CN103204928A - Preparation method for polyclonal antibody of cryptococcus neoformans - Google Patents
Preparation method for polyclonal antibody of cryptococcus neoformans Download PDFInfo
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- CN103204928A CN103204928A CN 201210011792 CN201210011792A CN103204928A CN 103204928 A CN103204928 A CN 103204928A CN 201210011792 CN201210011792 CN 201210011792 CN 201210011792 A CN201210011792 A CN 201210011792A CN 103204928 A CN103204928 A CN 103204928A
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- cryptococcus neoformans
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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 a cryptococcus neoformans capsular polysaccharide (Glucuronoxylomannan, GXM) antigen and a preparation method thereof. The invention provides a method comprising the steps of immunizing an animal with the cryptococcus neoformans capsular polysaccharide GXM 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 and high purity. The polyclonal antibody is the first polyclonal antibody directed against the cryptococcus neoformans capsular polysaccharide GXM 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 Cryptococcus neoformans capsular polysaccharide (Glucuronoxylom-annan, GXM) a kind of polyclonal antibody of antigen and preparation method thereof.
Background technology
Cryptococcus neoformans (Crytococcus Neoformans) is a kind of important conditioned pathogen, and the patient of the low or immunological competence defective of normal infection immunity ability causes the deep fungal infection based on central nervous system infection, and case fatality rate is very high.
In recent years because the long-term widespread use of immunosuppressor after extensive pedigree antibiotic, adrenocortical hormone, chemotherapy of tumors, radiotherapy and the organ transplantation, and acquired immune deficiency syndrome (AIDS) is popular, the fungal meningitis showed increased.And Neoformans Meningitis meningitis (hereinafter to be referred as " latent brain ") is by the modal central nervous system disease due to the Cryptococcus neoformans.Latent brain accounts for all Cryptococcus neoformans infection and causes about 80% of disease.Latent brain complicated clinical manifestation, atypical symptom, thereby be difficult to diagnosis, about 80% latent brain patient can be tuberculous meningitis by mistaken diagnosis.The detection method of carrying out in the world at present mainly contains following 3 big classes:
1. india ink method, this method susceptibility is poor, and positive rate is very low;
2. cultured method such as hemoculture or cerebrospinal fluid cultivation, this method susceptibility is poor, and positive rate is not high;
3. immunological detection: realize detection to Cryptococcus neoformans with specificity at the antibody of Cryptococcus neoformans capsular polysaccharide antigen GXM, the susceptibility that detects blood and cerebrospinal fluid can both reach more than 85%.Immunological detection method sensitivity and specificity all more preceding two kinds of methods have remarkable advantages, become the common method that Cryptococcus neoformans detects just gradually.
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 Cryptococcus neoformans, on the world market immunodetection product few in number is arranged at present, as: the latex agglutination of Meridian company and ELISA method detection kit product, the latex agglutination product of IMMY company and colloidal gold method product, the latex agglutination product of Bio-Rad company.The domestic inreal immunology detection product that is used for the Cryptococcus neoformans 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 Cryptococcus neoformans capsular polysaccharide GXM antigen.
Polysaccharose substance is difficult to as the easy monoclonal antibody that obtains of protein immunogen because structure is special, immunogenicity is poor, similar, much is haptens, often need carry out chemically modified to epitope, makes it become complete antigen and just can carry out immunity.The domestic report of not seeing at present about the polyclonal antibody preparation of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM.
In sum, in Cryptococcus neoformans clinical detection field, press for the antibody of preparing anti-Cryptococcus neoformans capsular polysaccharide antigen GXM.
The present invention describes in detail
The object of the present invention is to provide the polyclonal antibody of a kind of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM.
The object of the present invention is to provide a kind of method for preparing the polyclonal antibody of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM.
Cryptococcus neoformans capsular polysaccharide antigen GXM used in the present invention makes a gift of promise fine jade biotechnology company limited by Tianjin (number of patent application: 201110240065.X) is provided.
Technical solution of the present invention is as follows:
Step:
1. use the GXM 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 2, as: intrasplenic injection method, intraperitoneal injection etc.Immunizing dose is decided by concrete animal species.Animal for the preparation of the GXM 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 3 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 4 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 the polyclonal antibody of a kind of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM, and the Polyclonal Antibody Preparation method of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM.In preparation method of the present invention, GXM antigen is to make (number of patent application: 201110240065.X) from the culture of Cryptococcus neoformans, this antigen is domestic the first Cryptococcus neoformans capsular polysaccharide antigen of purifying, thereby the polyclonal antibody that the present invention obtains is the polyclonal antibody of the anti-Cryptococcus neoformans capsular polysaccharide of domestic the first antigen, filled up domestic blank.
Experiment showed, that the polyclonal antibody that makes with accompanying method of the present invention has the height of tiring, the characteristics that specificity is good 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.
One, Polyclonal Antibody Preparation
1. immune animal
GXM 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 0.01-1mg.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.
2. 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-Cryptococcus neoformans capsular polysaccharide GXM 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.Detected result is seen Fig. 2.Can find out that from the result this antibody titer is very high, greater than 1: 1 * 10
6
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.
Claims (7)
1. the polyclonal antibody of an anti-Cryptococcus neoformans capsular polysaccharide antigen GXM is characterized in that with Cryptococcus neoformans capsular polysaccharide antigen GXM as immunogen, the polyclonal antibody that obtains through immune animal, separation and purifying antiserum(antisera).
2. the polyclonal antibody of an anti-Cryptococcus neoformans capsular polysaccharide antigen GXM 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-Cryptococcus neoformans capsular polysaccharide antigen GXM as claimed in claim 1 is characterized in that wrapping quilt with GXM, and indirect method ELISA detect to show that it is tired and is not less than 1: 1 * 10
6
4. the Polyclonal Antibody Preparation method of an anti-Cryptococcus neoformans capsular polysaccharide antigen GXM as claimed in claim 1 may further comprise the steps:
(a) use GXM 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-10mg; 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-Cryptococcus neoformans capsular polysaccharide antigen GXM of the arbitrary described method preparation of claim 1-6.
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| CN 201210011792 CN103204928A (en) | 2012-01-16 | 2012-01-16 | Preparation method for polyclonal antibody of cryptococcus neoformans |
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| CN 201210011792 CN103204928A (en) | 2012-01-16 | 2012-01-16 | Preparation method for polyclonal antibody of cryptococcus neoformans |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105646703A (en) * | 2016-02-29 | 2016-06-08 | 丹娜（天津）生物科技有限公司 | Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody |
| CN105784987A (en) * | 2016-02-29 | 2016-07-20 | 丹娜（天津）生物科技有限公司 | Preparation method of cryptococcus neoformans capsular polysaccharide GXM |
| CN107001450A (en) * | 2014-11-18 | 2017-08-01 | 巴斯德研究所 | It is specific to the sero-group X of Neisseria meningitidis polyclonal antibody and its purposes in diagnosis |
| CN109880805A (en) * | 2019-03-26 | 2019-06-14 | 天津喜诺生物医药有限公司 | Anti- cryptococcus capsular polysaccharide monoclonal antibody and its hybridoma cell strain preparation and application |
-
2012
- 2012-01-16 CN CN 201210011792 patent/CN103204928A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107001450A (en) * | 2014-11-18 | 2017-08-01 | 巴斯德研究所 | It is specific to the sero-group X of Neisseria meningitidis polyclonal antibody and its purposes in diagnosis |
| CN105646703A (en) * | 2016-02-29 | 2016-06-08 | 丹娜（天津）生物科技有限公司 | Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody |
| CN105784987A (en) * | 2016-02-29 | 2016-07-20 | 丹娜（天津）生物科技有限公司 | Preparation method of cryptococcus neoformans capsular polysaccharide GXM |
| CN105646703B (en) * | 2016-02-29 | 2020-05-26 | 丹娜（天津）生物科技有限公司 | Cryptococcus neoformans capsular polysaccharide GXM polyclonal antibody and preparation method thereof |
| CN109880805A (en) * | 2019-03-26 | 2019-06-14 | 天津喜诺生物医药有限公司 | Anti- cryptococcus capsular polysaccharide monoclonal antibody and its hybridoma cell strain preparation and application |
| CN109880805B (en) * | 2019-03-26 | 2022-09-06 | 天津喜诺生物医药有限公司 | Cryptococcus resistant capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof |
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Application publication date: 20130717 |