CN101149377B - Antibody for detecting aspergillus - Google Patents
Antibody for detecting aspergillus Download PDFInfo
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- CN101149377B CN101149377B CN2007100311091A CN200710031109A CN101149377B CN 101149377 B CN101149377 B CN 101149377B CN 2007100311091 A CN2007100311091 A CN 2007100311091A CN 200710031109 A CN200710031109 A CN 200710031109A CN 101149377 B CN101149377 B CN 101149377B
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Abstract
This invention provides the capture antibody and the detection antibody to detect the aspergillus, the two antibody is the single clone antibody of the cross oncocyte CCTCC NO.C200731 and CCTCC NO.C200730, they can unites the glucoprotein whose molecular weight is 25-100kDa in the cell wall of the aspergillus, the reaction with the Penicillium marneffei, white oidiomycosis, smoothness oidiomycosis, tropic oidiomycosis, close smoothness oidiomycosis, Candida krusei are all negative. The antibody in this invention can make into the immunity reagent to detect the aspergillus in common use.
Description
Technical field
The present invention relates to biochemical field, be specifically related to a kind of new antibody that is used to detect aspergillus.
Background technology
Aspergillus is the modal saprophytic bacteria of occurring in nature, and kind more than 100 is arranged, and wherein at least 10 kinds of aspergillus are conditioned pathogens, often pollutes grain and food, environment and can suppress crowd, serious threat human health by infection immunity.(Invasive Aspergillosis, IA) owing to lack early stage effectively clinical means, mortality ratio is very high, reaches 95% for the aggressive aspergillosis that is caused by aspergillus.IA mainly causes by aspergillus fumigatus, aspergillus flavus, and few part is caused that by Aspergillus terreus, aspergillus niger, aspergillus nidulans wherein, aspergillus fumigatus is main pathogenic bacteria, accounts for human infection's due to the aspergillus class fungi 80%～90%.
Aspect food inspection, the method that aspergillus pollutes in the detection food of domestic and foreign current is to detect aspertoxin, mainly comprises TLC, high performance liquid chromatography, immune analysis method etc.Wherein TLC does not need special instrument and equipment; Common laboratory can be carried out; But the contamination hazard to experimenter and environment is bigger, is not suitable for field quick detection, and complex operation, is subject to other component and disturbs; Because of its poor sensitivity, can't satisfy the mycotoxin limit standard of current increasingly stringent.This method is still more in developing country and China's application, and is replaced by high performance liquid chromatography and immune analysis method gradually in developed country.High performance liquid chromatography, it is highly sensitive, and accuracy is good; But instrument is expensive, requires the sample degree of purification high, and sample pretreatment process is loaded down with trivial details; Length consuming time, requiring has the professional to operate, because of the expensive instrument of needs; Detection cost height is difficult to apply in China, thereby has influenced effective monitoring of mycotoxin in China's oil and foodstuffs.And said method only can detect a kind of aspertoxin at every turn, is not suitable for extensive examination that harmful aspergillus in the grain and food is polluted, thereby further cuts off pollution section.Therefore, lack method quick, special, that wide spectrum detects various harmful aspergillus in the oil and foodstuffs at present.
The method that aspect clinical diagnosis, detects aspergillus mainly is to use the ELISA sandwich method to CAg galactomannan (galactomannan; GM) detection; GM is the aspergillus fumigatus antigen that the researcher finds in infected animal model and aggressive aspergillus fumigatus patient serum; Also be the polysaccharide antigen in a kind of certified aspergillus fumigatus source, this be a kind of can the antigenemic effective marker of early detection IA.France Pasteur Sanofi diagnostic center with the monoclonal antibody EB-A2 of rat anti GM as capture antibodies and detection antibody; Set up the double-antibody sandwich elisa single stage method; Be Platelia Aspergillus; Detecting blood and urine sample, is present unique a kind of commercial kit that is used for early diagnosis IA.Though this kit is very responsive to Detection of antigen; But discover in a large number when serum that detects suspicious aspergillus patient and urine sample; And used PCs, like the patients serum of piperacillin with tazobactam very high false positive rate is arranged, up to 74%.In addition; False-positive problem equally also is present in the pediatric population; Up to 83%; This possibly be because the β (1,5) of EB-A2 identification aspergillus GM-galactofuranoside side chain residue the time, but also identification to be arranged in the intersection epi-position of Bifidobacterium fat muramic acid matter and other microorganisms and food of other fungies (like Penicillium) cell wall polysaccharides, neonate's enteron aisle relevant.Therefore, applying of this kit is restricted.
Summary of the invention
The object of the present invention is to provide new antibody.
A purpose more of the present invention is to provide the application of said antibody.
The new monoclonal antibody of the present invention is:
A kind of capture antibody that is used to detect aspergillus, this antibody are the monoclonal antibodies that is produced by hybridoma cell line CCTCC NO.C200731.
A kind of detection antibody that is used to detect aspergillus, this antibody are the monoclonal antibodies that is produced by hybridoma cell line CCTCC NO.C200730.The chemical reagent that described detection antibody can marker enzyme, biotin or luminescent substance etc. can produce detectable signal difference.
Capture antibody of the present invention all is to obtain with aspergillus fumigatus filament antigen immune mouse with detecting antibody; And obtaining hybridoma cell line 9D47A1 and the hybridoma cell line 7E11A1 that two strains produce these two kinds of antibody respectively with cell-fusion techniques, this two strain of hybridoma is the IgG1 positive.
Hybridoma cell line 9D47A1 and hybridoma cell line 7E11A1 are preserved in Chinese typical culture collection center (CCTCC) on October 18th, 2007, and preserving number is respectively C200731 and C200730.
Statement for ease, above-mentioned by hybridoma cell line CCTCC NO.C200731 generation monoclonal antibody called after monoclonal antibody 9D47A1, produce monoclonal antibody called after monoclonal antibody 7E11A1 by hybridoma cell line CCTCC NO.C200730.
It is 25～100kDa and the glycoprotein that combines with ConA that monoclonal antibody 9D47A1 according to the invention and monoclonal antibody 7E11A1 can specificity combine aspergillus cell membrane molecular weight; Positive with various aspergillus reactions in clinical and the environment, like aspergillus such as aspergillus fumigatus, aspergillus flavus, Aspergillus terreus, aspergillus nidulans, aspergillus niger, aspergillus versicolor, excellent aspergillus, aspergillus candidus, aspergillus sydowii, aspergillus flavipes, aspergillus ustus, Aspergillus ochraceus, aspergillus oryzaes; But all negative with the reaction of other common fungies, like Marni Fei Shi Penicillium notatum, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, candida krusei etc.
Two monoclonal antibodies pairing of the present invention is used to detect aspergillus; It mainly is the detection that combines the double-antibody sandwich method; Detection like double-antibodies sandwich ELISA, colloidal gold immunochromatographimethod or immunochemiluminescence method; Wherein monoclonal antibody 9D47A1 is as capture antibody, and monoclonal antibody 7E11A1 perhaps combines the various materials that can send detectable signal as detecting antibody separately.Based on above-mentioned principle, but monoclonal antibody of the present invention is used to be prepared into the reagent of the detection aspergillus of industrialization, and these reagent can be the forms of kit, also can be the forms of test paper.Described kit can be double-antibody sandwich elisa kit, the immunochemistry kit etc. of giving out light, and described test paper can be that the collaurum fast immune chromatographic detects test paper etc.
The double-antibody sandwich elisa kit of detection aspergillus of the present invention; This kit is made up of following reagent: the micro reaction plate, sample preparation liquid, enzyme conjugates, positive control, negative control, concentrated washing lotion, colour developing liquid, the stop buffer that encapsulate capture antibody; It is characterized in that capture antibody is monoclonal antibody 9D47A1; Enzyme conjugates is that monoclonal antibody 7E11A1 is prepared with enzyme labeling, and described enzyme is oxidase or alkaline phosphatase, like the HRPO mark.
The collaurum fast immune chromatographic of detection aspergillus of the present invention detects test paper; This test paper is by sample pad; Polyester cellulose pad, nitrocellulose filter and the adsorptive pads composition of overlapping successively is characterized by and is coated with one deck collaurum-monoclonal antibody 9D47A1 compound on the polyester cellulose film; Be distributed with on the nitrocellulose filter and detect band and quality control band, wherein detecting band is that spraying monoclonal antibody 7E11A1 forms on nitrocellulose filter, and quality control band is that spraying goat anti-mouse igg 1 antibody forms on nitrocellulose filter; Wherein said collaurum-monoclonal antibody 9D47A1 compound is coated on the colloid gold particle of being processed by gold chloride by the 9D47A1 of purifying and makes, and its grain size is 18～20nm, is peony.
Immunochemiluminescence kit of the present invention, the composition of this kit is similar with the double-antibody sandwich elisa kit, just wherein monoclonal antibody 7E11A1 mark be chemiluminescent substance such as luciferin.
Test paper/the kit of the said detection aspergillus of the present invention is simple to operate; Fast; Specificity is high, with other fungi no cross reaction, both can be used for the detection of aspergillus in the quality control of grain or food; Can be used in the environment detection of polluting again, can also foundation be provided for patient's IA early diagnosis like aspergillus such as air, wood, clothings.Test paper/kit of the present invention has following advantage with the existing compared with techniques that detects aspergillus:
1, is used for food quality control; With the existing aspertoxin compared with techniques that detects; Enzyme-multiplied immune technique kit of the present invention capable of using or colloidal gold strip technology; The suspicious pollution sample that receives aspergillus is carried out large-scale preliminary examination, can detect aspergillus antigen in the sample simple and easy, quickly and accurately;
2, be used for environment aspergillus contamination monitoring; Enzyme-multiplied immune technique kit of the present invention capable of using or colloidal gold strip technology; The suspicious pollution wood that receives aspergillus, clothing etc. are carried out large-scale preliminary examination, can detect aspergillus antigen in the sample simple and easy, quickly and accurately;
3, be used to diagnose the early diagnosis kit of IA to compare at present, have similar sensitivity, but specificity is higher, with the reaction of other fungi non-false positives such as Penicillium notatum, candida albicans, cost is low, is fit to domestic grass-roots unit and uses.
Description of drawings
Fig. 1 be monoclonal antibody 9D47A1 of the present invention and monoclonal antibody 7E11A1 to carry out the result of Western blotting through the aspergillus cell wall sugar albumen of ConA column purifying, it combines the molecular weight of band is 25-100kDa; Wherein band 1 is Marker, and band 2 is monoclonal antibody 7E11A1, and band 5 is monoclonal antibody 9D47A1, and band 3,4 is irrelevant monoclonal antibody.
Fig. 2 is the structural representation of collaurum fast immune chromatographic test paper of the present invention, and wherein 1 is sample pad, the 2nd, and polyester cellulose pad, the 3rd, nitrocellulose filter, the 4th, adsorptive pads, the 5th detects band, the 6th, quality control band.
Fig. 3 is that the present invention detects the testing result of the collaurum fast immune chromatographic test paper of aspergillus to 19 kinds of fungies, and wherein 1 is Aspergillus terreus, the 2nd, and aspergillus flavipes, the 3rd, aspergillus flavus, the 4th, aspergillus nidulans; The 5th, aspergillus niger (ATCC 10864), the 6th, aspergillus niger, the 7th, aspergillus ustus, the 8th, aspergillus versicolor; The 9th, excellent aspergillus, the 10th, aspergillus candidus, the 11st, aspergillus sydowii, the 12nd, Aspergillus ochraceus; The 13rd, aspergillus oryzae, the 14th, aspergillus fumigatus, the 15th, Candida albicans, the 16th, Candida tropicalis; The 17th, Candida glabrata, the 18th, Candida parapsilosis, the 19th, candida krusei, the 20th, Marni Fei Shi Penicillium notatum.
Embodiment
Example 1 MONOCLONAL ANTIBODIES SPECIFIC FOR of the present invention and evaluation
1, the preparation of monoclonal antibody 9D47A1 and monoclonal antibody 7E11A1
1) aspergillus fumigatus filament antigen preparation:
The aspergillus fumigatus bacterial strain on the Sha Shi flat board 37 ℃ the growth a couple of days to forming single bacterium colony.With typical single bacterium colony results, add in the 500ml conical flask that contains 50ml RPMI-1640,37 ℃ were shaken isolated by filtration mycelium and filtered fluid bacterium 3-7 days.The mycelium of collecting is resuspended among the PBS, ultrasonic degradation, supernatant is collected in centrifugal back, and be stored in-80 ℃ subsequent use.
2) immune mouse
Get female BALB/c mouse in 4-6 age in week; Adopt for the first time Freund's complete adjuvant and the emulsification of equal-volume aspergillus fumigatus filament antigen mixing; Hypodermic injection 100 a μ g/ mouse, later per 10 days with incomplete Freund and 50 μ g antigen equal-volume emulsifications, abdominal cavity and subcutaneous multi-point injection; Behind the mouse immune 4 times, strengthen 100 a μ g/ antigen in merging preceding 3 days veins.
3) immune serum titration
Adopting indirect elisa method to measure immune serum tires.Prepare the 50mMpH9.6 carbonate buffer solution of 10 μ g/ml aspergillus fumigatus filament antigens, encapsulate little 96 orifice plates of polystyrene, 100 μ l/ holes, 4 ℃ are spent the night.Next day, spend the night for 4 ℃ in the confining liquid 300 μ l/ holes that contain 0.25% casein (Sigma), dries coated slab, and vacuum drying 12～24h with the vacuum-packed 4 ℃ of preservations of aluminum foil bag, is used for the titration of mouse immune serum.In eye socket blood sampling in immune back 10 days for the third time, the mouse immune serum was with containing 1%BSA 10mM PBS with 10
3～10
6Doubly dilution adds 96 orifice plates, the 37 ℃ of 30min in 100 μ l/ holes; After 10mM PBS contains the 0.1%Tween-20 cleansing solution and washes plate four times, add 1: 1000 times of dilution horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.); The 37 ℃ of 30min in 100 μ l/ holes, the same wash plate after, add and to contain 0.05% (W/V) TMB and 0.06% (W/V) dioxygen pH5.0 citrate buffer solution; 100 μ l/ holes, room temperature lucifuge 10min adds 100 μ l/ hole 1M H
2SO
2Cessation reaction is surveyed the 450nm absorption value,, must compare>=2.1 with measured value and control value and positively judge tiring of immune serum as negative control with mice serum before the immunity.
4) hybridoma preparation
Select serum antibody titer to reach 1 * 10
5Mouse, in merging preceding 3 days tail vein injection 100 μ g aspergillus fumigatus filament antigens.The aseptic mouse spleen of getting, the murine myeloma cell strain NS-1 that processes splenocyte suspension and exponential phase was by 10: 1 mixed, and (PEG, MW4000 Sigma) merge under the effect with 45% polyglycol.By following step polyglycol solution is added cell.In 37 ℃ of water-baths; In 1-2min, slowly add 1.0ml PEG, the limit edged shakes up gently, respectively at adding 1ml, 2ml, 3ml, 4ml, 5ml serum-free RPMI-1640 nutrient culture media termination fusion in 1min, 2min, 3min, 4min, the 5min; Add 10ml at last and contain the two-in-one nutrient culture media of 15%FBS; The centrifugal 5min of room temperature 1000rpm abandons supernatant, and the nutrient culture media that contains 15% hyclone with 36ml has hanged cell gently.This cell suspension is added on 6 96 well culture plates, and temperature is in 37 ℃, the incubator of 5%CO2 in CO2gas incubator.After one day, add 100 μ l in every hole and contain hypoxanthine, aminopterin-induced syndrome-thymidine (HAT, Sigma) screening culture medium.Changed liquid once with this screening culture medium to culture in per 3 days later on, and formed up to clone cell.
4) hybridoma of screening secretion aspergillus antigen monoclonal antibody
Indirect elisa method screening cells and supernatant is selected strong positive clone hybridization oncocyte to carry out subcloning, and with the continuous cloning of limiting dilution assay 2-3 time, is obtained the hybridoma cell strain of 2 strain stably excreting antibody.Positive rate after the cloning is reached 100% cell amplification cultivate the back liquid nitrogen cryopreservation.
Inoculation positive hybridoma cell in the mouse body, preparation ascites, and adopt the antibody in sad-ammonium sulfate precipitation method purifying ascites.
2, the specificity of monoclonal antibody of the present invention is identified
(1) experiment material: aspergillus fumigatus strain (available from Peking University medical mycology research centre)
(2) antigen preparation:
The aspergillus fumigatus bacterial strain on the Sha Shi flat board 37 ℃ the growth a couple of days to forming single bacterium colony.With typical single bacterium colony results, add in the 500ml conical flask that contains 50ml RPMI-1640,37 ℃ were shaken isolated by filtration mycelium and filtered fluid bacterium 3-7 days.The mycelium of collecting is resuspended among the PBS, ultrasonic degradation, supernatant is collected in centrifugal back, and be stored in-80 ℃ subsequent use.
(3) antibody subgroup identification
The Ig subgroup identification adopts indirect ELISA, encapsulates aspergillus fumigatus filament antigen, and hatch with the Hybridoma Cell Culture supernatant sealing back, more respectively with the different subclass SIGs of the anti-mouse of rabbit that are 1: 1000 times of dilution HRP mark; These antibody comprise the anti-mouse IgG1 of rabbit (Sigma, Inc), the anti-mouse IgG2a of rabbit (Sigma, Inc); The anti-mouse IgG2b of rabbit (Sigma, Inc), the anti-mouse IgG3 of rabbit (Sigma; Inc), and the anti-mouse IgM of rabbit (Sigma, Inc).Testing result two strain of hybridoma strains are the IgG1 positive
(4) indirect elisa method carries out the monoclonal antibody specificity analyses:
With the antigen coated microwell plate of aspergillus fumigatus filament, detect according to the indirect elisa method of routine.In the microwell plate that encapsulates, add the Hybridoma Cell Culture supernatant of this patent invention, hatch 1h again for 37 ℃, add 1: 1000 dilution horseradish peroxidase-labeled goat anti-mouse igg (Sigma; Inc); The 37 ℃ of 30min in 100 μ l/ holes add TMB colour developing liquid chamber temperature lucifuge 10min, add 1M H
2SO
2Cessation reaction is surveyed 450nm absorption value (A
450).Table 1 result shows that the monoclonal antibody of this patent invention and aspergillus antigen produce very strong specific immune response.
Table 1: aspergillus monoclonal antibody and aspergillus antigen-reactive indirect ELISA result
| A 450 | The aspergillus monoclonal antibody | Irrelevant antibody | |
| 9D47A1 | 7E11A1 | ||
| 1.842 | 1.934 | 0.082 | |
(5) IIF is identified the specificity of monoclonal antibody of the present invention
1) experiment material: aspergillus fumigatus, aspergillus flavus, aspergillus niger and Aspergillus terreus, Candida albicans, candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Marni Fei Shi Penicillium notatum.
2) experimental technique:
Aspergillus strain 37 ℃ of growth a couple of days on the Sha Shi flat board are collected typical single bacterium colony to forming single bacterium colony, with 1 * PBS of precooling collection, to wash cell two times and adjust concentration be 1 * 10
6Individual cell/ml drips cell on the slide of aseptic drying then, after the drying, is prepared into smear; Fully dry, with the fixing 20min of precooling immobile liquid (acetone: the methyl alcohol volume ratio is 3: 7), dry up, it is 10 μ g/ml that monoclonal antibody is adjusted concentration; The fluorescence that adds to different Aspergillus strains is coated with in the film perforation, establishes feminine gender and positive control serum simultaneously, put 37 ℃ of water-bath 60min after, take out the antigen sheet be put in the staining jar with 0.01mM pH7.2 PBS cleaning 3 times; Dry up, add the fluorescence labeling goat anti-mouse igg antibody, behind 37 ℃ of water-bath 30min, the same method washing 5 times; After drying up, 0.25% Azo-Blue contrast dyeing, fluorescent microscope is observed fluoroscopic image down; Carry out the result with intensity of fluorescence with the dyeing form and judge, detect antibody intensity and count the positive with (+～++ ++), antibody intensity (±) and (-) count feminine gender.
3) experimental result:
The result is as shown in table 2, and monoclonal antibody specificity of the present invention combines aspergillus fumigatus, aspergillus flavus, aspergillus niger and Aspergillus terreus, and very strong binding ability is arranged, and does not combine with other fungi.
Table 2 indirect immunofluorescence detects the specificity of the monoclonal antibody of anti-aspergillus antigen to the different aspergillus of 4 strains
| Monoclonal antibody of the present invention | Monoclonal antibody 7E11A1 | Monoclonal antibody 9D47A1 | ||
| The antibody subclass | IgG1 | IgG1 | ||
| Immunofluorescent test (I F A) | Aspergillus fumigatus | Spore | + | + |
| Mycelia | ++++ | ++++ | ||
| Aspergillus flavus | Spore | ++ | + | |
| Mycelia | +++ | +++ | ||
| Aspergillus niger | Spore | ++ | + | |
| Mycelia | ++++ | ++ | ||
| Aspergillus terreus | Spore | +++ | +++ | |
| Mycelia | ++++ | ++++ | ||
| Candida albicans | - | - | ||
| Candida krusei | - | - | ||
| Candida glabrata | - | - | ||
| Candida tropicalis | - | - | ||
| Candida parapsilosis | - | - | ||
| Marni Fei Shi Penicillium notatum | - | - | ||
(6) Western blot is analyzed the specificity of monoclonal antibody of the present invention
Aspergillus fumigatus bacterial strain (available from Peking University medical mycology research centre) on the Sha Shi flat board 37 ℃ the growth a couple of days to forming single bacterium colony; Collect typical single bacterium colony; Add in the 500ml conical flask that contains 50ml RPMI-1640; 37 ℃ were shaken bacterium 3-7 days, filter, with filtrating through through ConA column purifying glycoprotein wherein.Protein band through the 8%SDS-PAGE electrophoretic separation is transferred on the nitrocellulose filter, after the confining liquid sealing, cellulose membrane in 7E11A1-HRP 37 ℃ hatch 1h; With the washing lotion cleaning many times that contains 0.5% (v/v) Tween-20; DAB (Amresco Inc, Solon, OH) colour developing.2 kinds of monoclonal antibodies among the present invention combine glycoprotein molecule amount band to be positioned at 25-100kDa place (Fig. 1), explain that these 2 groups of monoclonal antibody identification antigens are the glycoprotein of 25-100kDa for the molecular weight through ConA column purifying.
(7), the analysis of monoclonal antibody recognition site of the present invention
1) antigen preparation: the aspergillus fumigatus bacterial strain on the Sha Shi flat board 37 ℃ the growth a couple of days to forming single bacterium colony, collect typical single bacterium colony, add in the 500ml conical flask that contains 50ml RPMI-1640,37 ℃ were shaken isolated by filtration mycelium and filtered fluid bacterium 3-7 days.The mycelium of collecting is resuspended among the PBS, ultrasonic degradation, supernatant is collected in centrifugal back, and be stored in-80 ℃ subsequent use.
2) method and result: dilute above-mentioned antigen to 30 μ g/ml with 50mM pH9.6 carbonate buffer solution, encapsulate little 96 orifice plates of polystyrene, the 0.1ml/ hole, 4 ℃ are spent the night.Next day; The confining liquid 0.3ml/ hole that contains 0.25% casein (Sigma), 4 ℃ spend the night after, add earlier monoclonal antibody 10 μ g/ml, 50 μ l/ holes and after add 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 2,000 50 μ l/ of serial dilution HRP mark monoclonal antibody hole; 37 ℃, hatch 1h; PBST adds TMB (AmrescoInc) 100 μ l/ holes after washing five times, behind the 10min, and 1M H
2SO
4Cessation reaction is measured the 450nm absorption value.Is 100% with monoclonal antibody to the monoclonal antibody inhibiting rate of same HRP mark, to the negative contrast of the inhibition of mark monoclonal antibody, calculates the inhibiting rate between each monoclonal antibody with known irrelevant monoclonal antibody.Be that inhibiting rate is (1-measured value A450/ negative control value A450) * 100.Inhibiting rate＞75% is for relevant, and＞50% is not exclusively relevant, and＜50% is uncorrelated, and＜25% is uncorrelated for fully.The result finds that the inhibiting rate between the two strain monoclonal antibodies is 0, and 2 diverse antigen sites of 2 strain monoclonal antibodies identification are described.
3, the screening of double-antibody sandwich elisa method monoclonal antibody best pairing and ELISA Parameter Optimization
2 strain monoclonal antibodies of ascites purifying are used for carrying out one group of matrix format experiment to be suitable for setting up the sandwich ELISA method most to be used as the monoclonal antibody of catching with mark right to select.Briefly, derive and the ascites of coming encapsulates 96 orifice plates with 2 strain of hybridoma (being numbered 9D47A1,7E11A1) of sad-ammonium sulfate precipitation method purifying, right with the aspergillus fumigatus CA antigen of aforementioned preparation as antigen selection antibody.Horseradish peroxidase-labeled 2 strain monoclonal antibodies; Adopt matrix format; Just above-mentioned 2 strain monoclonal antibodies encapsulate every strain as catching or hybrid packet is used as and catches; Match with each strain enzyme labeling monoclonal antibody respectively, right to catch with the monoclonal antibody of mark in the rapid screening sandwich ELISA.Preliminary experiment shows that monoclonal antibody 9D47A1 as capture antibody, when matching with enzyme labeling 7E11A1 monoclonal antibody, can produce stronger signal.And on this basis; Make up the pairing of the antibody of antibody that above-mentioned 2 strain monoclonal antibodies catch and mark respectively, with signal intensity and specificity, with 9D47A1 as the monoclonal antibody of catching; Monoclonal antibody with 7E11A1 antibody serves as a mark produces the strongest signal.
Example 2 the present invention detect the double-antibody sandwich elisa kit of aspergillus
1, the double-antibody sandwich elisa kit that detects aspergillus is made up of following reagent:
Encapsulate the micro reaction plate of monoclonal antibody 9D47A1
Sample preparation liquid: 4% ethylenediamine tetraacetic acid (PH 7.0), promptly take by weighing ethylenediamine tetraacetic acid 40g, be dissolved in the 1L distilled water, NaOH transfers pH value to 7.0, is stored in 4 ℃;
Enzyme conjugates: the monoclonal antibody 7E11A1 of horseradish peroxidase-labeled;
Concentrate washing lotion: contain 20 * PBS of 2%Tween-20, promptly contain 4.56g NaH in the 1L solution
2PO
4, 58.02gNa
2HPO
4.12H
2O, 175.3g NaCl behind 15 pounds of 20min autoclavings, adds 20ml Tween-20 and stirs 20 times of dilutions during use;
Positive control: aspergillus fumigatus is cultivated filtered fluid dilution in 1: 1000,
Negative control: contain the 10mM PH7.4 PBS of 0.1%Tween-20, promptly contain 4.56g NaH in the 1L solution
2PO
4, 58.02gNa
2HPO
4.12H
2O, 175.3g NaCl, 15 pounds of 20min autoclavings add 0.1%Tween-20 after 20 times of dilutions;
Colour developing liquid: form by colour developing liquid A and B, get the two equivalent mixing during use and use.The constituent of liquid A, B of wherein developing the color is following:
Colour developing liquid A:
0.89g citric acid and 0.16g EDTA disodium are dissolved in the 1000ml water, behind 115 ℃ of 30min, add TMB 0.25g after reducing to 90 ℃, shake up in 4 ℃ of black outs and preserve;
Colour developing liquid B:
9.33g citric acid and 14.6g EDTA disodium are dissolved in the 1000ml water, behind 115 ℃ of 30min, add 0.75% hydrogen peroxide urea 12.8ml after reducing to 90 ℃, shake up in 4 ℃ of black outs and preserve;
Stop buffer: 1M H
2SO
4
Wherein,
The preparation method who encapsulates the micro reaction plate of monoclonal antibody 9D47A1 is: monoclonal antibody 9D47A1 of the present invention is diluted to 10 μ g/ml with 50mM carbonate buffer solution (pH9.6), encapsulates polystyrene 96 microwell plates with the 0.1ml/ hole, spend the night in 4 ℃.After bat was done, every hole added the confining liquid of 0.25% casein (Sigma) of 0.3ml, spent the night with the sealing nonspecific binding site in 4 ℃.Dry lath, vacuum drying 12～24h, subsequent use with the vacuum-packed 4 ℃ of preservations of aluminum foil bag;
The preparation method of enzyme conjugates is: the horseradish peroxidase-labeled Antibody Preparation adopts improvement sodium periodate method; With 5mg horseradish peroxidase stirring and dissolving in 1ml distilled water; Add 0.2ml and newly join 0.1M sodium periodate 30min, put in the 1mM pH4.4 sodium-acetate buffer 4 ℃ of dialysed overnight; Add the 10mg antibody of pH 9.5 dialysis equilibriums in the 0.01M carbonate buffer solution in advance after adding 20 μ l 0.2M pH9.5 carbonate buffer solutions next day; Stir 2～3h gently in the room temperature lucifuge, add 0.1ml and newly join the 4mg/ml sodium borohydride, 4 ℃ of lucifuges are spent the night; Lucifuge stirs and dropwise adds equal-volume saturated ammonium sulfate (ammonium sulfate is transferred pH to 7.0-7.2 with preceding elder generation with ammoniacal liquor) down on the ice bath, puts 4 ℃ of 6h.10000g, 4 ℃ of centrifugal 30min abandon supernatant, and deposition is dissolved in an amount of 10mM phosphate buffer, and with same liquid, 4 ℃ of dialysed overnight are changed liquid three times.Collect bond and add the agent of 2%BSAPBS50% glycerin for protecting, with 1000 times of phosphate buffer dilutions, get final product at last.
2, method of application:
After sample preparation liquid is handled various samples to be measured, add 100 μ l in the polystyrene 96 hole trace test plates that 9D47A1 encapsulates, every duplicate samples is established multiple hole, establishes cloudy contrast and positive control simultaneously; 37 ℃ of incubation 1h wash lath after 20 times of dilutions of concentrated cleaning solution, wash plate four times after, add enzyme conjugates; 100 μ l/ holes, 37 ℃ of 30min, the same wash plate eight times after, add colour developing liquid (colour developing liquid A and B mixed in equal amounts; Existing with join at present), 100 μ l/ holes are behind the room temperature lucifuge 10min; Add stop buffer, 100 μ l/ holes, cessation reaction.
3, the result judges:
With the blank well zeroing, measure the A value in the 450nm wavelength.
CUT OFF value=0.15+ negative control mean value
Like sample A450 value to be measured >=CUT OFF value, then be judged to the positive, otherwise, like sample A450 value to be measured＜CUT OFF value, then be judged to feminine gender.
When positive control A450 value be lower than 1.0 or negative control A450 value be higher than 0.3, testing result is invalid.
4, detect of specificity and the sensitivity analysis of the double-antibody sandwich elisa kit of aspergillus to various aspergillus and other common fungies
With clinical separation aspergillus and other common fungi (Marni Fei Shi Penicillium notatum, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, candida krusei) on the Sha Shi flat board 37 ℃ of growth a couple of days to forming single bacterium colony; Environment separation strain aspergillus in 28 ℃ cultivate 7 days after, collect single bacterium colony and be forwarded to that to grow to concentration among the RPMI-1640 be 2 * 10
6During individual cell/ml, collect the nutrient solution supernatant.After the sample diluting liquid diluted sample, detect above-mentioned various fungus culture medium supernatant according to the method for the 2nd of present embodiment.The result is as shown in table 3; Kit of the present invention separates the aspergillus strain specific reaction is all arranged with clinical 19 strain environment; And with other common fungies, like Marni Fei Shi Penicillium notatum, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, candida krusei no cross reaction.Be limited to 1: 1000 under the detection of kit of the present invention～1: 10000 extension rate, because of different types of mould different.
Table 3 kit of the present invention is to the specificity of various aspergillus and other common fungies
| Aspergillus specie | The culture extension rate | The result | |
| Positive | Negative | ||
| Aspergillus fumigatus-clinical | 1∶10000 | + | 0 |
| Aspergillus flavus-clinical | 1∶10000 | + | 0 |
| Aspergillus nidulans-clinical | 1∶1000 | + | 0 |
| Aspergillus terreus-clinical | 1∶10000 | + | 0 |
| Aspergillus niger-clinical | 1∶1000 | + | 0 |
| Aspergillus fumigatus- |
1∶10000 | + | 0 |
| Aspergillus flavus- |
1∶10000 | + | 0 |
| Aspergillus nidulans- |
1∶1000 | + | 0 |
| Aspergillus terreus- |
1∶1000 | + | 0 |
| Aspergillus niger- |
1∶100000 | + | 0 |
| |
1∶1000 | + | 0 |
| Aspergillus oryzae- |
1∶10000 | + | 0 |
| Aspergillus candidus- |
1∶1000 | + | 0 |
| Rod aspergillus- |
1∶1000 | + | 0 |
| Aspergillus versicolor- |
1∶1000 | + | 0 |
| Aspergillus flavipes- |
1∶10000 | + | 0 |
| Aspergillus sydowii- |
1∶1000 | + | 0 |
| Aspergillus ustus- |
1∶1000 | + | 0 |
| Aspergillus ochraceus- |
1∶10000 | + | 0 |
| Marni Fei Shi Penicillium notatum | Cultivate stoste | 0 | - |
| Candida albicans | Cultivate stoste | 0 | - |
| Candida krusei | Cultivate stoste | 0 | - |
| Candida glabrata | Cultivate stoste | 0 | - |
| Candida tropicalis | Cultivate stoste | 0 | - |
| Candida parapsilosis | Cultivate stoste | 0 | - |
5, the repeatability analysis of kit of the present invention
Experimental technique according to the 4th carries out repeated experiments, and the coefficient of variation of 10 repeated experiments is respectively 1.85% (1: 500), 4.12% (1: 1000) and 2.27% (1: 5000).With 20 days be one-period, detect sample every day one time, the coefficient of variation is respectively: 7.96% (1: 500), 10.46% (1: 1000) and 8.03% (1: 5000).
Example 3 detects the collaurum fast immune chromatographic test paper of aspergillus
1, the collaurum fast immune chromatographic test paper of detection aspergillus of the present invention constitutes as shown in Figure 2; This test paper is by sample pad 1; Polyester cellulose pad 2, nitrocellulose filter 3 and adsorptive pads 4 composition of overlapping successively; Wherein be coated with one deck collaurum-monoclonal antibody 9D47A1 compound on the polyester cellulose film 2, its grain size is 18～20nm, is peony; Be distributed with on the nitrocellulose filter 3 detect be with 5 with quality control band 6, wherein detect be with 5 be form at spraying monoclonal antibody 7E11A1 on the nitrocellulose filter 3, quality control band 6 is that spraying goat anti-mouse igg 1 antibody forms on nitrocellulose filter 3.
The preparation method of test paper of the present invention is: the aspergillus fumigatus monoclonal antibody 7E11A1 and the goat anti-mouse igg 1 that with concentration are 1-1.5mg/ml are attached on the nitrocellulose filter with the spray cloth amount spray of 0.8-1.5 μ l/cm; Respectively as detection line and nature controlling line; Seal with 1% bovine serum albumin(BSA) after the drying at room temperature; Be attached at after the oven dry on the base plate that scribbles double faced adhesive tape; Spray attaches that absorbance is the collaurum-monoclonal antibody 9D47A1 compound of the concentration of 0.5-0.8 under the 540nm on the overlapping 1-2mm polyester cellulose of right side and the adsorptive pads film, and sticks on the base plate, with the overlapping 1-2mm of the left end of cellulose nitrate pad; The base plate left end attaches sample pad, and the base plate that its right side and the overlapping 1-2mm of polyester cellulose pad will post cellulose nitrate pad, adsorptive pads, sample pad, polyester cellulose pad is cut into the rectangular of 4-6mm.
The preparation method of above-mentioned collaurum-monoclonal antibody 9D47A1 is: the preparation method who adopts conventional collaurum-monoclonal anti nanocrystal composition: get 0.1 gram sodium chloraurate and be dissolved in the deionized water of 1000ml, heating is boiled to 60 ℃, puts into 1% sodium citrate solution that 50ml newly joins; Wherein contain citric acid 0.2%, contain tannic acid 0.2%, be heated to 95 ℃ of 5min then; Present peony, formed colloid gold particle diameter is 18-20nm, with the centrifugal 30min of monoclonal antibody warp 36000 commentaries on classics/min of purifying; Get its supernatant through 0.2 μ m membrane filtration; When its concentration was transferred to 0.6mg/ml. liquid cold to be mixed to 15-30 ℃, the polyvinyl alcohol (PVA) PEG that adds 1ml1% concentration in the above-mentioned solution of every 100ml was to stop nonspecific agglutination, with the centrifugal 30min of 12000 commentaries on classics/min; Remove supernatant; Again with the centrifugal 1h of 12000 commentaries on classics/min, remove the not protein of absorption, the collaurum that encapsulates is diluted to absorbance is the concentration of 0.5-0.8 under the 540nm wavelength; With 0.2 μ m membrane filtration degerming, the i.e. collaurum of purifying-monoclonal antibody 9D47A1 compound.4 ℃ of preservations are subsequent use.
2, test paper of the present invention detects 19 kinds of aspergillus nutrient solutions.
Get the aspergillus nutrient solution 0.5ml (the aspergillus source is identical with example 2 with cultivation) of dilution in 1: 100, in centrifugal a moment, vertically insert test strips; Insertion depth is no more than sample pad; In sample, stop about 1-2min, make sample abundant up after, horizontal positioned 5-15min sentence read result.
The result judges:
Negative: test strips detects is with 5 not develop the color, and 1 red line appears in only quality control band 6 colour developings;
Positive: as, 2 red lines to occur if test strips detects band and quality control band all develops the color;
Invalid: if test strips detects band and quality control band does not all develop the color, no red line occurs, and then test invalidation representes that test strips lost efficacy.
The result is as shown in Figure 3, and collaurum fast immune chromatographic test paper of the present invention can detect various aspergillus, the reaction but other fungies all are negative.
Claims (4)
1. by the monoclonal antibody of hybridoma cell line CCTCC NO.C200731 generation and the application of monoclonal antibody in reagent, kit or the test paper of pairing preparation detection aspergillus that produces by hybridoma cell line CCTCC NO.C200730; The concrete mode that it is characterized by this application is; The monoclonal antibody that hybridoma cell line CCTCC NO.C200731 produces is used to detect the capture antibody of aspergillus, and the monoclonal antibody that hybridoma cell line CCTCC NO.C200730 produces is used to detect the detection antibody of aspergillus.
2. application according to claim 1; It is characterized in that described kit is a double antibodies sandwich ELISA kit; This kit is made up of the micro reaction plate that encapsulates capture antibody, sample preparation liquid, enzyme conjugates, positive control, negative control, concentrated washing lotion, colour developing liquid and stop buffer; Described capture antibody is the monoclonal antibody that hybridoma cell line CCTCC NO.C200731 produces; Enzyme conjugates is that the monoclonal anti body and function enzyme labeling that hybridoma cell line CCTCC NO.C200730 produces is made, and wherein said enzyme is oxidase or alkaline phosphatase.
3. application according to claim 2 is characterized in that described enzyme is a horseradish peroxidase.
4. application according to claim 1; It is characterized in that described test paper is that the collaurum fast immune chromatographic detects test paper; This test paper is by sample pad (1); Polyester cellulose pad (2), nitrocellulose filter (3) and adsorptive pads (4) composition of overlapping successively is coated with one deck collaurum-monoclonal antibody 9D47A1 compound on the polyester cellulose film (2); Be distributed with on the nitrocellulose filter (3) and detect band (5) and quality control band (6); Wherein detecting band (5) is to go up the monoclonal antibody that sprays hybridoma cell line CCTCC NO.C200730 generation at nitrocellulose filter (3) to form, and quality control band (6) is to go up the formation of spraying goat anti-mouse igg 1 antibody at nitrocellulose filter (3); The monoclonal antibody that wherein said collaurum-monoclonal antibody 9D47A1 compound is produced by hybridoma cell line CCTCC NO.C200731 is coated on the colloid gold particle and makes.
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| WO2019000267A1 (en) * | 2017-06-28 | 2019-01-03 | 丹娜（天津）生物科技有限公司 | Gm hybridoma, monoclonal antibody, kit, preparation method therefor and application thereof |
| CN108179114B (en) * | 2017-11-27 | 2021-08-20 | 南京晓庄学院 | Strain for producing anti-anaerobic bacteria compound, fermentation method, extraction and preparation method of anti-anaerobic bacteria compound and use method |
| EP3495819A1 (en) * | 2017-12-11 | 2019-06-12 | Euroimmun Medizinische Labordiagnostika AG | An improved method for diagnosing a fungal infection |
| CN109609466B (en) * | 2019-01-24 | 2022-03-01 | 天津一瑞生物科技股份有限公司 | Mouse aspergillus polysaccharide hybridoma cell strain, monoclonal antibody and application |
| CN109655614A (en) * | 2019-02-26 | 2019-04-19 | 广州维佰生物科技有限公司 | Ox brucellosis virus fluorescence micro-ball immune chromatography test paper and preparation method thereof and detection method |
| CN113791216A (en) * | 2021-08-31 | 2021-12-14 | 石家庄和亚生物技术有限公司 | ELISA detection method for detecting human serum candida glabrata enolase IgG antibody |
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