CN104402996A - Method for preparing specific egg yolk antibody against influenza A virus H3N2 - Google Patents
Method for preparing specific egg yolk antibody against influenza A virus H3N2 Download PDFInfo
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Abstract
The invention discloses a method for preparing a specific egg yolk antibody against influenza A virus H3N2. The method comprises the following steps: producing an influenza A virus H3N2 immune antigen and immune laying hens to obtain immune eggs; extracting a specific egg yolk antibody crude product against influenza A virus H3N2 from the yolks of the immune eggs based on a water dilution method; performing ultrafiltration purification, aseptic filtration and ultralow temperature spray drying on the crude product to obtain the specific egg yolk antibody against influenza A virus H3N2; detecting the purity and activity of the antibody. The method disclosed by the invention has the advantages of low cost and high yield, and the antibody obtained according to the method has high specificity, and can realize industrialized production.
Description
Technical field
The invention belongs to biomedicine technical field, a kind of preparation method of anti-A type H3N2 specific immunoglobulin of egg yolk.
Background technology
Influenza A virus can be divided into 16 hypotypes (H1-H16) according to the difference of hemagglutinin (HA) antigen on its surface, can be divided into 9 hypotypes (N1-N9) according to the difference of neuraminidase (NA) antigen.Influenza virus changes (antigenic drift) by the accumulation of surface protein antigen or various flows Influenza Virus producer is reset and produced the Immune discrimination that a kind of new influenza virus (antigenic shift) escapes infection host, thus is infecting propagation in colony, procreation.A type H3N2 influenza is a kind of respiratory system disease caused because of A type H3N2 influenza virus.The H3N2 influenza of Hong Kong outburst in 1968 loses one's life to making million people.At the beginning of 2013, the multiple state outburst in eastern united states H3N2 seasonal influenza, classic city government announced the public health emergency state January 9.Prevention and control measure at present for human world A type H3N2 flu outbreak mainly adopts chemicals and vaccine, but chemicals excessive use the raw side effect of fecund and vaccine development often lag behind epidemic outbreak and vaccine can only the drawback of the touching prophylactic effect of all rising of commute remarkable.
Chicken yolk antibody is that the IgY of hen in the growth course of ovum in serum effectively transfers in yolk and obtain.Yolk antibody has safe ready, simple and easy to get, output is high, with IgG, serological reaction does not occur to intersect; Do not combine with Rheumatoid factors, polyclonal or Fc acceptor, the advantage producing false negative or false positive results can be avoided in immunoassay procedures, and it is heat-resisting, acidproof, can not inactivation and also can keep stabilizing active when pH value is less than 3 under pasteurizing temperature, the advantage of these uniquenesses makes chicken yolk antibody play an important role in the application of clinical and basic science, therefore the application of yolk antibody in medical prophylaxis field more and more receives the concern of the mankind, the new breakthrough that the development of anti-A type H3N2 specific immunoglobulin of egg yolk also will bring A type H3N2 influenza to prevent.
Summary of the invention
The present invention relates to a kind of preparation method of anti-A type H3N2 specific immunoglobulin of egg yolk, it is characterized in that: preparation A type H3N2 influenza virus, determine TCID50 value, after dilution, A type H3N2 influenza virus immunization antigen is made in equal proportion mixing, emulsification; With this antigen injection immunity bird inlay, adaptive immune egg; Extract anti-A type H3N2 specific immunoglobulin of egg yolk crude product; Ultrafiltration purification is carried out to described crude product, retains relative molecular weight 30KD ~ 150KD, ultra-filter retentate is carried out filtration treatment, with the various bacterium of filtering, filtrate, through very low temperature spraying dry, obtains anti-A type H3N2 specific immunoglobulin of egg yolk finished product, 4 DEG C of preservations.And carry out purity, Activity determination.
Embodiment
Embodiment below further illustrates the present invention.
Embodiment one: the preparation of anti-A type H3N2 specific immunoglobulin of egg yolk.
(1) preparation of A type H3N2 influenza virus: A type H3N2 strains of influenza viruses is inoculated into and grows in the mdck cell of individual layer, CO
2gather in the crops when being cultured to cells showed cytopathic CPE++++ in incubator, ﹣ 20 DEG C of freeze thawing 3 times, centrifugation, add PBS and dilute virus liquid after purifying, determine TCID50 value, 56 DEG C of high-temperature inactivation 30min, 4 DEG C of preservations.
(2) preparation of A type H3N2 influenza virus immunization antigen: according to TCID50 value, by above-mentioned A type H3N2 influenza virus 0.01mol/L phosphate buffered saline buffer PBS(pH=7.2) dilute, and with Freund's complete adjuvant according to the fully emulsified rear obtained immunizing antigen of 1:1 (V/V).
(3) with A type H3N2 influenza virus immunization antigen immune bird inlay: get healthy of the right age bird inlay, the immunizing antigen that the position emulsification such as brachial triangle flesh, back under chicken dipteron axle bone is good carries out muscle, subcutaneous multi-point injection, so that laying hen produces better immune response; First immunisation adopts Freund's complete adjuvant, and all the other adopt Freund's incomplete adjuvant; Collect not immune egg before first immunisation as negative controls, immunity second time after 14 ~ 21 days, once every immunity in 14 ~ 21 days, each immunizing dose is 0.2 ~ 1ml/ later, altogether immunity 4 ~ 6 times; With tiring of anti-A type H3N2 specific immunoglobulin of egg yolk in ELISA tracing detection immune egg, collect high-titer egg (1:1024), the immune egg numbering of being collected every day, preserves at 4 DEG C.
(4) Isolation and purification of anti-A type H3N2 specific immunoglobulin of egg yolk.
The immune egg obtained is sterilized by 0.1% bromogeramine by S1, purified water is rinsed well, cracks, and removes egg white and membrane of yolk, leave and take egg yolk fractions, yolk volume collected by mensuration, water dilution method extracts: 1:6 ~ 12 ratio adds purified water in proportion, regulates PH to be 5.2 ~ 5.6 with 1mol/L hydrochloric acid soln, hold over night under 4 DEG C of conditions, 6000 ~ 10000r/min is centrifugal, gets supernatant liquor, measures the volume of collected supernatant liquor.
S2 in the supernatant liquor that S1 is obtained slightly jogging speed add PEG6000 or diatomite under stirring, after abundant stirring, be placed in high speed freezing centrifuge, with 6000 ~ 10000r/min r/min at 10 DEG C, centrifugal, incline supernatant liquor, and sediment fraction is anti-A type H3N2 specific immunoglobulin of egg yolk crude product.
S3 purified water dissolves anti-A type H3N2 specific immunoglobulin of egg yolk crude product to supernatant liquor just volume, ultrafiltration purification is carried out with ultra-filtration membrane, retaining relative molecular weight is 30KD ~ 150KD filtrate, ultra-filter retentate is carried out Sterile Filtration, with the various bacterium of filtering, anti-A type H3N2 specific immunoglobulin of egg yolk sterling can be obtained.
Anti-A type H3N2 specific immunoglobulin of egg yolk sterling is carried out very low temperature spraying dry by S4, i.e. obtained anti-A type H3N2 specific immunoglobulin of egg yolk finished product, 4 DEG C of preservations.
Embodiment two: the purity detecting of anti-A type H3N2 specific immunoglobulin of egg yolk.
Cellulose-acetate membrane electrophoresis is adopted to be separated and to measure the purity of anti-A type H3N2 specific immunoglobulin of egg yolk.Cellulose-acetate membrane electrophoresis take cellulose acetate film as the electrophoresis method of upholder.Cellulose acetate film is made up of secondary cellulose acetate, it has the structure of homogeneous foam sample, thickness only 120 microns, has strong perviousness, moves non-resistance to molecule, upholder as zone electrophoresis carries out protein electrophoresis to be had easy, quick, amount of samples is few, applied range, and it is clear to be separated, there is no the advantages such as adsorption phenomena, be widely used in the separation andpreconcentration of serum protein, lipoprotein, oxyphorase, glycoprotein, isozyme at present.Detected result shows: purity >=40%.
Embodiment three: the ELISA of anti-A type H3N2 specific immunoglobulin of egg yolk tires Activity determination.
The ELISA adopting " ELISA method " (enzyme linked immunosorbent assay) to detect the anti-A type H3N2 specific immunoglobulin of egg yolk that technique of the present invention is produced tires.Detected result shows, tires and reaches 1:1280.
Claims (1)
1. a preparation method for anti-A type H3N2 specific immunoglobulin of egg yolk, is characterized in that comprising the following steps:
(1) preparation of influenza virus complex immunity antigen: preparation A type H3N2 influenza virus, determines TCID50 value, carries out inactivation of virus; According to the TCID50 value of virus, with 0.01mol/L phosphate buffered saline buffer (PBS, pH=7.2) dilution, and with Freund's complete adjuvant according to 1:1(V/V) fully emulsified rear obtained A type H3N2 influenza virus immunization antigen;
(2) with A type H3N2 influenza virus immunization antigen immune bird inlay: get healthy of the right age bird inlay, the immunizing antigen that the position emulsification such as brachial triangle flesh, back under chicken dipteron axle bone is good carries out muscle, subcutaneous multi-point injection, produces better immune response to make laying hen; First immunisation adopts Freund's complete adjuvant, and all the other adopt Freund's incomplete adjuvant; Collect not immune egg before first immunisation as negative controls, immunity second time after 14d ~ 21d days, once every immunity in 14d ~ 21d days, each immunizing dose is 0.2 ~ 1ml/ later, altogether immunity 4 ~ 6 times; With tiring of anti-A type H3N2 specific immunoglobulin of egg yolk in ELISA tracing detection immune egg, collect high-titer egg (1:1024), the immune egg numbering of being collected every day, preserves at 4 DEG C;
(3) extraction, purifying: by the immune egg sterilization obtained, remove egg white and membrane of yolk, leave and take egg yolk fractions, obtain water soluble ingredient by water dilution method centrifugation, and extract the anti-A type H3N2 specific immunoglobulin of egg yolk crude product of acquisition with polyethylene glycol 6000 or diatomite; After crude product is dissolved by purified water, carry out ultrafiltration purification, retain relative molecular weight 30KD ~ 150KD filtrate, ultra-filter retentate is carried out Sterile Filtration, with the various bacterium of filtering, then through very low temperature spraying dry, obtained anti-A type H3N2 specific immunoglobulin of egg yolk finished product, 4 DEG C of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684103A (en) * | 2019-09-27 | 2020-01-14 | 广东京豪生物制药有限公司 | Preparation method of high-yield H3N2 influenza A virus antibody |
| CN111378032A (en) * | 2018-12-28 | 2020-07-07 | 埃格生物科技(常州)有限公司 | Preparation method of egg yolk antibody for clinical diagnostic reagent |
-
2014
- 2014-12-08 CN CN201410738947.2A patent/CN104402996A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 张雪梅,等: "抗甲3 型流感病毒卵黄抗体的研制", 《微生物学免疫学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111378032A (en) * | 2018-12-28 | 2020-07-07 | 埃格生物科技(常州)有限公司 | Preparation method of egg yolk antibody for clinical diagnostic reagent |
| CN110684103A (en) * | 2019-09-27 | 2020-01-14 | 广东京豪生物制药有限公司 | Preparation method of high-yield H3N2 influenza A virus antibody |
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Application publication date: 20150311 |