CN103204927A - Preparation method for monoclonal antibody of cryptococcus neoformans - Google Patents
Preparation method for monoclonal antibody of cryptococcus neoformans Download PDFInfo
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- CN103204927A CN103204927A CN 201210011791 CN201210011791A CN103204927A CN 103204927 A CN103204927 A CN 103204927A CN 201210011791 CN201210011791 CN 201210011791 CN 201210011791 A CN201210011791 A CN 201210011791A CN 103204927 A CN103204927 A CN 103204927A
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- cryptococcus neoformans
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Abstract
The invention relates to a preparation method for monoclonal antibody of cryptococcus neoformans. The invention relates to an antibody, a preparation method and an application thereof. Specifically, the invention relates to a mouse IgG monoclonal antibody BE-A6 directed against a cryptococcus neoformans capsular polysaccharide (Glucuronoxylomannan, GXM) antigen and a preparation method for the monoclonal antibody capable of resisting the cryptococcus neoformans capsular polysaccharide GXM antigen, and an application of the monoclonal antibody in antigen detection of the cryptococcus neoformans. The invention provides a method comprising the steps of preparing hybridoma cells by coupling protein molecules on the cryptococcus neoformans capsular polysaccharide GXM antigen and immunizing an animal with the cryptococcus neoformans capsular polysaccharide GXM antigen, then inoculating the animal with the hybridoma cells, separating and purifying. The monoclonal antibody BE-A6 prepared by the method has the characteristics of good specificity, high titer and high purity. Being applied in detections of the cryptococcus neoformans in the fields such as medical treatment and public health and scientific researches, the monoclonal antibody BE-A6 has the advantages of good sensitivity and good specificity, and has wide application prospects.
Description
Technical field
The present invention relates to antibody and preparation method thereof, especially for (Glucuronoxyloma-nnan, GXM) the MONOCLONAL ANTIBODIES SPECIFIC FOR method of a kind of mouse IgG type monoclonal antibody BE-A6 of antigen and anti-Cryptococcus neoformans capsular polysaccharide GXM antigen is used at the Cryptococcus neoformans capsular polysaccharide.
Background technology
Cryptococcus neoformans (Crytococcus Neoformans) is to one of very big condition pathogenic fungus of human health risk, mainly is present in birds droppings and by in the birds droppings Contaminated soil.The patient of the low or immunological competence defective of the normal infection immunity ability of Cryptococcus neoformans, and cause torulosis, normal because of discovery in time or mistaken diagnosis delay treatment, case fatality rate is very high.
In recent years owing to the long-term of immunosuppressor after broad-spectrum antifungal medicine, corticosteroid medication, monoclonal antibody drug, the organ transplantation used in a large number, and the increase of AIDS patient's quantity in recent years, the torulosis patient's that cryptococcus causes quantity increases year by year.Cryptococcus neoformans infects and often causes 3 big class diseases: cryptococcal meningitis, pulmonary cryptococosis and cryptococcosis cutis.Wherein, cryptococcal meningitis accounts for all Cryptococcus neoformans and infects and cause about 80% of disease.Neoformans Meningitis meningitis (hereinafter to be referred as " latent brain ") is by the central nervous system fungi infestation due to the Cryptococcus neoformans.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, positive rate about 55%;
2. body fluid cultured method such as hemoculture or cerebrospinal fluid cultivation, this method are the gold standard that cryptococcus detects, but susceptibility is poor, positive rate about 75%;
3. Cryptococcus neoformans capsular polysaccharide antigen immune is learned detection method: realize detection to Cryptococcus neoformans with specificity at the monoclonal antibody of Cryptococcus neoformans capsular polysaccharide antigen GXM, the susceptibility that detects blood and cerebrospinal fluid can reach 87% and 97% respectively.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 monoclonal 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.
In sum, in Cryptococcus neoformans clinical detection field, press for the monoclonal 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 mouse IgG type monoclonal antibody of the anti-Cryptococcus neoformans capsular polysaccharide antigen GXM of a kind of BE-A6 of numbering.
The object of the present invention is to provide a kind of method for preparing the hybridoma cell strain of numbering BE-A6, this cell strain can be secreted the monoclonal antibody BE-A6 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 sodium periodate method with GXM antigen and KLH (KeyHole Lympet Hemokyanin, KLH) albumen coupling.
With coupling the GXM antigen immune BALB/C mice of KLH albumen.
3. measure the serum titer of immunity back mouse, with the splenocyte taking-up of the mouse after the immunity, carry out cytogamy and screening with the myeloma cell, obtain hybridoma.
4. monoclonal hybridoma is expelled in the BALB/C mice body.
5. serum or the ascites that will obtain in the BALB/C mice body is carried out antibody purification, obtains the monoclonal antibody at GXM.
The method that is used for coupling in the above-mentioned steps 1 is diversified, except the sodium periodate oxidation style of using in the step 1, also comprises: carbodlimide method, glutaraldehyde method, activation fat method, mixed anhydride method etc.The kinds of protein that can be used for coupling is also a lot, except KLH can also be BSA (Bull Serum Albumin, BSA), TT (Tetanus Toxin, TT), OVA (Ovalbumin, OVA) etc.
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 monoclonal antibody can be the animal that mouse, rabbit, chicken, sheep, horse, pig, donkey etc. can be used for immunity.
Do not obtain immune effect preferably in the above-mentioned steps 3, should every about 7 days, carry out titration by the serum to immune animal.After immunity about 4 times, with sacrifice of animal, separating Morr. cell carries out cytogamy with tumour cell.The tumour cell kind that is used for cytogamy has a lot, as SP2/0-Ag14 (SP2/0), and F0, P3/X63-Ag8 (X63), U-266AR etc.
In the above-mentioned steps 3 after the cytogamy with carrying out cloning, the method for cloning is a lot, can be limiting dilution assay, soft agar culture method etc.
The method of injection cell strain can be abdominal injection in the above-mentioned steps 4, subcutaneous multi-point injection etc.
The method that is used for antibody purification in the above-mentioned steps 5 can be saturated ammonium sulphate salt precipitation method and affinity chromatography etc.
The monoclonal antibody of the mouse IgG type monoclonal antibody BE-A6 for preparing with aforesaid method and other kinds source and type needs the present invention to protect.
The invention provides the mouse IgG type monoclonal antibody of a kind of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM, and the MONOCLONAL ANTIBODIES SPECIFIC FOR 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 monoclonal antibody that the present invention obtains is the antibody of the anti-Cryptococcus neoformans capsular polysaccharide of domestic the first antigen, filled up domestic blank.
Experiment showed, that the mouse IgG type monoclonal antibody BE-A6 that makes with accompanying method of the present invention has the height of tiring, the characteristics that specificity is good are used it in scientific research and the clinical detection field, have highly sensitively, and the advantage that specificity is a lot of has very strong application prospect.
Accompanying drawing is described:
Fig. 1 has shown the result that the SDS-PAGE of mouse IgG type monoclonal antibody BE-A6 detects.
Fig. 2 has shown the result of the titration of mouse IgG type monoclonal antibody BE-A6.
Embodiment 1, preparation and the titration thereof of the mouse IgG type monoclonal antibody BE-A6 of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM
One, the coupling of GXM antigen and KLH
Concrete grammar is:
1) take by weighing 0.1mg KLH be dissolved in the 0.1ml carbonate buffer solution (pH9.5,0.01M) in.
2) taking by weighing 0.4mg GXM is dissolved in the 0.1ml deionized water.
3) with the NaIO of 0.02ml 0.1M
4Solution adds in the GXM solution, and room temperature concussion 20min carries out aldehyde reaction.
4) with the GXM solution of hydroformylation to pH4.4,4 ℃ of dialysed overnight of the acetate buffer of 1M.
5) use pH9.5, the carbonate buffer solution of 0.2M is transferred to pH9.0～9.5 with the GXM solution of hydroformylation, mixes stirring at room 2h with KLH solution.
6) add 0.1ml NaBH
4Solution, 4 ℃ of reaction 2h.
7) the even conjugate solution that obtains is to 4 ℃ of dialysed overnight of NaCl solution of 0.9%.
Two, the preparation of hybridoma cell strain BE-A6
1. immune animal
The GXM antigen of coupling KLH was mixed final volume 0.2ml in 1: 1 by volume with Freund's complete adjuvant.The abdominal injection immunity is carried out to 3 male BALB/C mice in fully emulsified back, and every mouse immune dosage is 50 μ g.Immunity was got tail blood in preceding 3 days, and separation of serum is done negative control.The 2nd, 4,6 weeks were carried out immunity respectively behind the initial immunity, and method is with for the first time identical.
2. cytogamy
1) preparation of feeder layer cells
Choose the 6-10 BALB/C mice in age in week, draw neck to put to death 5-6ml4 ℃ of precooling injected in the back to intraperitoneal with asepsis injector at aseptic condition RPMI1640 cell culture fluid (hereinafter referred " cell culture fluid ").With the nutrient solution sucking-off, in 1000rpm, 4 ℃ were descended centrifugal 5 minutes again.With the cell culture fluid re-suspended cell that contains 20% foetal calf serum, in cell transfer to 96 orifice plate, put into cell culture incubator and cultivate.Require 37 ℃ of culture temperature, CO
2Content 5.0%.
2) preparation immune spleen cell
Draw neck to put to death the mouse that the 4th time immunity is back 3 days, get spleen under the aseptic condition.Grind after washing 1 time with cell culture fluid, cross stainless steel mesh, will wash 2 times with cell culture fluid behind the cell centrifugation that obtain.
3) cytogamy
The take the logarithm SP2/0 myeloma cell of phase mixes with splenocyte.With the cell culture fluid that does not contain foetal calf serum wash once the back centrifugal, abandon supernatant.37 ℃ of constant temperature are about 90 seconds after adding polyglycol solution.With centrifugal after the cell culture fluid termination reaction that does not contain foetal calf serum, select nutrient solution resuspended with the HAT that contains 20% foetal calf serum again, cell is added in 96 orifice plates, put into cell culture incubator and cultivate.Require 37 ℃ of culture temperature, CO
2Content 5.0%.
4) cell monoclonalization and screening
Growth conditions good cell in 96 orifice plates is diluted to 1-3 cell/ml with cell culture fluid, joins in 96 orifice plates, put into cell culture incubator and cultivate.Require 37 ℃ of culture temperature, CO
2Content 5.0%.Each cell strain is numbered respectively, chooses the cell strain enlarged culturing that the nutrient solution supernatant is positive.Finally obtain the hybridoma cell strain of strain numbering BE-A6.
Three, the preparation of monoclonal antibody BE-A6
1. will number the hybridoma cell strain enlarged culturing of BE-A6, cell number reaches 10
6During/ml concentration, with resuspended with PBS behind the cell centrifugation, wash 2 times with PBS again, be expelled to the BALB/C mice intraperitoneal in 6 ages in week.2 weeks were injected pristane or whiteruss 0.5ml earlier before the injection cell strain in mouse peritoneal.The injection cell strain draws neck to put to death mouse after 2 weeks, extracts ascites.
2. get supernatant after ascites is centrifugal, carry out preliminary purification with saturated ammonium sulphate salt precipitation method.
3. load chromatography column with Protein A chromatographic stuffing, carry out purifying with the method for affinity chromatography.
Embodiment 2, the detection of the monoclonal antibody BE-A6 of anti-Cryptococcus neoformans capsular polysaccharide antigen GXM
1.SDS-PAGE electrophoresis detection
The antibody that step 3 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 BE-A6 swimming lane is BE-A6 antibody, 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-mouse 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 BE-A6 antibody titer is very high, 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.
Claims (12)
1. the monoclonal antibody BE-A6 of an anti-Cryptococcus neoformans capsular polysaccharide antigen GXM, it is characterized in that with after the Cryptococcus neoformans capsular polysaccharide antigen GXM coupling protein matter as immunogen, inoculate the mouse IgG type monoclonal antibody that animal, separation, purifying obtain through immunity, cytogamy, screening, hybridoma.
2. the monoclonal antibody BE-A6 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 monoclonal antibody BE-A6 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
5
4. the MONOCLONAL ANTIBODIES SPECIFIC FOR method of an anti-Cryptococcus neoformans capsular polysaccharide antigen GXM as claimed in claim 1 may further comprise the steps:
(a) use the sodium periodate method with Cryptococcus neoformans capsular polysaccharide antigen GXM and albumen coupling;
(b) GXM that obtains with (a) step and protein conjugate are as the immunogen immune animal;
(c) splenocyte and the tumour cell of step (b) gained animal carry out cytogamy, obtain hybridoma;
(d) hybridoma with step (c) gained is inoculated in the animal body;
(e) go out monoclonal antibody from the interior separation and purification of the animal body of step (d) gained.
5. as method as described in the claim 4, it is characterized in that in the step (a), the albumen that is used for coupling is keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxin (TT), the pure albumen of ovum gallinaceum (OVA).
6. method as claimed in claim 4 is characterized in that, in the step (b), the method for immunity is subcutaneous injection, intrasplenic injection, intravenous injection, abdominal injection; Immunizing dose is 10-1000 μ g/; The animal of immunity is rat, mouse, cavy, rabbit, chicken, sheep, horse, pig, donkey.
7. as method as described in the claim 4, it is characterized in that, in the step (c), the tumour cell kind that is used for cytogamy is: SP2/0, F0, X63, X63-Ag8.653, NS-1, P3U1, S194/5.XXO.BU.1, MPC11-45.6TG1.7,210.RCY3.Ag1.2.3, GM15006TG-A12, U-266AR.The method of cytogamy rear cloneization is limiting dilution assay, soft agar culture method.
8. as method as described in the claim 4, it is characterized in that in the step (c), the method for inoculating cell strain is abdominal injection, subcutaneous multi-point injection; The inoculation animal be rat, mouse, cavy, rabbit, chicken, sheep, horse, pig, donkey.
9. as method as described in the claim 4, it is characterized in that the method for the employed antibody purification of step (d) is saturated ammonium sulphate salt precipitation method and affinity chromatography.
10. use the monoclonal antibody of the anti-Cryptococcus neoformans capsular polysaccharide antigen GXM of the arbitrary described method preparation of claim 1-9.
11. an ELISA test kit that detects Cryptococcus neoformans capsular polysaccharide antigen GXM comprises the monoclonal antibody of the described anti-Cryptococcus neoformans capsular polysaccharide antigen GXM of claim 1-3.
12. test kit according to claim 11, it is characterized in that: described test kit also comprises the ELIAS secondary antibody of the monoclonal antibody of described anti-Cryptococcus neoformans capsular polysaccharide antigen GXM, the colour developing liquid of being made up of colour developing liquid A and colour developing liquid B or the single-component liquid that develops the color.
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| CN 201210011791 CN103204927A (en) | 2012-01-16 | 2012-01-16 | Preparation method for monoclonal antibody of cryptococcus neoformans |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740832A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Cryptococcus neoformans detecting kit |
| CN105651996A (en) * | 2016-02-29 | 2016-06-08 | 丹娜（天津）生物科技有限公司 | Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof |
| 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 |
| CN105968198A (en) * | 2016-06-27 | 2016-09-28 | 天津汇滨生物科技有限公司 | Monoclonal antibody of candida mannan and preparation method of monoclonal antibody |
| WO2017148330A1 (en) * | 2016-02-29 | 2017-09-08 | 丹娜（天津）生物科技有限公司 | Method for preparing crytococcus neoformans capsular polysaccharide gxm and gxm antigen immunoassay kit and use thereof |
| CN109879961A (en) * | 2019-03-26 | 2019-06-14 | 天津喜诺生物医药有限公司 | A kind of anti-cryptococcus capsular polysaccharide monoclonal antibody and its hybridoma cell strain preparation and application |
| CN110305184A (en) * | 2019-07-15 | 2019-10-08 | 武汉轻工大学 | A kind of buffer and its application for co-immunoprecipitation |
| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
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- 2012-01-16 CN CN 201210011791 patent/CN103204927A/en not_active Withdrawn
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740832A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Cryptococcus neoformans detecting kit |
| CN103740832B (en) * | 2014-01-15 | 2016-03-09 | 湖南圣湘生物科技有限公司 | A kind of Cryptococcus neoformans detection kit |
| CN105651996A (en) * | 2016-02-29 | 2016-06-08 | 丹娜（天津）生物科技有限公司 | Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof |
| 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 |
| WO2017148330A1 (en) * | 2016-02-29 | 2017-09-08 | 丹娜（天津）生物科技有限公司 | Method for preparing crytococcus neoformans capsular polysaccharide gxm and gxm antigen immunoassay kit and use thereof |
| CN105968198A (en) * | 2016-06-27 | 2016-09-28 | 天津汇滨生物科技有限公司 | Monoclonal antibody of candida mannan and preparation method of monoclonal antibody |
| CN109879961A (en) * | 2019-03-26 | 2019-06-14 | 天津喜诺生物医药有限公司 | A kind of anti-cryptococcus capsular polysaccharide monoclonal antibody and its hybridoma cell strain preparation and application |
| CN110305184A (en) * | 2019-07-15 | 2019-10-08 | 武汉轻工大学 | A kind of buffer and its application for co-immunoprecipitation |
| CN110305184B (en) * | 2019-07-15 | 2021-04-27 | 武汉轻工大学 | Buffer solution for co-immunoprecipitation and application thereof |
| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
| CN110954691B (en) * | 2019-12-17 | 2021-02-26 | 丹娜(天津)生物科技股份有限公司 | Sample pretreatment reagent and application thereof |
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Application publication date: 20130717 |