CN102109515A - Preparation method and using method of nodularin (NODLN) polyclonal antibody immunoaffinity column (IAC) - Google Patents
Preparation method and using method of nodularin (NODLN) polyclonal antibody immunoaffinity column (IAC) Download PDFInfo
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Abstract
The invention relates to a preparation method and a using method of a nodularin (NODLN) polyclonal antibody immunoaffinity column (IAC), and belongs to the technical field of immunoaffinity chromatography and algae toxin detection. In the prepared IAC, an NODLN polyclonal antibody is fixed in the column to adsorb NODLN in sample solution, enrichment and purification effects are achieved after elution, and qualitative and quantitative analysis is performed through high performance liquid chromatography (HPLC); in the pretreatment process, IAC enrichment is superior to the conventional solid-phase extraction (SPE) method; an HPLC graph shows that the NODLN polyclonal antibody IAC can separate the peak of the NODLN from the peak of other impurities so as not to influence a measurement result, and excessive impurities exist in SPE purification, so that the peak of the NODLN is even difficult to distinguish; and the NODLN polyclonal antibody can be specifically combined with the NODLN, so that the IAC has high specificity, and most interfering substances can be removed through one-time purification, while the SPE does not have specificity, and the interfering substances cannot be removed, so that the final measurement result is influenced.
Description
Technical field
A kind of preparation and using method that saves ball algae toxin polyclonal antibody immunoaffinity column belongs to immunoaffinity chromatography and joint ball algae toxin detection technique field.
Background technology
Human in recent years because produce, the development of life activity, a large amount of discharge waste waste water in water body make body eutrophication, algal bloom, wherein the quantity of poisonous algae also sharply increases.Lakes such as potable water seedbed of China such as Taihu Lake, Chaohu, Dian Chi, some reservoirs, river etc. all are subjected to the invasion and attack of blue-green alga bloom." the Changjiang river protection and the development report " of in April, 2007 issue claims, after the reservoir area of Three Gorges retaining to 135 in 2003 meter, the wawter bloom phenomenon appears in 12 the Changjiang river one-level tributary to some extent in the backwater zone, and the trend of aggravation was arranged in recent years.
Behind the toxic action of Francis reported first joint ball algaes in 1878 to livestock.All there is algal bloom to cause the report of domestic animal and wild animal death and crowd's incidents such as hepatic lesion, gastroenteritis, diarrhoea and dermatitis all over the world.
Joint ball algae toxin (hereinafter to be referred as NODLN) is to be released to one of toxin in the water after blue-green alga bloom rots.NODLN is a ring-type pentapeptide hepatotoxin, is the inhibitor of serine/threonine protein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A).Up-to-date studies show that, NODLN is tumor initiator and promoter simultaneously, and microcapsule algae toxin (hereinafter to be referred as MC-LR) only is a TPA.Because MC-LR toxicity is better than NODLN, so MC-LR research is more, and limiting the quantity of of MC-LR is 1 μ g/L in WHO and China GB 5749-2006 drinking water sanitary standard regulation potable water.And the limit standard of NODLN is not arranged also in the potable water.In view of the short function of tumor of NODLN is better than MC-LR, the limit standard of NODLN should be lower than MC-LR in the potable water.
Immune affinity column (IAC) is the compartment analysis prepacked column that utilizes the immunoaffinity chromatography principle to make.Because antibody and antigenic action have the height selectivity, therefore purify and to remove most impurity by IAC.The IAC technology is day by day ripe, with the detection of mycotoxin like the algae endotoxin in widespread use, wherein the immunoaffinity chromatography of aflatoxin has been put into (GB18980-2003 and GB18979-2003) in the GB.
Toxicity and the short function of tumor of NODLN also do not cause enough attention at present, and detection method only sees the foreign language report.Our early-stage Study shows, available HPLC of the detection of NODLN and LC-MS method in the water, because of content is low, before sample introduction, generally use solid-phase extraction column (SPE) enrichment, but SPE does not have specificity, impurity is many in the water, can introduce many impurity, interference detection results after the enrichment, HPLC method particularly, measured value and actual value greatly differ from each other sometimes.Though LC-MS method detectability is low, measurement result is more accurate, costs an arm and a leg, and common laboratory does not have.
The IAC that utilizes the present invention to develop can remove most impurity by primary purification, directly advances HPLC and just can obtain result accurately, can satisfy the demand of the daily monitoring of NODLN.
Summary of the invention
The preparation and the using method that the purpose of this invention is to provide a kind of NODLN polyclonal antibody immunoaffinity column, the IAC that is developed is how anti-being fixed in the post with NODLN, thereby the NODLN in the adsorption sample solution plays enrichment and purification effects behind the wash-out, advances HPLC then and carries out qualitative and quantitative analysis.As the pre-treatment process, the IAC enrichment is better than existing SPE method.
Technical scheme of the present invention: a kind of preparation method of NODLN polyclonal antibody immunoaffinity column:
(1) with NODLN and bovine serum albumin(BSA) (BSA) coupling, the conjugate that obtains (hereinafter to be referred as NODLN-BSA) is as artificial antigen, and the rabbit epidemic disease obtains the NODLN antiserum, and how anti-adopt two step of saturated ammonium sulfate precipitation method purifying to obtain NODLN again;
(2) activation of Ago-Gel Sepharose 4B: with cyanogen bromide method activated agarose gel Sepharose 4B, reactivation process is as follows:
(a) get 10mL Sepharose 4B and be placed in the Buchner funnel and drain,, add the NaHCO of a spot of 0.1mol/L pH 8.3 with draining after twice washing of 30mL moisture
3Washing changes in the 100mL beaker immediately, and ice bath slowly stirs down;
(b) take by weighing the 1g cyanogen bromide in fuming cupboard, add water 10mL dissolving, add then among the Sepharose 4B in batches, the limit edged stirs, survey the pH value simultaneously,, make pH remain on 10.5 by dripping 2mol/LNaOH, treat that cyanogen bromide reaction is complete, pH remains unchanged, and stops to stir;
(c) the Sepharose 4B with activation adds ice cube, pours into rapidly in the Buchner funnel, takes out with frozen water and washes neutrality, the rapid again NaHCO with the cold 0.1mol/L pH 8.3 of 150mL
3Take out and wash;
(3) preparation of NODLN polyclonal antibody immunoaffinity column:
NODLN resists with Ago-Gel Sepharose 4B coupling more obtains affinity adsorbent, inserts and makes the NODLN polyclonal antibody immunoaffinity column in the post, and operation steps is as follows:
(d) coupling
The NODLN that above-mentioned preparation purifying is good resists more places pH8.3 to contain the 0.1M NaHCO of 0.5M NaCl
3Coupling buffer in the 12h that dialyses; The Sepharose 4B that above-mentioned activation is good places sand core funnel to take out fast with coupling buffer and washes, and pours into rapidly then in the how anti-solution of NODLN and carries out coupling, UV scanning monitoring coupling process; The NODLN of coupling is how not anti-with the coupling buffer flush away more than 5 times of how anti-liquor capacities of NODLN, obtains agarose-antibody coupling compound; Collect whole eluents, calculate the amount that NODLN resists in the not coupling more by measuring its Protein content;
(e) sealing reactive group
Agarose-antibody coupling compound is changed in the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl of 5 times of volume pH8.0, keep 2h, with unnecessary reactive group in the sealing Ago-Gel;
(f) washing
The agarose that step (e) obtains-antibody coupling compound is successively with the 0.1M Tris-HCl damping fluid washing that contains 0.5M NaCl of the pH8.0 of the 0.1M acetate buffer solution that contains 0.5M NaCl of the pH4.0 of 5 times of coupled complex volumes and 5 times of coupled complex volumes, this washing process triplicate; Use the PBS washed twice of 5 times of coupled complex volumes then;
(g) preservative treatment
The agarose that step (f) prepares-antibody coupling compound such as need are placed a period of time, should use the PBS damping fluid that contains 1/10000 Sodium azide to soak, and drop removes to put into after the excessive solution packaging bag or bottle 4 ℃ of airtight preservations then;
(h) dress post
Step (f) or agarose-antibody coupling compound of (g) obtaining are packed in 2mL or the 5mL Solid-Phase Extraction void column pipe, under negative pressure, gel are compressed, promptly make the how anti-IAC of NODLN with PBS;
(i) preserve
Agarose-antibody coupling compound or the populated how anti-IAC of NODLN are sure not freezing, are kept in 4 ℃ the refrigerator;
(j) regeneration of post
After post uses, the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl with 4-10 bed volume pH8.5, clean IAC twice in turn with the 0.1M sodium-acetate buffer that contains 0.5M NaCl with 4-10 bed volume pH4.5, again with being kept at behind the abundant balance IAC of PBS damping fluid in 4 ℃ of refrigerators for using next time.
With the how anti-IAC of NODLN of described method preparation, be applied to the mensuration of NODLN in the actual water sample.Getting 0.2L is subjected to the water sample of blue-green algae pollution earlier by 0.45 μ m filtering with microporous membrane; IAC uses 5mL methyl alcohol, 5mL distilled water, 5mL PBS pre-service earlier successively; Filtrate is crossed the how anti-IAC of pretreated NODLN; Use 5mL PBS, 5mL distilled water, 5mL 10% methyl alcohol drip washing impurity more successively; Use the pure methanol-eluted fractions of 5mL at last; The eluent evaporated under reduced pressure, residue dissolves laggard HPLC analysis with 0.2mL PBS; Do control experiment with SPE simultaneously;
The condition determination of HPLC: instrument is that Waters 600 high performance liquid chromatographs cooperate Waters 996 diode array detector; Chromatographic column is Kromasil 100-5C
18, 150 * 4.6mm, 5 μ m; Moving phase is the deionized water that contains 0.05% formic acid: the acetonitrile volume ratio is 60: 40, and flow velocity is 1mL/min; Column temperature is 30 ℃; Sample size 20 μ L; The detection wavelength is 240nm.
Beneficial effect of the present invention: the NODLN polyclonal antibody immunoaffinity column can separate NODLN with other impurity, makes the unlikely measurement result that influences of impurity, and general SPE purifies because impurity is too many, makes NODLN be difficult to accurate mensuration.Because how anti-NODLN can specificity combine with NODLN make IAC that excellent specificity be arranged, primary purification can be removed most chaff interferences.And SPE does not have specificity, can not remove chaff interference, can influence last measurement result.
Embodiment
NODLN and bovine serum albumin(BSA) (BSA) conjugate is hereinafter to be referred as NODLN-BSA; NODLN and ovalbumin (OVA) conjugate is hereinafter to be referred as NODLN-OVA.
The preparation that embodiment 1:NODLN resists more
1, comlete antigen is synthetic
(1) NODLN-BSA is immunogenic synthetic
The synthetic employing water-soluble carbodiimide method of comlete antigen NODLN-BSA, utilize 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) as " bridging agent ", make the carboxyl of NODLN (COOH) generate intermediate product with the EDC reaction, again with the BSA protein molecular on amino reaction, make comlete antigen NODLN-BSA conjugate as immunogene.Synthetic method is as follows:
0.4mg NODLN is dissolved in 0.5mL methyl alcohol, is divided into two equal portions of 0.25mL, logical nitrogen evaporates into dried under 35 ℃.The residue that portion contains 0.2mg NODLN is dissolved in 1mL PBS (pH7.4) damping fluid, adds 5mg EDC, and vibration transfers to 5 with the 0.1M hydrochloric acid solution with the pH value to dissolving fully, slowly stirs 5min under the room temperature.1.4mg BSA is dissolved in the 3mL PBS damping fluid, and vibration dropwise adds in the above-mentioned NODLN solution then to dissolving fully, at room temperature keeps 1h.Spend the night under 4 ℃ then.With the potpourri 72h that in the PBS at 0.1mol/LpH7.4 under 4 ℃, dialyses, change dislysate 1 time every 6h.After the freeze drying in-20 ℃ of preservations.
(2) NODLN-OVA detects the synthetic of antigen
Finish the crosslinked of NODLN and carrier protein OVA with mixed anhydride method, synthetic NODLN-OVA comlete antigen is as detecting antigen.Synthetic method is as follows:
The residue that portion contains 0.2mg NODLN adds 0.5mL 1,4-dioxane, and vibration makes its dissolving, adds 7 μ L isobutyl chlorocarbonates and 12 μ L tri-n-butylamines again, and the slight vibration of this potpourri is kept 30min down at 4 ℃ then.0.9mg OVA is dissolved in the 3mL sodium bicarbonate buffer liquid (pH 9.0).Above-mentioned NODLN solution is dropwise joined among the OVA of dissolving, and in 4 ℃ of gentle agitation 4h, whole process is kept pH 9.0.Then, potpourri in the 72h that dialyses under 4 ℃, is changed dislysate 1 time every 6h in the PBS of 0.1mol/L pH7.4.After the freeze drying in-20 ℃ of preservations.
2, many anti-preparation and purifying of NODLN
As immunogene, immunity obtains the NODLN antiserum with synthetic NODLN-BSA.
The antiserum that adopts two step of saturated ammonium sulfate precipitation method purifying to obtain, operate as follows:
(1) get the 10mL antiserum and mix with isopyknic PBS (pH7.4), dropwise add the 10mL saturated ammonium sulfate under stirring, 4 ℃ are spent the night, and immunoglobulin (Ig) is fully precipitated.
(2) the centrifugal 20min of 4000g, supernatant discarded.With 10mL PBS dissolution precipitation, dropwise add 5mL saturated ammonium sulfate solution again, 4 ℃ are spent the night.
Repeat the second step process once.With the centrifugal gained sediment of the 10mL PBS dissolving last bag filter of packing into.Three days abundant desalinations (changing liquid every day 4 times) of 4 ℃ of dialysis.How anti-be NODLN after the protein freeze drying of dialysis purifying, it is kept under-20 ℃.
Embodiment 2: the activation of Ago-Gel Sepharose 4B
With cyanogen bromide method activated agarose gel Sepharose 4B.The cyanogen bromide method activation effect is good, the influence of coupling rate height, antagonist is little, and reactivation process is as follows:
(1) gets 10mL Sepharose 4B and be placed in the Buchner funnel and drain,, add the NaHCO of a spot of 0.1mol/LpH8.3 with draining after twice washing of 30mL moisture
3Washing changes in the 100mL beaker immediately, and ice bath slowly stirs down.
(2) take by weighing the 1g cyanogen bromide in fuming cupboard, add water 10mL dissolving, pour into then in the agarose in batches, the limit edged stirs, and surveys the pH value simultaneously, by dripping 2mol/L NaOH, pH is remained on about 10.5.Treat that the cyanogen bromide complete reaction is complete, pH remains unchanged substantially, can stop to stir.
(3) the Sepharose 4B with activation adds ice cube, pours into rapidly in the Buchner funnel, takes out with frozen water and washes neutrality, the rapid again NaHCO with the cold 0.1mol/L pH 8.3 of 150mL
3Take out and wash.
The preparation of the how anti-IAC of embodiment 3:NODLN
NODLN resists with Ago-Gel Sepharose 4B coupling more obtains affinity adsorbent, inserts and makes the how anti-IAC of NODLN in the post, and operation steps is as follows:
(1) coupling
The NODLN that above-mentioned preparation purifying is good resists more places pH 8.3 to contain the 0.1M NaHCO of 0.5M NaCl
312h dialyses in the coupling buffer of damping fluid.The Sepharose 4B gel that above-mentioned activation is good places sand core funnel to take out fast with coupling buffer and washes, pour into rapidly then in the how anti-solution of NODLN and carry out coupling, UV scanning monitoring coupling process.The anti-NODLN of coupling is how not anti-with 5 times of coupling buffer flush awaies more than the volume, obtains agarose-antibody coupling compound.Collect whole eluents, calculate the amount that NODLN resists in the not coupling more by measuring its Protein content.
(2) sealing reactive group
Agarose-antibody coupling compound is changed in the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl of 5 times of volume pH 8.0, keep 2h, with unnecessary reactive group in the sealing Ago-Gel.
(3) washing
The agarose that step (2) obtains-antibody coupling compound is with the 0.1M Tris-HCl damping fluid washing that contains 0.5M NaCl of the pH 8.0 of the 0.1M acetate buffer solution that contains 0.5M NaCl of the pH 4.0 of 5 times of coupled complex volumes and 5 times of coupled complex volumes, this washing process triplicate.Use the PBS washed twice of 5 times of coupled complex volumes then.
(4) preservative treatment
The agarose that step (3) prepares-antibody coupling compound such as need are placed a period of time, should use the PBS damping fluid that contains 1/10000 Sodium azide to soak, and drop removes to put into after the excessive solution packaging bag or bottle 4 ℃ of airtight preservations then.
(5) dress post
Agarose-antibody coupling compound that step (3) or (4) are obtained is packed in 2mL or the 5mL Solid-Phase Extraction void column pipe, with PBS gel is compressed under certain negative pressure, promptly makes the how anti-IAC of NODLN.
(6) preserve
Agarose-antibody coupling compound or populated algae toxin IAC are sure not freezing, are kept in 4 ℃ the refrigerator.
(7) regeneration of post
After post uses, the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl with 4-10 bed volume pH8.5, clean IAC twice in turn with the 0.1M sodium-acetate buffer that contains 0.5M NaCl with 4-10 bed volume pH4.5, again with being kept at behind the abundant balance IAC of PBS damping fluid in 4 ℃ of refrigerators for using next time.
Embodiment 4: the detection of NODLN in the water sample
1, the condition determination of HPLC
High performance liquid chromatograph is that Waters 600 cooperates Waters 996 diode array detector, and chromatographic column is Kromasil 100-5C
18(150 * 4.6mm, 5 μ m), moving phase is the deionized water that contains 0.05% formic acid: acetonitrile=60: 40 (v/v), flow velocity are 1mL/min, column temperature is 30 ℃, sample size 20 μ L, the detection wavelength is 240nm.
2, the mensuration of NODLN in the actual water sample
0.2L the water sample that polluted by blue-green algae passes through 0.45 μ m filtering with microporous membrane earlier, filtrate is crossed the how anti-IAC (pillar is used 5mL methyl alcohol respectively, 5mL distilled water, 5mL PBS pre-service) of the NODLN of preparation.Use 5mL PBS, 5mL distilled water, 5mL 10% methyl alcohol drip washing impurity respectively, use the pure methanol-eluted fractions of 5mL at last.The eluent evaporated under reduced pressure, residue dissolves laggard HPLC analysis with 0.2mL PBS.Do control experiment with SPE simultaneously.
The effect of primary purification, IAC obviously is better than SPE.The how anti-IAC of NODLN can separate the peak of NODLN with other impurity peaks in HPLC, the unlikely measurement result that influences, and SPE purifies because impurity is too many, NODLN peak even be difficult to differentiate.Because how anti-NODLN can specificity combine with NODLN make IAC that excellent specificity be arranged, primary purification can be removed most chaff interferences.And SPE does not have specificity, can not remove chaff interference, and influence last measurement result.
A water sample, the HPLC method detects the wherein content of NODLN.IAC purifies, and twice replicate determination result is 0.209 μ g/L and 0.224 μ g/L, and SPE purifying measuring result is 0.032 μ g/L, and the measurement result deviation is bigger.
Claims (2)
1. preparation method who saves ball algae toxin polyclonal antibody immunoaffinity column is characterized in that:
(1) with joint ball algae toxin, hereinafter to be referred as NODLN, with bovine serum albumin(BSA) BSA coupling, conjugate is hereinafter to be referred as NODLN-BSA, and as artificial antigen, the rabbit epidemic disease obtains the NODLN antiserum, and how anti-adopt two step of saturated ammonium sulfate precipitation method purifying to obtain NODLN again;
(2) activation of Ago-Gel Sepharose 4B: with cyanogen bromide method activation Sepharose 4B, reactivation process is as follows:
(a) get 10mL Sepharose 4B and be placed in the Buchner funnel and drain,, add the NaHCO of a spot of 0.1mol/LpH8.3 with draining after twice washing of 30mL moisture
3Washing changes in the 100mL beaker immediately, and ice bath slowly stirs down;
(b) take by weighing the 1g cyanogen bromide in fuming cupboard, add water 10mL dissolving, add then among the Sepharose 4B in batches, the limit edged stirs, survey the pH value simultaneously,, make pH remain on 10.5 by dripping 2mol/L NaOH, treat that cyanogen bromide reaction is complete, pH remains unchanged, and stops to stir;
(c) the Sepharose 4B with activation adds ice cube, pours into rapidly in the Buchner funnel, takes out with frozen water and washes neutrality, the rapid again NaHCO with the cold 0.1mol/LpH 8.3 of 150mL
3Take out and wash;
(3) preparation of joint ball algae toxin polyclonal antibody immunoaffinity column:
NODLN resists with Ago-Gel Sepharose 4B coupling more obtains affinity adsorbent, inserts and makes the NODLN polyclonal antibody immunoaffinity column in the post, and operation steps is as follows:
(d) coupling
The NODLN that above-mentioned preparation purifying is good resists more places pH 8.3 to contain the 0.1M NaHCO of 0.5M NaCl
3Coupling buffer in the 12h that dialyses; The Sepharose 4B that above-mentioned activation is good places sand core funnel to take out fast with coupling buffer and washes, and pours into rapidly then in the how anti-solution of NODLN and carries out coupling, UV scanning monitoring coupling process; The NODLN of coupling is how not anti-with the coupling buffer flush away more than 5 times of how anti-liquor capacities of NODLN, obtains agarose-antibody coupling compound; Collect whole eluents, calculate the amount that NODLN resists in the not coupling more by measuring its Protein content;
(e) sealing reactive group
Agarose-antibody coupling compound is changed in the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl of 5 times of volume pH 8.0, keep 2h, with unnecessary reactive group in the sealing Ago-Gel;
(f) washing
The agarose that step (e) obtains-antibody coupling compound is successively with the 0.1M Tris-HCl damping fluid washing that contains 0.5M NaCl of the pH 8.0 of the 0.1M acetate buffer solution that contains 0.5M NaCl of the pH 4.0 of 5 times of coupled complex volumes and 5 times of coupled complex volumes, this washing process triplicate; Use the PBS washed twice of 5 times of coupled complex volumes then;
(g) preservative treatment
The agarose that step (f) prepares-antibody coupling compound such as need are placed a period of time, should use the PBS damping fluid that contains 1/10000 Sodium azide to soak, and drop removes to put into after the excessive solution packaging bag or bottle 4 ℃ of airtight preservations then;
(h) dress post
Step (f) or agarose-antibody coupling compound of (g) obtaining are packed in 2mL or the 5mL Solid-Phase Extraction void column pipe, under negative pressure, gel are compressed, make the NODLN polyclonal antibody immunoaffinity column, i.e. the how anti-IAC of NODLN with PBS;
(i) preserve
Agarose-antibody coupling compound or the populated how anti-IAC of NODLN are sure not freezing, are kept in 4 ℃ the refrigerator;
(j) regeneration of post
After post uses, the 0.1M Tris-HCl damping fluid that contains 0.5M NaCl with 4-10 bed volume pH8.5, clean IAC twice in turn with the 0.1M sodium-acetate buffer that contains 0.5M NaCl with 4-10 bed volume pH4.5, again with being kept at behind the abundant balance IAC of PBS damping fluid in 4 ℃ of refrigerators for using next time.
2. the using method of the NODLN polyclonal antibody immunoaffinity column of the described method of claim 1 preparation is characterized in that the mensuration of NODLN in the actual water sample, gets 0.2L and is subjected to water sample that blue-green algae pollutes earlier by 0.45 μ m filtering with microporous membrane; IAC uses 5mL methyl alcohol, 5mL distilled water, 5mLPBS pre-service earlier successively; Filtrate is crossed the how anti-IAC of pretreated NODLN; Use 5mLPBS, 5mL distilled water, 5mL 10% methyl alcohol drip washing impurity more successively; Use the pure methanol-eluted fractions of 5mL at last; The eluent evaporated under reduced pressure, residue dissolves laggard HPLC analysis with 0.2mL PBS; Do control experiment with solid-phase extraction column simultaneously.
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| CN104117228B (en) * | 2013-04-28 | 2016-06-22 | 北京华安麦科生物技术有限公司 | A kind of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone immune affinity column and its production and use |
| CN104725506A (en) * | 2013-12-23 | 2015-06-24 | 上海复星医药（集团）股份有限公司 | Method for purifying human serum prealbumin polyclonal antibody by application of immunoaffinity column |
| CN105738628A (en) * | 2014-12-12 | 2016-07-06 | 上海复星长征医学科学有限公司 | Method of purifying goat-anti-human plasma apolipoprotein A-I monoclonal antibody |
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