CN103204930A - Preparation method for polyclonal antibody of aspergillus fumigatus - Google Patents
Preparation method for polyclonal antibody of aspergillus fumigatus Download PDFInfo
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- CN103204930A CN103204930A CN 201210011794 CN201210011794A CN103204930A CN 103204930 A CN103204930 A CN 103204930A CN 201210011794 CN201210011794 CN 201210011794 CN 201210011794 A CN201210011794 A CN 201210011794A CN 103204930 A CN103204930 A CN 103204930A
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Abstract
The invention relates to an antibody and a preparation method thereof. Specifically, the invention relates to a polyclonal antibody directed against an aspergillus fumigatus galactomannan (GM) antigen and a preparation method thereof. The invention provides a method comprising the steps of immunizing an animal with the aspergillus fumigatus galactomannan (GM) antigen to obtain antiserum of the animal, and separating and purifying the antiserum of the animal to obtain the antibody. The polyclonal antibody prepared by the method has the characteristics of high titer, high purity and good specificity. The polyclonal antibody is the first polyclonal antibody directed against the aspergillus fumigatus galactomannan (GM) antigen in China, and has wide application prospects in the fields such as medical treatment and scientific researches.
Description
Technical field
The present invention relates to antibody and preparation method thereof, especially at aspergillus fumigatus polygalactomannan (Glucomannan, GM) a kind of polyclonal antibody of antigen and preparation method thereof.
Background technology
Because the increasing of extensive pedigree antibiotic use in a large number, tumour and organ transplantation patient, sickness rate and the case fatality rate of deep fungal infection increase year by year in recent years.Aspergillus fumigatus (Aspergillus fumigatus) is a kind of important conditioned pathogen, and the patient of the low or immunological competence defective of normal infection immunity ability becomes a kind of important pathomycete just gradually.
The gold standard of aggressive aspergillin infection detection at present is that biopsy and aseptic body fluid are cultivated.Yet should the disease PD very fast, that traditional cultured method positive detects be low, culture cycle is long, often cause fail to pinpoint a disease in diagnosis, mistaken diagnosis, cause affecting adversely the state of an illness.
Immunoassay technology is to utilize energy specificity association reaction between antigen-antibody, by the marker of certification mark on reactant, antigen or antibody is realized qualitative or quantitative detection method.According to the difference of mark substance, be divided into the enzyme immunoassay technology (Enzyme-Linked Immunosorbant Assay, ELISA), immunofluorescence detection technique, chemiluminescence immunoassay technology, immune microsphere technology, immune colloidal gold technique etc.Wherein elisa technique has simple to operately, highly sensitive, and the characteristics that detection time is short are widely used in clinical detection and scientific research.
In the clinical detection of aspergillus fumigatus, the Aspergillus fumigatus GM Detection of antigen product of Bio-Rad company is only arranged on the world market at present.The domestic inreal product that is used for the aspergillus fumigatus clinical detection of China.And can the key of immunology detection product be obtain suitable antibody, and therefore, preparation becomes the key of this Detection of antigen product of exploitation at the antibody of aspergillus fumigatus GM antigen.
The polygalactomannan structure is special, be incomplete antigen, similar with the polysaccharide component of a variety of microorganisms, be difficult to as easy specificity height, the monoclonal antibody that avidity is good of obtaining of protein immunogen.Use aspergillus fumigatus GM antigen crude extract as immunogen in the document 1, gone out monoclonal antibody with aspergillus fumigatus GM antigen crude extract, Aspergillus fumigatus conidium, Aspergillus fumigatus reorganization polygalactomannan albumen respectively as immunogen preparing in the document 2.But GM antigen crude extract or Aspergillus fumigatus conidium all have shortcomings such as immunogen molecular composition complexity, albumen and polypeptide class foreign matter content be too many as immunogen, and reorganization GM antigen is not polysaccharide but glycoprotein, so domestic not really with the precedent of pure aspergillus fumigatus GM antigen prepd antibody.
In sum, in Cryptococcus neoformans clinical detection field, press for the antibody of preparing anti-aspergillus fumigatus bacterium GM antigen.
The present invention describes in detail
The object of the present invention is to provide a kind of polyclonal antibody of anti-aspergillus fumigatus bacterium GM antigen.
The object of the present invention is to provide a kind of method for preparing the polyclonal antibody of anti-aspergillus fumigatus bacterium GM antigen.
Aspergillus fumigatus GM antigen used in the present invention is made a gift of promise fine jade biotechnology company limited by Tianjin (number of patent application: 201110289099.8) is provided.
Technical solution of the present invention is as follows:
Step:
1. use the GM antigen-immunized animal.
2. measure the serum titer of immunity back animal, get blood in the animal body after the immunity.
3. with saturated ammonium sulphate salting-out process and affinity chromatography serum is carried out purifying, obtain the polyclonal antibody of purifying.
The method of immunity is diversified in the above-mentioned steps 1, as: intrasplenic injection method, intraperitoneal injection etc.Immunizing dose is decided by concrete animal species.Animal for the preparation of the GM polyclonal antibody can be the animal that mouse, rabbit, chicken, sheep, horse, pig, donkey etc. can be used for immunity.
Animal in the above-mentioned steps 2 after the immunity can be put to death the back blood sampling, also can not put to death, and adopts a certain amount of blood in the feeding process at every turn.
The method that is used for antibody purification in the above-mentioned steps 3 can be saturated ammonium sulphate salt precipitation method and affinity chromatography etc.
The polyclonal antibody in the polyclonal antibody for preparing with aforesaid method and other kinds source needs the present invention to protect.
The invention provides a kind of polyclonal antibody of anti-aspergillus fumigatus bacterium GM antigen, and the Polyclonal Antibody Preparation method of anti-aspergillus fumigatus bacterium GM antigen.In preparation method of the present invention, GM antigen is to make (number of patent application: 201110289099.8) from the culture of aspergillus fumigatus, this antigen is domestic the first aspergillus fumigatus GM antigen of purifying, thereby the polyclonal antibody that the present invention obtains is the polyclonal antibody of domestic the first anti-aspergillus fumigatus bacterium GM antigen, filled up domestic blank.
It is good to experiment showed, that the polyclonal antibody that makes with accompanying method of the present invention has specificity, and the high characteristics of tiring have very strong application prospect.
Accompanying drawing is described:
Fig. 1 has shown the result that the SDS-PAGE of rabbit igg type polyclonal antibody detects.
Fig. 2 has shown the result of the titration of rabbit igg type polyclonal antibody.
Embodiment 1, the Polyclonal Antibody Preparation of anti-aspergillus fumigatus bacterium GM antigen
One, immune animal
GM antigen and Freund's complete adjuvant equal-volume are mixed to suitable volumes.Subcutaneous multi-point injection is carried out to new zealand rabbit in fully emulsified back, and every rabbit immunizing dose control is at 0.01-0.8mg.Immunity was got ear blood in preceding 3 days, and separation of serum is done negative control.Per 2 week immunity are 1 time behind the initial immunity, and method is identical with the 1st time.
Two, the acquisition of polyclonal antibody
1) titration: in the immunologic process, the immunity back is surveyed every blood sampling in several days and is tired 1 time, and immune time is no less than 3 times.
2) separate antiserum(antisera): when serum titer reaches the highest, take a blood sample in a large number with the method for carotid artery bloodletting.Treat blood coagulation, after serum was isolated, high speed centrifugation was got supernatant ,-20 ℃ of preservations.
3) carry out preliminary purification with the saturated ammonium sulphate salting-out process
(1) get 2ml antiserum(antisera) sample, add isopyknic physiological saline, add 4ml saturated ammonium sulphate solution again, 4 ℃ of precipitations are spent the night.
(2) the 10000g low-temperature centrifugation is 10 minutes, abandons supernatant, will precipitate the dissolving with 2ml PBS, slowly drips 1ml saturated ammonium sulphate solution, and 4 ℃ left standstill 1 hour.
(3) the 10000g low-temperature centrifugation is 10 minutes, abandons supernatant, will precipitate the dissolving with 1ml PBS, with 4 ℃ of dialysed overnight of PBS solution.
4) method with affinity chromatography is further purified
(1) washes post with the elution buffer of 5-10 times of column volume;
(2) wash post with the coupling buffer of 5-10 times of column volume;
(3) sample on the sample that will cross with saturated ammonium sulphate salting-out process preliminary purification;
(4) wash post with the coupling buffer of 5-10 times of column volume;
(5) with the elution buffer wash-out of 2-5 times of column volume, obtain the polyclonal antibody of anti-aspergillus fumigatus bacterium GM antigen.
Embodiment 2, detection of antibodies
1.SDS-PAGE electrophoresis detection
The antibody that embodiment 1 is made carries out the SDS-PAGE electrophoresis, and the gel that obtains is carried out coomassie brilliant blue staining.Experimental result is seen Fig. 1 (the pAb swimming lane is for how anti-, and the M swimming lane is albumen Marker).By finding out among the figure, at 25KD and 50KD molecular weight area clear tangible band is arranged, illustrate that antibody purity is very high.
2. titration
Tire with the indirect elisa method antagonist and to measure.Used ELIAS secondary antibody is the goat anti-rabbit igg of horseradish peroxidase-labeled, and negative control is PBS solution, with the 2 times of positive criterion greater than negative control OD value of the OD value of antibody-solutions.Detected result is seen Fig. 2.Can find out that from the result this antibody titer is higher, greater than 1: 1 * 10
5
Should know that just invention has been described with exemplifying embodiment, the improvement of making on basis of the present invention still belongs to category of the present invention.
Reference (it is for referencial use to fit into this paper in it)
[1] Che Xiaoyan etc., (2002) anti-aspergillus fumigatus bacterium monoclonal antibody is identified and Preliminary Applications. Chinese Journal of Immunology .18:175-177
[2] Hao Wei etc., the mould MONOCLONAL ANTIBODIES SPECIFIC FOR of (2008) 1 suites and evaluation. Chinese mycology magazine .3 (3): 129-133
Claims (7)
1. the polyclonal antibody of an anti-aspergillus fumigatus bacterium GM antigen is characterized in that with aspergillus fumigatus GM antigen as immunogen, the polyclonal antibody that obtains through immune animal, separation and purifying antiserum(antisera).
2. the polyclonal antibody of an anti-aspergillus fumigatus bacterium GM antigen as claimed in claim 1 is characterized in that being shown as homogeneous antibody product with SDS-PAGE.
3. the polyclonal antibody of an anti-aspergillus fumigatus bacterium GM antigen as claimed in claim 1 is characterized in that with aspergillus fumigatus GM antigen coatedly, and indirect method ELISA detects and shows that it is tired and be not less than 1: 1 * 10
5
4. the Polyclonal Antibody Preparation method of an anti-aspergillus fumigatus bacterium GM antigen as claimed in claim 1 may further comprise the steps:
(a) use aspergillus fumigatus GM antigen as the immunogen immune animal;
(b) serum that (a) step is obtained carries out separation and purification, obtains antibody.
5. method as claimed in claim 4 is characterized in that, in the step (a), the method for immunity is subcutaneous injection, intrasplenic injection, intravenous injection, abdominal injection; Immunizing dose is 0.1mg-0.8mg; The animal of immunity is rat, mouse, cavy, rabbit, chicken, sheep, horse, pig, donkey.
6. as method as described in the claim 4, it is characterized in that the method for the employed antibody purification of step (b) is saturated ammonium sulphate salt precipitation method and affinity chromatography.
7. use the polyclonal antibody of the anti-aspergillus fumigatus bacterium GM antigen of the arbitrary described method preparation of claim 1-6.
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| CN 201210011794 CN103204930A (en) | 2012-01-16 | 2012-01-16 | Preparation method for polyclonal antibody of aspergillus fumigatus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104628861A (en) * | 2015-02-03 | 2015-05-20 | 丹娜（天津）生物科技有限公司 | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof |
| CN105866407A (en) * | 2016-04-22 | 2016-08-17 | 丹娜（天津）生物科技有限公司 | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof |
| CN106220730A (en) * | 2016-08-15 | 2016-12-14 | 丹娜（天津）生物科技有限公司 | A kind of preparation method of Aspergillus fumigatus galactomannan polyclonal antibody |
| CN110887966A (en) * | 2019-12-17 | 2020-03-17 | 丹娜（天津）生物科技有限公司 | Chemiluminescent detection kit for aspergillus and application thereof |
| CN110894236A (en) * | 2019-12-17 | 2020-03-20 | 丹娜（天津）生物科技有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
-
2012
- 2012-01-16 CN CN 201210011794 patent/CN103204930A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104628861A (en) * | 2015-02-03 | 2015-05-20 | 丹娜（天津）生物科技有限公司 | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof |
| CN105866407A (en) * | 2016-04-22 | 2016-08-17 | 丹娜（天津）生物科技有限公司 | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof |
| CN106220730A (en) * | 2016-08-15 | 2016-12-14 | 丹娜（天津）生物科技有限公司 | A kind of preparation method of Aspergillus fumigatus galactomannan polyclonal antibody |
| CN110887966A (en) * | 2019-12-17 | 2020-03-17 | 丹娜（天津）生物科技有限公司 | Chemiluminescent detection kit for aspergillus and application thereof |
| CN110894236A (en) * | 2019-12-17 | 2020-03-20 | 丹娜（天津）生物科技有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
| CN110894236B (en) * | 2019-12-17 | 2021-01-01 | 丹娜(天津)生物科技股份有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
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Application publication date: 20130717 |