CN104628861A - 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof - Google Patents
1, 3-beta-D-glucan polyclonal antibody and preparation method thereof Download PDFInfo
- Publication number
- CN104628861A CN104628861A CN201510054177.4A CN201510054177A CN104628861A CN 104628861 A CN104628861 A CN 104628861A CN 201510054177 A CN201510054177 A CN 201510054177A CN 104628861 A CN104628861 A CN 104628861A
- Authority
- CN
- China
- Prior art keywords
- callose
- polyclonal antibody
- buffer
- immunogen
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a polyclonal antibody of a 1, 3-beta-D-glucan antigen, a preparation method and application of the polyclonal antibody in preparation of an ELISA detection kit. The preparation method of the antibody comprises the following steps: (1) preparing a 1, 3-beta-D-glucan immunogen; (2) carrying out animal immunization by using the 1, 3-beta-D-glucan immunogen; (3) collecting blood from an immunized animal body; and (4) purifying blood serum by sequentially using a saturated ammonium sulfate salting out method and a chromatography to obtain the polyclonal antibody. The antibody prepared by the method provided by the invention has the characteristics of high potency, high specificity and high purity and has a broad application prospect in the field of clinical diagnosis of invasive fungus diseases.
Description
Technical field
The present invention relates to immunogen and antibody and preparation method thereof, especially for for 1,3-callose immunogen and anti-polyclonal antibody of 1,3-callose and preparation method thereof.
Background technology
Invasive fungi disease (Invasive fungal disease, IFD) also known as deep fungal infection, refer to that fungi invades tissue, blood, and growth and breeding causes tissue injury, organ dysfunction, the pathological change of inflammatory reaction and pathophysiological process wherein.
In recent years, due to the long-term widespread use of immunosuppressor after extensive pedigree antibiotic, adrenocortical hormone, chemotherapy of tumors, radiotherapy and organ transplantation and the popular of acquired immune deficiency syndrome (AIDS), the sickness rate of invasive fungi disease raises year by year, this disease early symptom is without specificity, the course of disease is long, find more late, mortality ratio is high.Therefore, invasive fungi disease treatment success or failure key is to early diagnosis, gives antifungal therapy in early days.
The ordinary method of current invasive fungi medical diagnosis on disease, positive rate is low, be difficult to make a definite diagnosis, and nconventional method testing cost is high, experiment condition is harsh is difficult at hospital's popularity.Conventional tachypleus amebocyte lysate detects 1,3-callose, false positive many factors, and other antigen, antibody and enzyme testing product are general only for specific several fungi, are not suitable for a large amount of screening work of invasive fungi disease.And enzyme linked immunosorbent assay (ELISA) technology maturation, advantage of lower cost, diagnosis fast, is convenient to clinical labororatory and is used.Therefore, the ELISA detection system for fungi 1,3-callose has very important clinical value, and anti-1, the 3-callose antibody of preparation is the gordian technique setting up this ELISA system.
Non-patent literature " (1; 3)-callose ELISA detection method is set up and Preliminary Clinical " (great variety of modern medical examining magazine, 2003,5th phase) in disclose with galactosyl ceramide be encrusting substance, mouse-anti (1,3)-callose monoclonal antibody, biotinylation against murine IgG and Horseradish peroxidase-conjugated avidin is that detection system sets up ELISA method.
Non-patent literature " Development of a two-site enzyme immunoassay based on monoclonal antibodies to measure airborne exposure to (1 → 3)-β-d-glucan " (Journal of Immunological Methods, V.337, No.1) to be oxidized laminarin immune mouse, obtain 1,3-callose monoclonal antibody, on this basis, a kind of double antibody sandwich ELISA is established.But use anti-(1,3)-callose monoclonal antibody in aforesaid method, preparation method is comparatively complicated.
Although disclose the preparation method of dextran polyclonal antibody in prior art, usually adopt dextran and BSA cross-linking agent to be antigen, after carrying out animal immune, obtain dextran polyclonal antibody.But above-mentioned dextran polyclonal antibody or do not possess the purity of specificity or antibody and tire and can not meet the needs of disease detection, is not suitable for ELISA detection system.
In order to overcome the deficiencies in the prior art, the invention provides a kind of 1,3-callose polyclonal antibody and preparation method thereof.
Summary of the invention
One object of the present invention is to provide a kind of 1,3-callose polyclonal antibody and preparation method thereof, solves 1,3-callose antibody complicated process of preparation, tires low, the defect of poor specificity.
Another object of the present invention is to provide a kind of 1, the application of 3-callose polyclonal antibody in invasive fungi disease detection, advantage of lower cost, diagnosis is fast, reduce the false positive of detection, be suitable for the screening of a large amount of clinical sample of invasive fungi disease.
Therefore, first aspect present invention provides a kind of 1,3-callose polyclonal antibody, and the preparation method of described antibody comprises:
(1) 1,3-callose immunogen is prepared;
(2) animal immune is carried out by 1,3-callose immunogen;
(3) in the animal body after immunity, blood is got;
(4) with saturated ammonium sulphate salting-out process and affinity chromatography, serum is purified successively, obtain polyclonal antibody.
In step of the present invention (1), the immunogenic method of preparation 1,3-callose comprises: 1,3-callose and coupling protein coupling are prepared 1,3-callose immunogen; Or, the broken liquid of broken acquisition after fungal spore and/or yeast culture is prepared 1,3-callose immunogen.How the key of anti-preparation is the synthesis of immunizing antigen, and immunizing antigen needs purity good, and can keep the chemical structure of 1,3-callose, is conducive to the generation of follow-up animal internal antibody.
Described step (1) is 1, 3-callose and coupling protein coupling prepare 1, during 3-callose immunogen, 1, 3-callose can be well known in the art 1, 3-callose, such as laminarin (Laminarin), Pachymose (Pachyman), carboxymethylation Pachymose (Carboxymethyl pachyman), curdlan (Curdlan), carboxymethylation curdlan (Carboxymethyl curdlan), algal polysaccharide (Paramylon), lentinan (Lentinan), lichenstarch (Pustulan), yeast glucan (Yeast glucan), barley (Barley glucan), oat beta-glucan (Oat glucan), dextran (Dextran) etc.Preferably, 1,3-described callose is selected from laminarin, Pachymose, carboxymethylation Pachymose, curdlan, carboxymethylation curdlan.Most preferably, 1,3-described callose is gelling polysaccharide or carboxymethylation curdlan.Described coupling protein can be bovine serum albumin (Bull serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), tetanus toxin (Tetanus toxin, TT), chicken ovalbumin (Ovalbumin, OVA), bovine serum albumin is preferably.The coupling method of 1,3-callose and albumen can select iodate oxidation style, DMTMM reagent method, carbodlimide method, glutaraldehyde method, activation fat method, mixed anhydride method etc.
In a specific embodiment of the present invention, described coupling step comprises: by laminarin solution and NaIO
4solution mixes, and room temperature lucifuge reaction 0.5-3 hour, preferred reaction 1 hour, uses chromatographic column desalination, adds bovine serum albumin and NaBH by the solution obtained
3cN solution, shaken at room temperature reacts, and adds NaBH
4solution, shaken at room temperature reacts, with phosphate buffered saline buffer dialysis, centrifugal removing precipitation.In another embodiment of the present invention, described coupling step comprises: by carboxymethylation Pachymose and/or carboxymethylation curdlan solution and DMTMM (4-(4,6-dimethoxy-triazine-2-base)-4-methyl morpholine hydrochloride) solution mixing, oscillatory reaction, ultrapure water dialysed overnight, add BSA, room temperature lucifuge shaken overnight, centrifugal removing precipitation.
Described step (1) prepares 1 for fragmentation after fungal spore and/or yeast culture obtains broken liquid, during 3-callose immunogen, described fungi is the pathomycete that this area is suitable for, and comprises aspergillus tubigensis (Aspergillus), candidiasis (Candida albaican), cryptococcus (Crytococcus), mucormycosis (Mucor), Penicillium marneffei (Penicillium marneffei), sickle-like bacteria (Fusarium), Pneumocystis carinii (Penumocystis carinii bacteria).Described mycothallus can adopt collects thalline through solution culture fermentation; Described fungal spore can collect spore through plate solid culture wash-out.Described destruction step can adopt grinding fragmentation and/or ultrasonication.In the present invention's embodiment, described step (1) can comprise: pathomycete is collected thalline through solution culture fermentation, with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, thalline pours liquid nitrogen grinding fragmentation into, collects supernatant liquor; Or pathomycete is collected spore through plate solid culture wash-out, and with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, spore ultrasonication, collects supernatant liquor.
Preferably, in described step (1), 1,3-callose and coupling protein coupling prepare 1, after 3-callose immunogen, the optional authentication step comprised protein modified rear 1,3-callose, described authentication method method comprises high performance size exclusion chromatography method, ultraviolet/infrared spectroscopy, polyacrylamide gel electrophoresis etc.
In step of the present invention (2), the animal of preparing polyclonal antibody that described animal can use for this area, be selected from: one or more in mouse, rat, cavy, rabbit, chicken, sheep, horse, pig, donkey, be preferably mouse, rat, rabbit.The method of described immunity is selected from hypodermic injection, intrasplenic injection method, intravenous injection, intraperitoneal injection etc.Immunizing dose in step (2) immune step is determined by concrete animal species, and preferred immunizing dose is 10-1000 μ g//time.In the present invention one preferably embodiment, described step (2) comprising: step (1) obtained 1,3-callose immunogen mixes with Freund's complete adjuvant equal-volume, fully emulsifiedly rear subcutaneous multi-point injection is carried out to animal, immunizing dose is every animal 10-1000 μ g, every 2-4 week immunity 1 time after initial immunity, immune time >=3 time.Preferably, immunity in every 2 weeks 1 time after described initial immunity, immune time is 3-5 time, and preferred immune time is 3 times.
In step of the present invention (3), preferably, get blood in the animal body after immunity before, also comprise the step of the serum titer measuring animals following immunization, preferred, the serum titer step of described mensuration animals following immunization was carried out once every 1-7 days after immunity.In the animal body after immunity, get blood in described step (3) take a blood sample after putting to death, also can not put to death.Preferred, described in the animal body after immunity, get blood step comprise: with the method blood sampling of carotid artery bloodletting, treat blood coagulation, after serum is isolated, centrifugal, get supernatant.
In step of the present invention (4), described purification process for first to carry out preliminary purification by saturated ammonium sulphate salt precipitation method, then is further purified with affinity chromatography.Existing antibody purification process exists multiple, such as ammonium sulfate precipitation method, caprylic acid precipition, DEAE ion exchange chromatography, light base phosphatic rock chromatography, gel chromatography, affinity chromatography.The purification process that antibody of different nature is suitable for is different, need to filter out technique simple, can high-titer antibody be obtained.
Saturated ammonium sulphate salt precipitation method of the present invention comprises: get the serum that step (3) obtains, add isopyknic physiological saline, then add saturated ammonium sulphate solution, 0-10 DEG C of precipitates overnight, is preferably 4 DEG C of precipitates overnight; The centrifugal 5-20min of 10000G, preferred centrifugal 10min, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, slowly drips saturated ammonium sulphate solution, 0-10 DEG C of standing 0.5-2h, preferably 4 DEG C of standing 1h; The centrifugal 5-20min of 10000G, preferred centrifugal 10min, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, by phosphate buffered saline buffer 0-10 DEG C dialysed overnight, preferably 4 DEG C of dialysed overnight.
Affinity chromatography of the present invention comprises: wash post with the elution buffer of 5-10 times of column volume; Post is washed with the coupling buffer of 5-10 times of column volume; By the sample loading crossed with saturated ammonium sulphate salting-out process preliminary purification; Post is washed with the coupling buffer of 5-10 times of column volume; With the elution buffer wash-out of 2-5 times of column volume, obtain anti-1,3-callose polyclonal antibody.Preferably, described elution buffer is the one in glycine-HCI damping fluid (pH 4.2), citrate buffer solution (pH 4.5) etc., described coupling buffer is the one in 0.1mol/L phosphate buffered saline buffer (pH 7.4), citrate buffer (pH 6.0) etc., described affinity chromatographic column is as column packing with the crosslinked solid phase carrier of 1,3-callose antigen, staphylococcal protein A,SPA or streptococcal protein G.
Another aspect provides a kind of 1,3-callose preparation method of polyclonal antibody, comprising:
(1) 1,3-callose immunogen is prepared;
(2) animal immune is carried out by 1,3-callose immunogen;
(3) in the animal body after immunity, blood is got;
(4) with saturated ammonium sulphate salting-out process and affinity chromatography, serum is purified successively, obtain polyclonal antibody.
Preferably, described step (1) comprising:
By 1,3-callose and NaIO
4solution mixes, room temperature lucifuge reaction 0.5-3 hour, and preferred reaction 1 hour, uses chromatographic column desalination, is antigen 1 in mass ratio by adding in the solution obtained, 3-callose 1-10 BSA and NaBH doubly
3cN solution, shaken at room temperature reacts, and adds NaBH
4solution, shaken at room temperature reacts, with phosphate buffered saline buffer dialysis, centrifugal removing precipitation;
Or, by 1,3-callose immunogen and DMTMM (4-(4,6-dimethoxy-triazine-2-base)-4-methyl morpholine hydrochloride) solution mixing, oscillatory reaction, ultrapure water dialysed overnight, add is antigen 1 in mass ratio, 3-callose 1-10 BSA doubly, room temperature lucifuge shaken overnight, centrifugal removing precipitation;
Or, pathomycete is collected thalline through solution culture fermentation, with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, thalline pours liquid nitrogen grinding fragmentation into, collects supernatant liquor;
Or by pathomycete after plate solid culture, wash-out collects spore, with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, spore ultrasonication, collects supernatant liquor.
1,3-described callose is preferably one or more the combination in laminarin, Pachymose, carboxymethylation Pachymose or methylolation curdlan.
Preferably, described step (2) comprising: step (1) obtained 1,3-callose immunogen mixes with Freund's complete adjuvant equal-volume, fully emulsifiedly rear subcutaneous multi-point injection is carried out to animal, immunizing dose is every animal 10-1000 μ g, every 2-4 week immunity 1 time after initial immunity, immune time >=3 time.
Preferably, described step (3) comprising: after measuring the serum titer of animals following immunization, take a blood sample, treat blood coagulation by the method for carotid artery bloodletting, after serum is isolated, centrifugal, gets supernatant.
Preferably, described step (4) comprising: described purification process for first to carry out preliminary purification by saturated ammonium sulphate salt precipitation method, then is further purified with affinity chromatography.Described saturated ammonium sulphate salt precipitation method comprises: get the serum that step (3) obtains, add isopyknic physiological saline, then add saturated ammonium sulphate solution, 0-10 DEG C of precipitates overnight, is preferably 4 DEG C of precipitates overnight; The centrifugal 5-20min of 10000G, preferred centrifugal 10min, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, slowly drips saturated ammonium sulphate solution, 0-10 DEG C of standing 0.5-2h, preferably 4 DEG C of standing 1h; The centrifugal 5-20min of 10000G, preferred centrifugal 10min, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, by phosphate buffered saline buffer 0-10 DEG C dialysed overnight, preferably 4 DEG C of dialysed overnight.Described affinity chromatography comprises: wash post with the elution buffer of 5-10 times of column volume; Post is washed with the coupling buffer of 5-10 times of column volume; By the sample loading crossed with saturated ammonium sulphate salting-out process preliminary purification; Post is washed with the coupling buffer of 5-10 times of column volume; With the elution buffer wash-out of 2-5 times of column volume, obtain anti-1,3-callose polyclonal antibody.Preferably, described elution buffer is the one in glycine-HCI damping fluid (pH4.2), citrate buffer solution (pH 4.5) etc., described coupling buffer is the one in 0.1mol/L phosphate buffered saline buffer (pH 7.4), citrate buffer (pH 6.0) etc., described affinity chromatographic column is as column packing with the crosslinked solid phase carrier of 1,3-callose antigen, staphylococcal protein A,SPA or streptococcal protein G.
Another aspect provides a kind of 1, the 3-described application of callose polyclonal antibody in the test kit for the preparation of invasive fungi disease detection.Described detection refers to and utilizes the specific binding between antigen-antibody to react, and is preferably MBP enzyme linked immuno-adsorbent assay, the detection of immune colloid, Immunofluorescence test detection, chemiluminescence immune assay detection, is more preferably MBP enzyme linked immuno-adsorbent assay.The disease that invasive fungi disease and invasive infections with fungi cause, invasive fungi comprises candidiasis, aspergillus tubigensis, cryptococcus, mucormycosis, lung pityrosporion ovale, sickle-like bacteria, Dematiaceous fungi, trichosporon bacteria, histoplasma capsulatum, coccidioides immitis, blastomycete, Ma Nifei mould, fibrillae of spores etc., preferably aspergillus fumigatus, Candida albicans and Cryptococcus neoformans.
Another aspect of the present invention provides a kind of ELISA detection kit for invasive fungi disease detection, comprises envelope antigen, 1,3-callose polyclonal antibody and ELIAS secondary antibody.Described envelope antigen is 1,3-callose, such as laminarin (Laminarin), Pachymose (Pachyman), carboxymethylation Pachymose (Carboxymethyl pachyman), curdlan (Curdlan), carboxymethylation curdlan (Carboxymethyl curdlan), algal polysaccharide (Paramylon), lentinan (Lentinan), lichenstarch (Pustulan), yeast glucan (Yeast glucan), barley (Barley glucan), oat beta-glucan (Oat glucan), dextran (Dextran) etc., preferably, described 1,3-callose is selected from laminarin (Laminarin), Pachymose (Pachyman), carboxymethylation Pachymose (Carboxymethyl pachyman), curdlan (Curdlan), carboxymethylation curdlan (Carboxymethyl curdlan), most preferably, 1,3-described callose is carboxymethylation curdlan (Carboxymethyl curdlan).1,3-described callose polyclonal antibody is preferably 1,3-callose polyclonal antibody of the present invention.Described ELIAS secondary antibody is that horseradish peroxidase (HRP) marks IgG antibody, and preferably, HRP marks goat anti-rabbit igg antibody.
In an embodiment of the invention, also comprise in the described ELISA detection kit for invasive fungi disease detection: one or more in washings, substrate nitrite ion, antigen standard, stop buffer.Described washings is preferably PBST washings, and described substrate nitrite ion is TMB nitrite ion.Described antigen standard is 1,3-callose antigen.Described stop buffer is preferably H
2sO
4, be more preferably 2mol/L H
2sO
4.
The detection method also providing a kind of invasive fungi disease on the one hand of the present invention, comprising: (1) is that envelope antigen prepares antigen coated enzyme plate with 1,3-callose; (2) the antigen coated enzyme plate using (1) to prepare, adds antigen standard or detected sample, then adds 1,3-callose polyclonal antibody, mix latter 37 DEG C and hatch; (3) wash plate 1-5 time with washings, add ELIAS secondary antibody, hatch 1-3h for 37 DEG C, washings washes plate 1-5 time, adds the colour developing of substrate nitrite ion, adds stop buffer and stops; (4) OD is measured
450nm.Described envelope antigen is 1,3-callose, such as laminarin (Laminarin), Pachymose (Pachyman), carboxymethylation Pachymose (Carboxymethyl pachyman), curdlan (Curdlan), carboxymethylation curdlan (Carboxymethyl curdlan), algal polysaccharide (Paramylon), lentinan (Lentinan), lichenstarch (Pustulan), yeast glucan (Yeast glucan), barley (Barley glucan), oat beta-glucan (Oat glucan), dextran (Dextran) etc., preferably, described 1,3-callose is selected from laminarin (Laminarin), Pachymose (Pachyman), carboxymethylation Pachymose (Carboxymethyl pachyman), curdlan (Curdlan), carboxymethylation curdlan (Carboxymethyl curdlan), most preferably, 1,3-described callose is carboxymethylation curdlan (Carboxymethyl curdlan).1,3-described callose polyclonal antibody is preferably 1,3-callose polyclonal antibody of the present invention.Described ELIAS secondary antibody is that horseradish peroxidase (HRP) marks IgG antibody, and preferably, HRP marks goat anti-rabbit igg antibody.Described detected sample is serum to be detected.
In an embodiment of the invention, the detection method of described use invasive fungi disease, comprising: (1) is that envelope antigen prepares antigen coated enzyme plate with 1,3-callose; (2) the antigen coated enzyme plate using (1) to prepare, adds 50 μ L antigen standard or detected samples, then adds 50 μ L1,3-callose polyclonal antibody, mix latter 37 DEG C and hatch 2h; (3) wash plate 3 times with PBST washings, add the goat anti-rabbit igg antibody that 100 μ L HRP mark, hatch 30min for 37 DEG C, PBST washings washes plate 3 times, adds the colour developing of tmb substrate nitrite ion, adds stop buffer and stops; (4) microplate reader reads OD
450nm.
The detection method of a kind of invasive fungi disease of the present invention can be therapeutic purpose, can be non-therapeutic purpose.Thus, present invention also offers a kind of method of the detection invasive fungi disease for non-treatment object.
The invention provides 1,3-callose polyclonal antibody and preparation method thereof, immunogen is that chemically modified and fungi extract acquisition, purity is good, and can keep the chemical structure of 1,3-callose, the polyclonal antibody of preparation has high-titer, the feature of high specific, is shown as homogeneous antibody products with SDS-PAGE, indirect ELISA detection display its tire and be not less than 1:1.28 × 10
5, this antibody, in invasive fungi disease clinical diagnosis field, has a extensive future.
Accompanying drawing explanation
Figure 1 shows the high performance size exclusion chromatography method detected result of the laminarin after BSA modification.
Figure 2 shows the SDS-PAGE detected result of the rabbit igg type polyclonal antibody Ab-3B that the carboxymethylation curdlan immunity after BSA modification obtains.
Figure 3 shows the titration result of the rabbit igg type polyclonal antibody Ab-3B that the carboxymethylation curdlan immunity after BSA modification obtains.
Figure 4 shows the specific detection result of ELISA competition law system to various thalline.
Figure 5 shows the sensitivity technique result of ELISA competition law system.
Embodiment
The immunogenic preparation of embodiment 11,3-callose
(1) immunogen is prepared in laminarin and BSA coupling
By 10mL 20mg/mL laminarin solution and 0.5mL 5mol/LNaIO
4solution mixes, and room temperature lucifuge reaction 60min, with the desalination of Sephadex-G25 post.Antigen laminarin quality 1-10 BSA and NaBH is doubly added in above-mentioned solution
3cN solution, shaken at room temperature reacts.Add NaBH
4solution, shaken at room temperature reacts.By 0.01mol/L phosphate buffered saline buffer dialysed overnight.Last centrifugal removing precipitation.
(2) immunogen is prepared in carboxymethylation Pachymose or methylolation curdlan and BSA coupling
By carboxymethylation Pachymose or methylolation curdlan solution and the mixing of DMTMM solution, oscillatory reaction, ultrapure water dialysed overnight, adds antigen carboxymethylation Pachymose or methylolation curdlan quality 1-10 BSA doubly, room temperature lucifuge shaken overnight, last centrifugal removing precipitation.
(3) mycothallus and spore is adopted to prepare immunogen
Common pathomycete is collected thalline through solution culture fermentation, spore is collected through plate solid culture wash-out, with the physiological saline deactivation containing formaldehyde, repeatedly washing removing formaldehyde, thalline pours liquid nitrogen grinding fragmentation into, collects supernatant liquor, and spore is with after blood cell plate method counting, ultrasonication, obtains immunogen.
The immunogenic qualification of embodiment 21,3-callose
Use high performance size exclusion chromatography method, 1,3-callose after the BSA prepared according to the method for (1) and (2) in Example 1 modifies suitably dilutes, with the BSA solution of 5mg/mL in contrast.Chromatographic condition: chromatographic column is TSK-GELG3000SWXL, sampling volume is 20 μ L, and column temperature is normal temperature, and moving phase is 0.3mol/L sodium-chlor-0.05mol/L phosphate buffered saline buffer (pH6.8) solution, and flow velocity is 1mL/min, and determined wavelength is 280nm.
The qualification result of the laminarin wherein after BSA modification is shown in accompanying drawing 1.
The preparation of anti-1, the 3-callose polyclonal antibody of embodiment 3
(1) immune animal
1, the 3-callose immunogen obtained in embodiment 1 (1) (laminarin through BSA modifies) is mixed to suitable volumes with Freund's complete adjuvant equal-volume.Fully emulsifiedly carry out subcutaneous multi-point injection to new zealand rabbit afterwards, every rabbit immunizing dose controls at 0.01-1mg.Immunity gets ear blood in first 3 days, and separation of serum does negative control.Immunity in every 2 weeks 1 time after initial immunity, method is identical with the 1st time.
(2) polyclonal antibody purification
1) titration: in immunologic process, tire 1 time every blood sampling survey in several days after immunity, immune time is no less than 3 times.
2) antiserum(antisera) is separated: when serum titer reaches the highest, take a blood sample in a large number by the method for carotid artery bloodletting.Treat blood coagulation, after serum is isolated, high speed centrifugation, gets supernatant ,-20 DEG C of preservations.
(3) preliminary purification is carried out with saturated ammonium sulphate salting-out process
1) get 2mL antiserum(antisera) sample, add isopyknic physiological saline, then add 4mL saturated ammonium sulphate solution, 4 DEG C of precipitates overnight.
2) 10000G low-temperature centrifugation 10min, abandons supernatant, is dissolved by precipitation 2mL phosphate buffered saline buffer, slowly drips 1mL saturated ammonium sulphate solution, 4 DEG C of standing 1h.
3) 10000G low-temperature centrifugation 10min, abandons supernatant, dissolves, precipitation 1mL phosphate buffered saline buffer by phosphate buffered saline buffer 4 DEG C of dialysed overnight.
(4) be further purified by the method for affinity chromatography
1) post is washed with the elution buffer of 10 times of column volumes;
2) post is washed with the coupling buffer of 10 times of column volumes;
3) the sample loading will crossed with saturated ammonium sulphate salting-out process preliminary purification;
4) post is washed with the coupling buffer of 10 times of column volumes;
5) with the elution buffer wash-out of 5 times of column volumes, UV-detector detects, and collects protein peak and is anti-1,3-callose polyclonal antibody;
6) be adjusted to pH value neutrality with 1mol/L Tris-HCl damping fluid (pH 9.0), use phosphate buffered saline buffer dialysed overnight.
Wherein, in chromatography column with the crosslinked solid phase carrier of staphylococcal protein A,SPA as column packing, elution buffer is glycine-HCI damping fluid (pH 4.2), and coupling buffer is 0.1mol/L phosphate buffered saline buffer (pH 7.4).
The preparation of anti-1, the 3-callose polyclonal antibody of embodiment 4
(1) immune animal
1, the 3-callose immunogen obtained in embodiment 1 (2) (the methylolation Pachymose through BSA modifies) is mixed to suitable volumes with Freund's complete adjuvant equal-volume.Fully emulsifiedly carry out subcutaneous multi-point injection to new zealand rabbit afterwards, every rabbit immunizing dose controls at 0.01-1mg.Immunity gets ear blood in first 3 days, and separation of serum does negative control.Immunity in every 2 weeks 1 time after initial immunity, method is identical with the 1st time.
(2) polyclonal antibody purification
1) titration: in immunologic process, tire 1 time every blood sampling survey in several days after immunity, immune time is no less than 3 times.
2) antiserum(antisera) is separated: when serum titer reaches the highest, take a blood sample in a large number by the method for carotid artery bloodletting.Treat blood coagulation, after serum is isolated, high speed centrifugation, gets supernatant ,-20 DEG C of preservations.
(3) preliminary purification is carried out with saturated ammonium sulphate salting-out process
1) get 2mL antiserum(antisera) sample, add isopyknic physiological saline, then add 4mL saturated ammonium sulphate solution, 4 DEG C of precipitates overnight.
2) 10000G low-temperature centrifugation 10min, abandons supernatant, is dissolved by precipitation 2mL phosphate buffered saline buffer, slowly drips 1mL saturated ammonium sulphate solution, 4 DEG C of standing 1h.
3) 10000G low-temperature centrifugation 10min, abandons supernatant, dissolves, precipitation 1mL phosphate buffered saline buffer by phosphate buffered saline buffer 4 DEG C of dialysed overnight.
(4) be further purified by the method for affinity chromatography
1) post is washed with the elution buffer of 7.5 times of column volumes;
2) post is washed with the coupling buffer of 7.5 times of column volumes;
3) the sample loading will crossed with saturated ammonium sulphate salting-out process preliminary purification;
4) post is washed with the coupling buffer of 7.5 times of column volumes;
5) with the elution buffer wash-out of 4 times of column volumes, UV-detector detects, and collects protein peak and is anti-1,3-callose polyclonal antibody;
6) be adjusted to pH value neutrality with 1mol/L Tris-HCl damping fluid (pH 9.0), use phosphate buffered saline buffer dialysed overnight.
Wherein, in chromatography column with the solid phase carrier of 1,3-callose antigen cross-linking as column packing, elution buffer is citrate buffer solution (pH 4.5), and coupling buffer is 0.1mol/L citrate buffer (pH 6.0).
The preparation of anti-1, the 3-callose polyclonal antibody of embodiment 5
(1) immune animal
1, the 3-callose immunogen obtained in embodiment 1 (2) (the methylolation curdlan through BSA modifies) is mixed to suitable volumes with Freund's complete adjuvant equal-volume.Fully emulsifiedly carry out subcutaneous multi-point injection to new zealand rabbit afterwards, every rabbit immunizing dose controls at 0.01-1mg.Immunity gets ear blood in first 3 days, and separation of serum does negative control.Immunity in every 2 weeks 1 time after initial immunity, method is identical with the 1st time.
(2) polyclonal antibody purification
1) titration: in immunologic process, tire 1 time every blood sampling survey in several days after immunity, immune time is no less than 3 times.
2) antiserum(antisera) is separated: when serum titer reaches the highest, take a blood sample in a large number by the method for carotid artery bloodletting.Treat blood coagulation, after serum is isolated, high speed centrifugation, gets supernatant ,-20 DEG C of preservations.
(3) preliminary purification is carried out with saturated ammonium sulphate salting-out process
1) get 2mL antiserum(antisera) sample, add isopyknic physiological saline, then add 4mL saturated ammonium sulphate solution, 4 DEG C of precipitates overnight.
2) 10000G low-temperature centrifugation 10min, abandons supernatant, is dissolved by precipitation 2mL phosphate buffered saline buffer, slowly drips 1mL saturated ammonium sulphate solution, 4 DEG C of standing 1h.
3) 10000G low-temperature centrifugation 10min, abandons supernatant, dissolves, precipitation 1mL phosphate buffered saline buffer by phosphate buffered saline buffer 4 DEG C of dialysed overnight.
(4) be further purified by the method for affinity chromatography
1) post is washed with the elution buffer of 5 times of column volumes;
2) post is washed with the coupling buffer of 5 times of column volumes;
3) the sample loading will crossed with saturated ammonium sulphate salting-out process preliminary purification;
4) post is washed with the coupling buffer of 5 times of column volumes;
5) with the elution buffer wash-out of 2 times of column volumes, UV-detector detects, and collects protein peak and is anti-1,3-callose polyclonal antibody;
6) be adjusted to pH value neutrality with 1mol/L Tris-HCl damping fluid (pH 9.0), use phosphate buffered saline buffer dialysed overnight.
Wherein, in chromatography column with the crosslinked solid phase carrier of staphylococcal protein A,SPA as column packing, elution buffer is citrate buffer solution (pH 4.5), and coupling buffer is 0.1mol/L phosphate buffered saline buffer (pH 7.4).
The checking of anti-1, the 3-callose polyclonal antibody of embodiment 6
(1) SDS-PAGE electrophoresis detection
The antibody A b-3B obtained to embodiment 5 carries out SDS-PAGE electrophoresis, and the gel obtained carries out coomassie brilliant blue staining.
Experimental result is shown in accompanying drawing 2, can find out by figure, has clear obvious band, prove that antibody purity is high in 25kD and 50kD molecular weight area.
(2) antibody titer measures
The antibody A b-3B antibody titer obtained to embodiment 5 with indirect elisa method measures.ELIAS secondary antibody used is the goat anti-rabbit igg of horseradish peroxidase-labeled, and negative control is phosphate buffered saline buffer.Detected result is shown in accompanying drawing 3, can find out from result, and this antibody titer is greater than 1:1.28 × 10
5, show that antibody titer is high.
The foundation of embodiment 7 ELISA competition law system and checking
Using carboxymethylation curdlan as envelope antigen, antibody A b-3B and HRP marks goat anti-rabbit igg antibody as detection antibody, establishes indirect ELISA competition law detection system.Detection method is as follows: use antigen coated enzyme plate, add 50 μ L antigen standard (or the serum after process) and 50 μ L antibody A b-3B respectively successively, mix latter 37 DEG C and hatch 2h, PBST washes plate 3 times, add the goat anti-rabbit igg antibody that 100 μ L HRP mark, hatch 30min for 37 DEG C, PBST washes plate 3 times, stop after finally adding the colour developing of tmb substrate solution, microplate reader reads OD
450nm.
(1) specificity verification of ELISA competition law system
Preparation containing bacterium serum sample: the thalline dry powder (thalline comprises aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, mycobacterium tuberculosis, intestinal bacteria, Salmonellas, klebsiella and streptococcus aureus) weighing 10mg inactivation treatment respectively, dissolve with Healthy Human Serum and dilute, make the bacterium powder containing 100000,10000,1000,100,10 and 1ng/mL in serum sample, put into 37 DEG C of thermostat containers and hatch 2d.
Serum sample process: by serum sample and sample treatment solution mixing, incubation with heat, high speed centrifugation collects supernatant liquor.Sample treatment solution is the one in EDTA treatment solution, glycine treatment solution and protease treatment liquid etc.
Finally detect by indirect ELISA competition law system after the process of different concns containing bacterium serum sample.
Experimental result is shown in accompanying drawing 4, found out by figure, this antibody capable is effectively from detecting 3 kinds of common fungal pathogen bacterium (comprising aspergillus fumigatus, Candida albicans and Cryptococcus neoformans) serum sample, and strong to 5 kinds of common bacterial pathogen (comprising mycobacterium tuberculosis, intestinal bacteria, Salmonellas, klebsiella and streptococcus aureus) immunity from interferencies.Prove that antibody is used for invasive fungi disease detection and has high specific.
(2) the sensitivity checking of ELISA competition law system
1, the preparation of 3-callose standard substance: laminarin, Pachymose and curdlan are diluted to 10000,1000,100,10,1,0.1 and 0.01ng/mL with Sample dilution, Sample dilution is the one in 0.1mol/L Tris-HCl damping fluid (pH 8.5), 0.1mol/L phosphate buffered saline buffer (pH 7.4), 0.1mol/L citrate buffer (pH 6.0) etc.
1,3-callose antigen standard of above-mentioned different concns is finally detected by indirect ELISA competition law system.
Experimental result is shown in accompanying drawing 5, is found out by figure, and this indirect ELISA competition law system at least can reach 1,0.1 and 0.01ng/mL to the Monitoring lower-cut of laminarin, Pachymose and curdlan.
This specification sheets above in conjunction with embodiment to invention has been explaination, but should be understood that these describe and explaination just in order to understand the present invention better, and not form any restriction of the present invention.Those skilled in the art can carry out necessary change to the specific embodiment of the present invention and not depart from the spirit and scope of the present invention after having read present specification.Protection scope of the present invention is limited by the accompanying claims, and covers the equivalents of claim.
Claims (14)
1. anti-1, a 3-callose polyclonal antibody, it is characterized in that, its preparation method comprises:
(1) 1,3-callose immunogen is prepared;
(2) animal immune is carried out by 1,3-callose immunogen;
(3) in the animal body after immunity, blood is got;
(4) with saturated ammonium sulphate salting-out process and affinity chromatography, serum is purified successively, obtain polyclonal antibody.
2. anti-1, the 3-callose polyclonal antibody of one according to claim 1, it is characterized in that, described step (1) comprising: 1,3-callose and coupling protein coupling are prepared 1,3-callose immunogen; 1,3-described callose is selected from laminarin, Pachymose, carboxymethylation Pachymose, curdlan, carboxymethylation curdlan, algal polysaccharide, lentinan, lichenstarch, yeast glucan, barley, oat beta-glucan, dextran; Described coupling protein is selected from bovine serum albumin, keyhole limpet hemocyanin, tetanus toxin, chicken ovalbumin;
Or described step (1) comprising: the broken liquid of broken acquisition after fungal spore and/or yeast culture is prepared 1,3-callose immunogen; Described fungi is selected from aspergillus tubigensis (Aspergillus), candidiasis (Candida albaican), cryptococcus (Crytococcus), mucormycosis (Mucor), Penicillium marneffei (Penicillium marneffei), sickle-like bacteria (Fusarium), Pneumocystis carinii (Penumocystis carinii bacteria).
3. anti-1, the 3-callose polyclonal antibody of one according to claim 1, it is characterized in that, in described step (2), animal is selected from one or more in mouse, rat, cavy, rabbit, chicken, sheep, horse, pig, donkey; Described immunity adopts the one in hypodermic injection, intrasplenic injection method, intravenous injection, intraperitoneal injection; The immunizing dose of described immunity is 10-1000 μ g//time.
4. anti-1, the 3-callose polyclonal antibody of one according to claim 1, is characterized in that, in described step (3), get blood in the animal body after immunity before, also comprises the step of the serum titer measuring animals following immunization.
5. one according to claim 1 anti-1,3-callose polyclonal antibody, it is characterized in that, in described step (4), described saturated ammonium sulphate salt precipitation method comprises: get the serum that step (3) obtains, add isopyknic physiological saline, then add saturated ammonium sulphate solution, 0-10 DEG C of precipitates overnight; The centrifugal 5-20min of 10000G, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, slowly drips saturated ammonium sulphate solution, 0-10 DEG C of standing 0.5-2h; The centrifugal 5-20min of 10000G, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, by phosphate buffered saline buffer 0-10 DEG C dialysed overnight;
And/or in described step (4), described affinity chromatography comprises: wash post with the elution buffer of 5-10 times of column volume; Post is washed with the coupling buffer of 5-10 times of column volume; By the sample loading crossed with saturated ammonium sulphate salting-out process preliminary purification; Post is washed with the coupling buffer of 5-10 times of column volume; With the elution buffer wash-out of 2-5 times of column volume, obtain anti-1,3-callose polyclonal antibody.
6. the one described in any one of claim 1-5 resists 1,3-callose polyclonal antibody, it is characterized in that, with 1,3-callose bag quilt, indirect ELISA detection display its tire and be not less than 1:1.28 × 10
5.
7. the preparation method of anti-1, a 3-callose polyclonal antibody, is characterized in that, comprising:
(1) 1,3-callose immunogen is prepared;
(2) animal immune is carried out by 1,3-callose immunogen;
(3) in the animal body after immunity, blood is got;
(4) with saturated ammonium sulphate salting-out process and affinity chromatography, serum is purified successively, obtain polyclonal antibody.
8. the preparation method of anti-1, the 3-callose polyclonal antibody of one according to claim 7, is characterized in that, comprising: (1) prepares 1,3-callose immunogen: by 1,3-callose and NaIO
4solution mixes, and room temperature lucifuge reaction 0.5-3 hour, uses chromatographic column desalination, add bovine serum albumin and NaBH by the solution obtained
3cN solution, shaken at room temperature reacts, and adds NaBH
4solution, shaken at room temperature reacts, with phosphate buffered saline buffer dialysis, centrifugal removing precipitation;
Or, by 1,3-callose immunogen and the mixing of 4-(4,6-dimethoxy-triazine-2-base)-4-methyl morpholine hydrochloride solution, oscillatory reaction, ultrapure water dialysed overnight, adds BSA, room temperature lucifuge shaken overnight, centrifugal removing precipitation;
Or, pathomycete is collected thalline through solution culture fermentation, with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, thalline pours liquid nitrogen grinding fragmentation into, collects supernatant liquor;
Or by pathomycete after plate solid culture, wash-out collects spore, with the physiological saline deactivation containing volume percent 1-10% formaldehyde, repeatedly washing removing formaldehyde, spore ultrasonication, collects supernatant liquor;
(2) with 1,3-callose immunogen carries out animal immune: step (1) obtained 1,3-callose immunogen mixes with Freund's complete adjuvant equal-volume, fully emulsifiedly rear subcutaneous multi-point injection is carried out to animal, immunizing dose is every animal 10-1000 μ g, every 2-4 week immunity 1 time after initial immunity, immune time >=3 time;
(3) in the animal body after immunity, blood is got: after measuring the serum titer of animals following immunization, take a blood sample by the method for carotid artery bloodletting, treat blood coagulation, after serum is isolated, centrifugal, get supernatant;
(4) with saturated ammonium sulphate salting-out process and affinity chromatography, serum is purified successively, obtains polyclonal antibody:
Described saturated ammonium sulphate salt precipitation method comprises: get the serum that step (3) obtains, add isopyknic physiological saline, then add saturated ammonium sulphate solution, 0-10 DEG C of precipitates overnight; The centrifugal 5-20min of 10000G, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, slowly drips saturated ammonium sulphate solution, 0-10 DEG C of standing 0.5-2h; The centrifugal 5-20min of 10000G, abandons supernatant, is dissolved by precipitation phosphate buffered saline buffer, by phosphate buffered saline buffer 0-10 DEG C dialysed overnight;
Described affinity chromatography comprises: wash post with the elution buffer of 5-10 times of column volume; Post is washed with the coupling buffer of 5-10 times of column volume; By the sample loading crossed with saturated ammonium sulphate salting-out process preliminary purification; Post is washed with the coupling buffer of 5-10 times of column volume; With the elution buffer wash-out of 2-5 times of column volume, obtain anti-1,3-callose polyclonal antibody.
9. the anti-application of 1,3-callose polyclonal antibody in the test kit for the preparation of invasive fungi disease detection of one according to claim 1.
10. according to claim 9 anti-1, the application of 3-callose polyclonal antibody in the test kit for the preparation of invasive fungi disease detection, it is characterized in that, described invasive fungi be selected from candidiasis, aspergillus tubigensis, cryptococcus, mucormycosis, lung pityrosporion ovale, sickle-like bacteria, Dematiaceous fungi, trichosporon bacteria, histoplasma capsulatum, coccidioides immitis, blastomycete, Ma Nifei mould, fibrillae of spores one or more.
11. 1 kinds, for the ELISA detection kit of invasive fungi disease detection, is characterized in that, comprise envelope antigen, 1,3-callose polyclonal antibody and ELIAS secondary antibody according to claim 1; Described envelope antigen is 1,3-callose; Described ELIAS secondary antibody is that horseradish peroxidase (HRP) marks IgG antibody.
12. a kind of ELISA detection kit for invasive fungi disease detection according to claim 11, is characterized in that, also comprise in described test kit: one or more in washings, substrate nitrite ion, antigen standard, stop buffer.
13. 1 kinds of methods utilizing the ELISA detection kit described in claim 11-12 to detect invasive fungi disease, is characterized in that, comprising: (1) is that envelope antigen prepares antigen coated enzyme plate with 1,3-callose; (2) the antigen coated enzyme plate using (1) to prepare, adds antigen standard or detected sample, then adds 1,3-callose polyclonal antibody, mix latter 37 DEG C and hatch; (3) wash plate 1-5 time with washings, add enzyme labelled antibody, hatch 1-3h for 37 DEG C, washings washes plate 1-5 time, adds the colour developing of substrate nitrite ion, adds stop buffer and stops; (4) OD is measured
450nm.
14. ELISA detection kit according to claim 13 detect the method for invasive fungi disease, and it is characterized in that, the method for described detection invasive fungi disease is non-therapeutic purpose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510054177.4A CN104628861A (en) | 2015-02-03 | 2015-02-03 | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510054177.4A CN104628861A (en) | 2015-02-03 | 2015-02-03 | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104628861A true CN104628861A (en) | 2015-05-20 |
Family
ID=53208140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510054177.4A Pending CN104628861A (en) | 2015-02-03 | 2015-02-03 | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104628861A (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001337A (en) * | 2015-07-22 | 2015-10-28 | 丹娜（天津）生物科技有限公司 | Rheumatoid factor adsorbent |
| 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 |
| CN108299558A (en) * | 2018-02-02 | 2018-07-20 | 暨南大学 | Human cytomegalovirus anti-pUL146 polyclonal antibodies and the preparation method and application thereof |
| CN108663509A (en) * | 2017-06-16 | 2018-10-16 | 江苏诺鬲生物科技有限公司 | A kind of immune colour reagent of label fungi |
| CN109633164A (en) * | 2018-12-14 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | A kind of thyroglobulin antibody detection kit calibration object and its preparation process |
| CN110954689A (en) * | 2019-11-25 | 2020-04-03 | 芜湖森爱驰生物科技有限公司 | Deep fungal infection rapid diagnosis kit and preparation method thereof |
| CN111208294A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Fungus quantum dot fluorescence immunoassay kit |
| CN112083167A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | S100A4 biomarker for evaluating intervention of methotrexate in psoriasis and application thereof |
| CN113574065A (en) * | 2019-03-08 | 2021-10-29 | 积水医疗株式会社 | Immunoassay method for beta-D-glucan in biological sample, and assay kit for beta-D-glucan |
| CN113980092A (en) * | 2021-12-09 | 2022-01-28 | 上海药明生物技术有限公司 | Protein affinity purification method |
| WO2022170977A1 (en) * | 2021-02-09 | 2022-08-18 | 丹娜(天津)生物科技股份有限公司 | Antifungal 1,3-beta-d-glucan monoclonal antibody and use thereof |
| CN116239706A (en) * | 2022-10-24 | 2023-06-09 | 湖北中医药大学 | Linear poria beta-glucan and extraction method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893618A (en) * | 2010-06-17 | 2010-11-24 | 中国科学院海洋研究所 | Pathogen-associated molecular pattern immune detection chip, preparation method and application thereof |
| CN103204930A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for polyclonal antibody of aspergillus fumigatus |
-
2015
- 2015-02-03 CN CN201510054177.4A patent/CN104628861A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893618A (en) * | 2010-06-17 | 2010-11-24 | 中国科学院海洋研究所 | Pathogen-associated molecular pattern immune detection chip, preparation method and application thereof |
| CN103204930A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for polyclonal antibody of aspergillus fumigatus |
Non-Patent Citations (6)
| Title |
|---|
| MATHIEU HOUDE等: "Streptococcus suis Capsular Polysaccharide Inhibits Phagocytosis through Destabilization of Lipid Microdomains and Prevents Lactosylceramide-Dependent Recognition", 《INFECT IMMUN.》 * |
| SONELA A. SCHLOTTMANN等: "A novel chemistry for conjugating pneumococcal polysaccharides to Luminex microspheres", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| WEI HAO等: "Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity", 《CLIN VACCINE IMMUNOL.》 * |
| 何力志等: "1，3-β-D-葡聚糖和抗白念珠菌芽管抗体检测对侵袭性念珠菌病诊断的临床价值", 《检验医学》 * |
| 曾练强等: "葡聚糖多克隆抗体制备与纯化", 《广西轻工业》 * |
| 闻平等: "( 1 , 3 )一β一D一葡聚糖ELISA检测方法建立及初步临床应用", 《现代检验医学杂志》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001337A (en) * | 2015-07-22 | 2015-10-28 | 丹娜（天津）生物科技有限公司 | Rheumatoid factor adsorbent |
| 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 |
| CN108663509A (en) * | 2017-06-16 | 2018-10-16 | 江苏诺鬲生物科技有限公司 | A kind of immune colour reagent of label fungi |
| CN108299558A (en) * | 2018-02-02 | 2018-07-20 | 暨南大学 | Human cytomegalovirus anti-pUL146 polyclonal antibodies and the preparation method and application thereof |
| CN109633164A (en) * | 2018-12-14 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | A kind of thyroglobulin antibody detection kit calibration object and its preparation process |
| CN113574065A (en) * | 2019-03-08 | 2021-10-29 | 积水医疗株式会社 | Immunoassay method for beta-D-glucan in biological sample, and assay kit for beta-D-glucan |
| CN112083167B (en) * | 2019-06-14 | 2024-03-26 | 复旦大学附属华山医院 | S100A4 as biomarker for evaluating methotrexate intervention psoriasis and application thereof |
| CN112083167A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | S100A4 biomarker for evaluating intervention of methotrexate in psoriasis and application thereof |
| CN110954689A (en) * | 2019-11-25 | 2020-04-03 | 芜湖森爱驰生物科技有限公司 | Deep fungal infection rapid diagnosis kit and preparation method thereof |
| CN111208294A (en) * | 2020-02-29 | 2020-05-29 | 济南德亨医学科技有限公司 | Fungus quantum dot fluorescence immunoassay kit |
| WO2022170977A1 (en) * | 2021-02-09 | 2022-08-18 | 丹娜(天津)生物科技股份有限公司 | Antifungal 1,3-beta-d-glucan monoclonal antibody and use thereof |
| CN113980092A (en) * | 2021-12-09 | 2022-01-28 | 上海药明生物技术有限公司 | Protein affinity purification method |
| CN113980092B (en) * | 2021-12-09 | 2024-05-14 | 上海药明生物技术有限公司 | Protein affinity purification method |
| CN116239706A (en) * | 2022-10-24 | 2023-06-09 | 湖北中医药大学 | Linear poria beta-glucan and extraction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104628861A (en) | 1, 3-beta-D-glucan polyclonal antibody and preparation method thereof | |
| Weiner et al. | Immunodiagnosis of systemic candidiasis: mannan antigenemia detected by radioimmunoassay in experimental and human infections | |
| Fernandes et al. | Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens | |
| Wenzel et al. | Immunoassay of tryptase from human mast cells | |
| US10428139B2 (en) | GM hybridoma cell, monoclonal antibody, kit and preparation method and use thereof | |
| CN113156106B (en) | Detection card for identifying bungarus venosus venom by using colloidal gold immunochromatography, preparation method and application thereof | |
| CN105866407A (en) | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof | |
| US10670598B2 (en) | Preparation method of Cryptococcus neoformans capsular polysaccharide GXM as well as GXM antigen immunoassay kit and application thereof | |
| Matthews et al. | Characterisation and cellular localisation of the immunodominant 47-Kda antigen of Candida albicans | |
| Taborda et al. | Diagnosis of paracoccidioidomycosis by passive haemagglutination assay of antibody using a purified and specific antigen-gp43 | |
| CN103204927A (en) | Preparation method for monoclonal antibody of cryptococcus neoformans | |
| CN105646703A (en) | Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody | |
| CN105277423B (en) | A kind of immunomagnetic beads for vomitoxin enrichment purification and its preparation method and application | |
| CN105001337A (en) | Rheumatoid factor adsorbent | |
| CN103454411B (en) | The preparation method of a kind of biotin labeling rabbit anti-tilapia IgM polyclonal antibody and application | |
| CN106220730A (en) | A kind of preparation method of Aspergillus fumigatus galactomannan polyclonal antibody | |
| CN105651996A (en) | Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof | |
| CN104745534A (en) | Procalcitonin monoclonal antibody hybrid tumor 2H4 and monoclonal antibody | |
| Sarfati et al. | Antigens of Aspergillus fumigatus produced in vivo | |
| CN103509760A (en) | Hybridoma cell capable of secreting anti-beta2-microglobulin monoclonal antibody, and application thereof | |
| CN101477116A (en) | Preparation of multi-organophosphor universal antibody and universal envelope antigen | |
| CN1580772A (en) | SARS virus antibody double antigen sandwich ELISA detecting method | |
| CN103204928A (en) | Preparation method for polyclonal antibody of cryptococcus neoformans | |
| CA1303979C (en) | Diagnostic test for staphylococcal mastitis | |
| CN105296520A (en) | Preparation method and enzyme-linked immunosorbent assay kit of human tumor antigen 3H11Ag |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150520 |