).
Hybridoma is centrifugal, and Eddy diffusion is in the frozen storing liquid of precooling, and concentration is 10
6~ 10
7/ ml, is transferred in cryopreservation tube, every bottle of 1ml, and be placed in-70 DEG C of refrigerators, next day proceeds in liquid nitrogen.
Embodiment 3: the preparation of monoclonal antibody
Cultivate the hybridoma obtained in above-mentioned (6) with serum free medium, carry out purifying by proteinA affinity column chromatography method and obtain a small amount of antibody.
In the present invention, the scheme of a large amount of manufacture order clonal antibody is mouse ascites method, and step is as follows:
The hybridoma of above-mentioned acquisition is inoculated in mouse peritoneal by this programme, in mouse peritoneal, grow hybridoma, and produces ascites, obtains a large amount of ascites monoclonal antibodies.
Concrete grammar is: BALB/c mouse intraperitoneal inoculation whiteruss, every mouse 0.5ml, after two weeks, and the hybridoma that intraperitoneal inoculation dilutes with serum free medium, every mouse 1 × 10
6individual hybridoma, every day, after 5 days, is observed mouse ascites production in interval, treats that mouse web portion obviously expands as seen, repeatedly extracts ascites several times.Room temperature leaves standstill 30min, 1000r/min, centrifugal 10min, and collect supernatant, packing ,-70 DEG C frozen for subsequent use.
Embodiment 4: anti-human H-FABP is monoclonal antibody-purified
The ascites monoclonal antibody of acquisition is carried out purifying by proteinA affinity column chromatography method, and concrete steps are:
Balance: balance chromatography column with the level pad (20mMPB+0.15MNaCl, pH7.0) of 5 ~ 10CV, to effluent liquid conductance constant with pH (consistent with balance liquid);
Upper prop: sample buffer should be consistent with balance liquid as far as possible, solid sample can dissolve preparation with balance liquid, low concentration sample solution can be dialysed with balance liquid, enriched sample solution can dilute with balance liquid, in order to avoid blocking chromatography column, sample should process through centrifugal or micro-filtration (0.45 μm), and inlet amount is according to the cubage of target protein in the carrying capacity of medium and feed liquid;
Washing: after loading continuation with level pad drip washing to baseline;
Wash-out: with elution buffer (20mM sodium acetate, pH3.0 ~ 4.0 or 0.1M glycine, pH3.0) wash-out, collect effluent liquid, after wash-out, should use ealkaline buffer (as 1MTris/HCl, pH9.0) that the antibody-solutions collected is neutralized neutrality at once, avoid antibody inactivation, maintain the biological activity of antibody.
Regeneration, situ cleaning: after medium uses for several times (5 ~ 10 times), need to regenerate medium, situ cleaning:
A) with the acetic acid of 0.1M or the acetic acid/20% ethanol rinse pillar 3 ~ 5CV of 0.1M, then neutrality and reusable is washed with damping fluid;
B) also can with 0.05MNaOH+1MNaCl or 6M Guanidinium hydrochloride drip washing pillar 3 ~ 5CV, and by the pure water rinsing of 3 ~ 10CV, then wash neutrality and reusable with damping fluid.
Embodiment 5:ELISA identifies anti-H-FABP antibody titer
Tiring of H-FABP monoclonal antibody is measured with indirect elisa method.Envelope antigen is H-FABP recombinant protein (5 μ g/ml), every hole 100 μ l, and negative control is PBS damping fluid, and antibody concentration carries out 10 times of gradient dilutions to 1:10 from 1:1000
7.As shown in Figure 3,4, anti-H-FABP monoclonal antibody 4A8 and anti-the tiring of H-FABP monoclonal antibody 4C2 of detecting purifying are 1:10 to result
5above.
Embodiment 6: anti-H-FABP pairing antibody screening
The present invention is with double antibody sandwich ELISA preliminary screening H-FABP pairing antibody pair.Be coated on enzyme plate by antibody 4A8, wrapping by concentration is 5 μ g/ml, 0.05mol/LPBS bag quilt, every hole 100 μ l, and hatch 1h for 37 DEG C, PBST washes 2 times, pats dry; Then the PBS (tween content is 0.05%) containing 1% gelatin is used to close 1h at 37 DEG C, every hole 150 μ l, PBST washes 3 times, pat dry, add biotin labelled antibodies 4C2 and H-FABP recombinant antigen, every hole 100 μ l, hatch 1h for 37 DEG C, PBST washes 3 times, pats dry, and adds streptavidin enzyme every hole 100 μ l, hatch 30min for 3737 DEG C, PBST washes 5 times, pats dry, and adds nitrite ion, color development stopping after 10min, microplate reader reading, OD value is lower, illustrates that the antigen recognition epi-position between two strain antibodies is more close; OD value is higher, and the antigen associative list phasic difference of both explanations is far away.Experimental result OD value is very high, and show that 4A8 antibody and antibody 4C2 are different antibody, the epitope of their identification is different.Meanwhile, this result shows, monoclonal antibody 4A8 and 4C2 pairing can be used for developing the diagnostic reagent detecting H-FABP.
Embodiment 7: anti-H-FABP monoclonal antibody hypotype qualification
Use sigma antibody subtype identification kit (article No. is ISO2) qualification, the hypotype of result anti-H-FABP monoclonal antibody 4A8 and 4C2 is as shown in Figure 5,6 IgG1.
Claims (8)
1. the preparation method of a hybridoma, comprise animal immune, cytogamy, filtering hybridoma, hybridoma cell clone, it is characterized in that in filtering hybridoma process, get rid of the interference of non-specific antibody, select object cell to clone, concrete steps are as follows: by non-specific antibody specific combination antigen coupled bead, adding 96 orifice plates after dilution is placed in above magnet, adsorption antigen ~ magnetic bead is bottom plate, add cell conditioned medium, non-specific antibody and antigen ~ magnetic bead are combined in bottom plate, get rid of its interference, determine positive hole again, carry out object cell clone and obtain hybridoma.
2. preparation method according to claim 1, is characterized in that in animal immune process, and immunogen has specific antigen epitope and heterogenetic antigen epi-position, produces the cell of secreting specificity antibody and the cell of secretion non-specific antibody after immunity.
3. preparation method according to claim 1, it is characterized in that described non-specific antibody interference is due to when detecting positive hole, non-specific antibody and specific antibody all can produce positive reaction, only exist in the hole of non-specific antibody there is not specific antibody, detected result also judges into positive hole, make the mistake, disturb the judgement in normal positive hole.
4. utilize hybridoma prepared by the method described in claim 1, for secreting the hybridoma of anti-H-FABP monoclonal antibody, called after 4A8 and 4C2 respectively, deposit number is CCTCCNO:C2015111 and CCTCCNO:C2015112.
5. a hybridoma as claimed in claim 4, when it is characterized in that preparing this cell, with H-FABP recombinant protein for immunogen, aminoacid sequence table is SEQINNO:1, there is specific antigen epitope SEQINNO:2 and SEQINNO:3 and heterogenetic antigen epi-position SEQINNO:4, after heterogenetic antigen epitopic immune generate secretion non-specific antibody cell interference cell screening, utilize described cell screening method exclusive PCR, complete screening and cell clone obtain object cell.
6. hybridoma cell strain according to claim 4 secretes a monoclonal antibody of anti-H-FABP, called after 4A8 and 4C2, and monoclonal antibody hypotype is IgG1.
7. an application for monoclonal antibody according to claim 6, utilizes antibody 4A8 and/or 4C2 prepared to detect respectively and capture antibody, the double antibodies sandwich detection method of Criterion, detects the H-FABP in natural sera.
8. the application in a monoclonal antibody immunity diagnostic test kits according to claim 6, comprise in chemiluminescence immunoassay kit, enzyme-linked immunologic detecting kit, colloidal gold immunoassay kit, fluorescence immunoassay kit, antibody all adopts described anti-H-FABP monoclonal antibody.
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| CN105936892A (en) * | 2015-03-06 | 2016-09-14 | 艾博生技抗体股份有限公司 | Method for screening hybridoma cells with antigen specificity |
| CN105936892B (en) * | 2015-03-06 | 2019-11-22 | 艾博生技抗体股份有限公司 | The method of screening tool antigenic specificity hybridoma cell |
| CN105606596A (en) * | 2016-01-26 | 2016-05-25 | 河南生生医疗器械有限公司 | Kit for chemiluminiscent immunodetection of brain fatty acid binding protein and preparation method thereof |
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| CN107119023B (en) * | 2016-12-27 | 2018-01-12 | 南京诺唯赞医疗科技有限公司 | The monoclonal antibody of the anti-human cardic fatty acid binding protein of resistance to aliphatic acid interference and its application |
| CN109504661A (en) * | 2017-09-15 | 2019-03-22 | 上海开拓者生物医药有限公司 | A kind of T lymphocyte hybridoma of antigentic specificity and its preparation method and application |
| CN109504661B (en) * | 2017-09-15 | 2022-04-26 | 上海昀怡健康科技发展有限公司 | Antigen-specific T lymphocyte hybridoma and preparation method and application thereof |
| CN110218703A (en) * | 2018-03-02 | 2019-09-10 | 南京大学 | A kind of antigen-specific b cells screening technique and its application in monoclonal antibody preparation |
| CN108531459B (en) * | 2018-03-30 | 2020-08-11 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
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| CN110016462A (en) * | 2019-02-20 | 2019-07-16 | 优睿赛思(武汉)生物科技有限公司 | The method of single antigen-specific b lymphocyte is efficiently separated from spleen cell |
| CN110016462B (en) * | 2019-02-20 | 2020-04-24 | 优睿赛思(武汉)生物科技有限公司 | Method for efficiently separating single antigen-specific B lymphocytes from spleen cells |
| CN113892463A (en) * | 2021-09-24 | 2022-01-07 | 北京艾德摩生物技术有限公司 | Peritoneal irrigation fluid tumor model for screening peritoneal and pelvic malignant tumor drugs, construction method and application |
| CN113892463B (en) * | 2021-09-24 | 2022-11-25 | 北京艾德摩生物技术有限公司 | Peritoneal irrigation fluid tumor model for screening peritoneal and pelvic malignant tumor drugs, construction method and application |
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