CN101724069B - Preparation method of yolk antibody IgY of high-abundance protein in anti-human serum/blood plasma and application thereof - Google Patents
Preparation method of yolk antibody IgY of high-abundance protein in anti-human serum/blood plasma and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a yolk antibody IgY of high-abundance protein in anti-human serum/blood plasma and application thereof; and eggs of a hen immunized by high-abundance protein are used as a source for extracting antibody. The preparation method comprises the steps of: detecting the serum potency of the immunized hen through an indirect ELISA method, obtaining an egg containing antibody with high potency after the potency meets the standard; acid hydrolyzing, freeze thawing and filtering the egg yolk liquid, and depositing by using ammonium sulphate; and further purifying through DEAE ion exchange layer chromatograph to obtain the yolk antibody IgY. The high-abundance protein of human serum/blood plasma is a high-abundance protein with albumin, IgG, transferrin, C3 alexin, IgA, IgM, trypsin, globin, lipoprotein, F factor, C9 alexin, C4 alexin, ceruloplasmin, apolipoprotein B, apolipoprotein A-1, alpha-1-acid glycoprotein, H factor, alexin factor B, prealbumin and C1q alexin, and the like, and the concentration of which are higher than 10 mug/ml.
Description
Technical field
The present invention relates to preparation method and the application thereof of high-abundance proteins yolk antibody IgY in a kind of AHS/slurry.
Background technology
Contain organism tissue emiocytosis in serum human/biological samples such as slurry, drop to the multiple low abundance proteins in the blood, they may be the key proteins of some signal conduction and regulation and control.For example, in necrosis, apoptosis or the haemolysis process, all can there be some intracellular protein to be discharged in the blood.Carry out the serum photeomics research of disease, seek low abundance proteins, will be conducive to illustrate pathogeny, and might find the significant protein that some are relevant with the disease particular state, thereby provide novel method for the diagnosis of disease.Yet the existence of some high-abundance proteins matter, especially albumin and antibody have had a strong impact on the Identification and detection of low abundance proteins.
Often utilize the high-abundance proteins matter in the antibody affinity chromatography method removal sample in the proteomics research, to improve sensitivity and the resolving power that low-abundance protein is detected.The key of utilizing antibody affinity chromatography removal technology is to obtain the high and antibody cheap and easy to get of specificity, and the antibody affinity chromatography fado adopts recombinant monoclonal or the polyclonal antibody in Mammals source now.The normal operation rabbit, the Mammalss such as mouse are as the animal-origin that produces high-abundance proteins antibody in antiserum(antisera)/blood plasma.Mammiferous monoclonal antibody preparation is complicated, and output is little, and cost is high; There are the problems referred to above equally in its polyclonal antibody that derives from blood plasma, and can have the shortcoming of non-specific binding.
Summary of the invention
The object of the present invention is to provide preparation method and the application thereof of high-abundance proteins antibody in a kind of AHS/slurry, it has solved and has used rabbit in the prior art, the Mammalss such as mouse are as the animal-origin that produces high-abundance proteins antibody in antiserum(antisera)/blood plasma, use mammalian blood serum as extracting antibody sources, cause the preparation process of antibody complicated, output is little, and cost is high, the problems such as antibody has non-specific binding, and it is low and unstable to tire; And solved Mammals source antibody in albumen sepn, the non-specific separation problem that causes in the extraction.Mammals source antibody non-specific adsorption and assorted effect of signals problem of causing in immunology detection have been solved.
The thinking that the present invention addresses the above problem is: bird kind is being that generation has the sibship farther with Mammals, can have the more antibody of high specific for a large amount of conservative protein antigens generations in the Evolution of Mammals, and it is little to produce the required antigen amount of effective immune response.The egg of use hen after immunity replaces animal serum, as the source of extracting antibody.In recent years, Yolk immunoglobulin (immunoglobulin of yolk, be called for short IgY), because of output is large, high specificity, obtain conveniently, low cost and other advantages, more and more be applied to diagnosis, prevention and the treatment of disease, become a kind of reliable and stable antibody sources.The specific IgY of anti-human blood plasma high-abundance proteins can be covalently bonded in solid support, removes the high-abundance proteins in the blood plasma, such as albumin, Transferrins,iron complexes, Fibrinogen, immunoglobulin (Ig) (IgA, IgM, IgG) etc.
The technical scheme that the present invention addresses the above problem is: the preparation method of high-abundance proteins antibody IgY may further comprise the steps in a kind of AHS who provides/slurry:
1, collects the egg that contains high-titer antibody that produces through hen after the high-abundance proteins immunity.The described egg that contains high-titer antibody is to determine like this: detect through the tiring of high-abundance proteins immunity stepmother chicken serum with indirect elisa method, tiring is the high-titer antibody egg after up to standard;
2, separation and Extraction high-abundance proteins yolk antibody IgY from the high-titer antibody egg: the yolk liquid of described high-titer antibody egg is dissolved through sour water, freeze thawing, filter, ammonium sulfate precipitation and DEAE anion-exchange chromatography are the separable high-abundance proteins yolk antibody IgY that obtains purifying.
The separation and Extraction of step 2 high-abundance proteins yolk antibody the steps include:
(1) albumen and the vitelline membrane of removal high-titer egg obtain yolk liquid, with ultrapure water dilution yolk liquid, press ultrapure water and yolk liquid 1: 2-1: 15 volume ratio, adjust the pH of diluent in the 4.5-6.0 scope, and 4 ℃ are stirred 9h to spending the night.After yolk diluent is frozen, place 4 ℃ of slowly dissolvings, filter.Filtrate is collected supernatant through the centrifugal 20min of 11000rpm.
(2) above-mentioned supernatant liquor saturated ammonium sulphate 2 times, ultimate density is respectively 50% and 35%.The centrifugal 25min of 11000rpm abandons supernatant, and precipitation is dissolved with PBS.4 ℃ of dialysis 24h, liquid is changed 4 times in the centre.The yolk antibody crude product of dialysing to get.
(3) under 6.8～7.0 nearly neutral environment, yolk antibody is carried out purifying with the DEAE anion-exchange column, 1mol/L NaCl gradient elution, flow velocity 1mL/min.Collect elutriant, be yolk antibody solution.
The step 1 pair serum through hen after the immunity carries out bioactivity, obtains the egg that contains high-titer antibody, the steps include:
(1.1) detect immune serum with indirect elisa method and tire, detect the absorbance value of 450nm with microplate reader, tire take the absorbance value of the positive serum 450nm minimum extent of dilution of serum during greater than 2.1 times of negative control mean values as ELISA.
(1.2) detect immune serum with indirect elisa method and tire, ELISA collects egg after tiring and reaching 1: 10000,4 ℃ of preservations.
High-abundance proteins described in the present invention is albumin, IgG, Transferrins,iron complexes, C3 complement, IgA, IgM, Trypsin, globin, lipoprotein, F-factor, C9 complement, the C4 complement, copper-protein, apolipoprotein B, aPoA-I, α-1-acid glycoprotein, the H factor, complement factor B, prealbumin, C1q complement isoconcentration is higher than the high-abundance proteins of 10 μ g/ml.
Among the present invention in AHS/slurry the high-abundance proteins yolk antibody IgY use and to comprise: the immune affinity extraction of high-abundance proteins, the immune affine separation of high-abundance proteins, the immunology detection of high-abundance proteins.
Advantage of the present invention is:
When 1, preparing the high-abundance proteins yolk antibody IgY, it is convenient to collect egg, need not blood sampling, and not damaged meets modern protection of animal rule.
2, antibody production is high, and the time that maintains high-titer is long, and is with low cost.Egg-laying bird can produce more efficient valency and the length of holding time soon in egg after immunity.Yolk antibody output is large, and each egg approximately contains the above IgY of 100mg, can reach 3g in one month, is equivalent to the 10-12 of rabbit doubly.Because the breeding expenses of bird is with cheap, and can produce high-titer antibody for a long time, its cost is well below extract corresponding antibodies from mammalian plasma.
3, antibody separation and Extraction convenience and purity are high.The Ig that does not have other in the yolk is easy to purify.The IgY crude product purity of utilizing ammonium sulfate precipitation method to be separated to just reaches more than 90%.
4, IgY is more stable, is easy to store.Because be subject to the protection of yolk, egg is stored in lower 6 months of 4 ℃ of conditions, the IgY activity only has very small loss.And IgY is more acidproof than Mammals antibody, and is alkaline-resisting, heat-resisting.
5, yolk antibody the height of tiring that the proteantigen in Mammals source is produced, high specificity, and it is little to produce the required antigen amount of effective immune response.
6, the yolk antibody of anti-high-abundance proteins is applied to the immune affinity extraction of high-abundance proteins, the immune affine separation of high-abundance proteins, the immunology detection of high-abundance proteins.The immune affinity extraction of high-abundance proteins, the immune affine separation of high-abundance proteins have been solved, the non-specific problem in the immunology detection of high-abundance proteins.
Description of drawings
Fig. 1 is yolk antibody IgY separation and Extraction SDS-PAGE electrophorogram, the high-abundance proteins IgY that swimming lane 1 obtains for purifying, and swimming lane 2 is high-abundance proteins IgY mark product.
Embodiment
Embodiment 1 is the IgY preparation process of anti-Transferrins,iron complexes chicken:
1, the egg through hen after the Transferrins,iron complexes immunity is detected, obtain the egg that contains high-titer antibody.Concrete steps are:
(1) detects immune serum with indirect elisa method and tire, detect the absorbance value of 450nm with microplate reader, tire take the absorbance value of the positive serum 450nm minimum extent of dilution of serum during greater than 2.1 times of negative control mean values as ELISA.
(2) detect immune serum with indirect elisa method and tire, ELISA collects egg after tiring and reaching 1: 10000,4 ℃ of preservations.
2, separation and Extraction high-abundance proteins yolk antibody IgY from the high-titer antibody egg: the yolk liquid of described high-titer antibody egg is dissolved through sour water, freeze thawing, filter, ammonium sulfate precipitation and DEAE-fast-flow ion exchange chromatography are the separable high-abundance proteins yolk antibody IgY that obtains purifying.Concrete steps are:
(1) albumen and the vitelline membrane of removal high-titer egg obtain yolk liquid, with ultrapure water and 1: 9 volume ratio of yolk liquid dilution yolk liquid, diluent are adjusted to about PH=5.5, and 4 ℃ are stirred 9h to spending the night.After yolk diluent is frozen, place 4 ℃ of slowly dissolvings, filter.Filtrate is collected supernatant through the centrifugal 20min of 11000rpm.
(2) above-mentioned supernatant liquor saturated ammonium sulphate 2 times, ultimate density is respectively 50% and 35%.The centrifugal 25min of 11000rpm abandons supernatant, and precipitation is dissolved with PBS.4 ℃ of dialysis 24h, liquid is changed 4 times in the centre.The yolk antibody crude product of dialysing to get.
(3) under 6.8～7.0 nearly neutral environment, yolk antibody is carried out purifying with the DEAE anion-exchange column, 1mol/L NaCl gradient elution, flow velocity 1mL/min.Collect elutriant, be yolk antibody solution.
(4) as can be seen from Figure 1, in swimming lane 1 and the swimming lane 2, same strap occurs in same position, illustrate to extract to have obtained yolk antibody IgY.
Embodiment 2 is the IgY preparation process of antialbumin chicken:
1, the serum through hen after the albumin immunity is carried out bioactivity, obtain the egg that contains high-titer antibody.Concrete steps are:
(1) detects immune serum with indirect elisa method and tire, detect the absorbance value of 450nm with microplate reader, tire take the absorbance value of the positive serum 450nm minimum extent of dilution of serum during greater than 2.1 times of negative control mean values as ELISA.
(2) detect immune serum with indirect elisa method and tire, ELISA collects egg after tiring and reaching 1: 10000,4 ℃ of preservations.
2, separation and Extraction high-abundance proteins yolk antibody IgY from the high-titer antibody egg: the yolk liquid of described high-titer antibody egg is dissolved through sour water, freeze thawing, filter, ammonium sulfate precipitation and DEAE ion exchange chromatography are the separable high-abundance proteins yolk antibody IgY that obtains purifying.
(1) albumen and the vitelline membrane of removal high-titer egg obtain yolk liquid, with ultrapure water and 1: 9 volume ratio of yolk liquid dilution yolk liquid, diluent are adjusted to about PH=5.5, and 4 ℃ are stirred 9h to spending the night.After yolk diluent is frozen, place 4 ℃ of slowly dissolvings, filter.Filtrate is collected supernatant through the centrifugal 20min of 11000rpm.
(2) above-mentioned supernatant liquor saturated ammonium sulphate 2 times, ultimate density is respectively 50% and 35%.The centrifugal 25min of 11000rpm abandons supernatant, and precipitation is dissolved with PBS.4 ℃ of dialysis 24h, liquid is changed 4 times in the centre.The yolk antibody crude product of dialysing to get.
(3) under 6.8～7.0 nearly neutral environment, yolk antibody is carried out purifying with the DEAE anion-exchange column, 1mol/L NaCl gradient elution, flow velocity 1mL/min.Collect elutriant, be yolk antibody solution.
(4) as can be seen from Figure 1, in swimming lane 1 and the swimming lane 2, same strap occurs in same position, illustrate to extract to have obtained highly purified yolk antibody IgY.
Claims (3)
1. the preparation method of high-abundance proteins yolk antibody IgY in AHS/slurry, it is characterized in that: described preparation process may further comprise the steps
(1) collects the egg that contains high-titer antibody that produces through hen after the high-abundance proteins immunity; The described egg that contains high-titer antibody is to determine like this: detect through the tiring of high-abundance proteins immunity stepmother chicken serum with indirect elisa method, tiring is the high-titer antibody egg after up to standard;
(2) imitate the anti-high-abundance proteins yolk antibody IgY of separation and Extraction the antibody egg from high price: the yolk liquid of described high-titer antibody egg is diluted through sour water, through freeze thawing, filtration obtains containing the aqueous solution of yolk antibody IgY, ammonium sulfate precipitation is slightly pure, and the DEAE ion exchange chromatography is the separable high-abundance proteins yolk antibody IgY that obtains purifying;
Step (2) specifically operates according to following steps:
(2.1) albumen and the vitelline membrane of removing the high-titer egg obtain yolk liquid, with ultrapure water dilution yolk liquid, the volume ratio of pressing yolk liquid and ultrapure water 1:2-1:15, and diluent are adjusted pH in the 4.5-6.0 scope, and 4 ℃ of stirring 9h are to spending the night; After yolk diluent is frozen, place 4 ℃ of slowly dissolvings, filter; Filtrate is collected supernatant and is the aqueous solution that contains yolk antibody IgY through the centrifugal 20min of 11000rpm;
(2.2) aqueous solution supernatant that contains yolk antibody IgY is with saturated ammonium sulphate 2 times, and final concentration is respectively 50% and 35%; The centrifugal 25min of 11000rpm abandons supernatant, and precipitation is dissolved with PBS; 4 ℃ of dialysis 24 h, liquid is changed 4 times in the centre; Dialysis obtains the yolk antibody crude product;
(2.3) under the nearly neutral environment of pH 6.8～7.0, yolk antibody is carried out purifying with the DEAE anion-exchange column, 1 mol/L NaCl gradient elution, flow velocity 1mL/min; Collect elutriant, obtain yolk antibody solution.
2. the preparation method of high-abundance proteins yolk antibody IgY in AHS according to claim 1/slurry, it is characterized in that: step (1) detects tiring through high-abundance proteins immunity stepmother chicken serum, obtain the egg that contains high-titer antibody, the steps include:
(1.1) detect immune serum with indirect elisa method and tire, detect the absorbance value of 450nm with microplate reader, tire take the absorbance value of the positive serum 450nm minimum extent of dilution of serum during greater than 2.1 times of negative control mean values as ELISA;
(1.2) detect immune serum with indirect elisa method and tire, ELISA collects egg after tiring and reaching 1:10 000,4 ℃ of preservations.
3. the preparation method of high-abundance proteins yolk antibody IgY in AHS according to claim 1 and 2/slurry, it is characterized in that: described high-abundance proteins is albumin, IgG, Transferrins,iron complexes, the C3 complement, IgA, IgM, Trypsin, globin, lipoprotein, F-factor, C9 complement, the C4 complement, copper-protein, apolipoprotein B, aPoA-I, α-1-acid glycoprotein, the H factor, complement factor B, prealbumin, the C1q complement, the concentration of described high-abundance proteins is higher than 10 μ g/ml.
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| CN1268643C (en) * | 2003-11-25 | 2006-08-09 | 中国人民解放军军事医学科学院微生物流行病研究所 | Antibody against SARS-CoV IgY and its preparing method |
| CN1724569A (en) * | 2005-06-30 | 2006-01-25 | 上海交通大学 | Preparation method of vitelline antibody for resisting pstudorabies |
| CN101343320A (en) * | 2008-07-01 | 2009-01-14 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
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