CN109970854A - Hybridoma cell strain AntiTput-13 and its AntiTput-DP10 monoclonal antibody of secretion - Google Patents

Abstract

The present invention relates to the relevant genetic engineering fields of dust mite allergen, disclose a strain of hybridoma strain AntiTput-13, the monoclonal antibody of the anti-tyrophagus putrescentiae allergen Der p10 of secretion that can be stable, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.16298, preservation address are as follows: BeiChen West Road, Chaoyang District, BeiJing City institute number, Institute of Microorganism, Academia Sinica.Test proves that specific reaction can occur for monoclonal antibody antiTPUT-DP10 secreted by the hybridoma cell strain and tyrophagus putrescentiae allergen Der p10.Based on the hybridoma secrete monoclonal antibody, the double sandwich-ELISA method of detection tyrophagus putrescentiae is established, the diagnostic method for precisely detecting the dust mites such as tyrophagus putrescentiae for environment such as house, storage, fields lays the foundation.

Description

Hybridoma cell strain AntiTput-13 and its AntiTput-DP10 monoclonal of secretion Antibody

Technical field

The invention belongs to the relevant genetic engineering fields of dust mite allergen, and in particular to a strain of hybridoma strain and its point The monoclonal antibody for the anti-tyrophagus putrescentiae allergen Der p10 albumen secreted.

Background technique

Tyrophagus putrescentiae (Tyrophagus putrescentiae) belongs to Arachnoidea (Arachinida), Acari (Acari), Acariformes (Acariformes) flour mite suborder (Acaridida) is universal storage harmful mite, Ji Zhufan It encloses extensively, is also widely present in human lives and working environment.Its mite body and metabolin, excreta etc. all have stronger allergen Property, the type Ⅰ allergies disease such as allergic asthma, dermatitis, allergic rhinitis can be caused, be the member of dust mite, dermatophagoides pteronyssinus One of.Research shows that about 50% or more type Ⅰ hypersensitivity Disease is caused by dust mite, population in the world sum 10~ 20% pair of dust mite allergy.Voorhort in 1964 etc. has found that mite has been in room dirt since main allergen, it has been discovered that dust mite Allergen is there are about 20 kinds, and there are the cross reactivities of height for dust mite and dermatophagoides pteronyssinus, and most dust mite patients are dermatophagoides pteronyssinus With the common sensitization of dust mite.Wherein, the first component allergen Der p1 of dermatophagoides pteronyssinus is primarily present in dermatophagoides pteronyssinus excreta, the 2 component allergen Der p2 are primarily present in mite body, and Der p1 and Der p2 are the masters of 80% or more dermatophagoides pteronyssinus autopath Want Allergen.The 1st group of allergic effect stock blend Der f1 of dust mite and the 2nd group of allergic effect stock blend Der f2 is main sensitization group Point.Der f1 and Der p1 can induce cross reaction, and Der f2 and Der p2 almost intersect.The 10th class of dermatophagoides pteronyssinus becomes Though answering the not main Allergen of former Der p10, the main allergic protein of cross reaction prompts Der p10 to can be used as room There are the monitoring Allergens of cross reaction with other allergens for dust mite.Research also shows that it is systemic that Der p10 is that one kind causes The important allergen of severe allergic reaction.Der p10 is compared with Der f10, there was only 3 amino acid differences, sequence in complete sequence Column consistency is widely present in dust mite muscle and nonmuscle cells up to 98.5%, and there are the conclusion of cross reaction is true for they It is fixed.

Der p10 protein structure and the tropomyosin in invertebrate are closely similar, such as shrimp, lobster, crab, roach Dung beetle and other mollusks.Der p10 is because of its extensive cross reactivity feature, except being positive in acarid autopath, and It also plays a significant role in the cross allergy of cockroach, Shrimp waste and shellfish food.In the inhibition test of cross reaction, dust mite Immersion liquid is strong inhibiting factor, shows that dust mite is main source in the allergen of sensitization.Therefore, Der p10 is by wide General concern, but as the tropomyosin Der p10 of dust mite, there are no detailed reports.

Antibody is biological and extremely wide medical domain purposes protein molecule.Monoclonal antibody is since immune B is thin Born of the same parents merge what the hybridoma to be formed generated with myeloma cell, which not only there is oncocyte constantly to divide Ability, and the ability with immunocyte generation antibody.Since monoclonal antibody is specific antibody, high specificity, therefore It is used widely.Test strips or ELISA kit etc. can be made for carrying out dust mite in environment in allergen monoclonal antibody The detection of allergen or the quantity that mite population is speculated according to the amount of allergen in detection environment.It can be with so filtering out one plant The hybridoma cell strain of the specific antibody of anti-Der p10 albumen is secreted, and the monoclonal antibody identified secreted by it is known Other Der p10 protein-specific B cell epitope polypeptide has positive effect to the diagnosis and prevention of Der p10 allergen, and Development for Der p10 diagnostic kit lays the foundation.

Summary of the invention

One of the object of the invention is to provide the hybridoma of one plant of secretion Der p10 allergen protein monoclonal antibody Strain；

The second object of the present invention is to providing a kind of monoclonal antibody antiTPUT- as secreted by above-mentioned hybridoma cell strain With Der p10 albumen specific reaction can occur for DP10, monoclonal antibody antiTPUT-DP10；

Its sequence are as follows:

MNHKVHHHHHHMEAIKKKMQAMKLEKDNAIDRAEIAEQKARDANLKSEKTEEEVRALQKKIQQIENELD QVQENLTQATTKLEEKEKALQTAEADVAALNRRIQLIEEDLERSEERLKVATAKLEEASHSADESERMRKMLEHRSI TDEERMDGLESQLKEARLMAEDADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEE KAQQREEAYEQQIRIMTSKLKEAEARAEFAERSVQKLQKEVDRLEDELVHEKEKYESISDELDQTFAELTGY

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention extracts tyrophagus putrescentiae total serum IgE using TRIZOL method, and tropomyosin gene (Der p10) is cloned in reverse transcription CDNA, which is inserted into prokaryotic expression carrier pCzn1, Der p10 gene is carried out using pCzn1 prokaryotic expression carrier former Nuclear expression regard the Der p10 of expressed inclusion bodies out as immunogene after HIS label affinity purification, SPF is immunized BALB/c mouse takes its splenocyte to merge with SP2/0 myeloma cell.In addition, the present invention also utilizes Bacillus coli expression System carries out prokaryotic expression to Der p10 gene, carries out inclusion body purification to the Der p10 albumen of expressed inclusion bodies It is used as detection antigen afterwards, establishes indirect ELISA detection method and positive hybridoma cell is screened, it is final to obtain one plant surely Surely the hybridoma cell strain of anti-Der p10 protein monoclonal antibody is secreted, microbial preservation number is: CGMCC 16298, life Entitled AntiTput-13, classification naming are: secreting the hybridoma cell strain of anti-Der p10 allergen monoclonal antibody；When preservation Between: on September 13rd, 2018；Depositary institution is: China General Microbiological culture presevation administrative center；Preservation address is: Beijing The institute of microbiology, the Chinese Academy of Sciences, institute of Chaoyang District North Star West Road 1.

The present invention also provides a kind of monoclonal antibodies as secreted by above-mentioned hybridoma cell strain, are named as AntiTput-DP10, Western-blot testing result show that monoclonal antibody AntiTput-DP10 can be with Der p10 albumen Specific reaction occurs, it is anti-that ELISA testing result shows that specificity occurs for monoclonal antibody AntiTput-DP10 and Der p10 It answers.

In one embodiment of the invention, with tyrophagus putrescentiae more resist for coated antibody, antibody label after monoclonal Antibody A ntiTput-DP10 is enzyme labelled antibody, establishes the double-antibody sandwich engineering method of detection tyrophagus putrescentiae, the dual anti-folder established with this Tyrophagus putrescentiae in the environment such as heart method detection detection house, storage, field, has practical application value.

Detailed description of the invention

Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:

Fig. 1 recombinates pCzn1-TM-T plasmid enzyme restriction as a result, wherein M:DNA Marker；1: plasmid before digestion；2: after digestion Plasmid；

Fig. 2 is to recombinate the SDS-PAGE analysis of pCzn1-TM-T albumen as a result, wherein M: standard protein Marker；1: Product before pCzn1-TM-T is induced；Product after 2:pCzn1-TM-T induction；3:pCzn1-TM-T purified product.

Fig. 3 is that SDS-PAGE is analyzed after purification for monoclonal antibody AntiTput-DP10, wherein 1: standard protein Marker；2: monoclonal antibody antiTPUT-DP10 heavy chain and light chain.

Fig. 4 is for application Western blot testing inspection monoclonal antibody antiTPUT-DP10 and Der p10 albumen WB Analysis, wherein 1: product after acarid cracking precipitating；M: standard protein Marker.

Specific embodiment

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings, it should be understood that preferred reality described herein Apply example only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.

Main experimental materials and source

1, cell, dust mite and serum

SP2/0 murine myeloma cell, HGPRT missing；Immunoglobulin is not generated and passes through detection of mycoplasma, by this Laboratory saves, and tyrophagus putrescentiae is raised in this laboratory population.Tyrophagus putrescentiae is positive, negative serum is prepared by laboratory, passes through ELISA experiment is detected as negative and positive.

2, main agents and drug

Fetal calf serum, DMEM culture medium are purchased from GIBCO company；Diaminobenzidine (DAB) colour reagent box, horseradish peroxide The goat anti-mouse IgG antibodies of compound enzyme (HRP) label, FITC label sheep anti-mouse igg antibody, plastic recovery kit are purchased from Bei Jingkang For century Biotechnology Co., Ltd；50%PEG, 50 × HAT, 50 × HT are purchased from Sigma company；O-phenylenediamine, pre-dyed albumen Marker is purchased from Fermentas company；SBA ClonotypingTM System/HRP Subclass of antibody identification kit is purchased from Southern Biotechnology company；Plasmid extraction kit is purchased from AXYGEN company, T-E1, reverse transcriptase, Ex Taq Archaeal dna polymerase, T4DNA ligase, HIS label purified reagent are purchased from Dalian treasured biotech firm.

3, experimental animal

BALB/c outbred mice is provided by this experimental animal of Changchun hundred million.Production licence: SCXK (Jilin) 2016- 0003

4, test equipment

The surgical instrument of sterilizing: three scissors, three tweezers, a cell sieve, syringe inner core, a plate Wet box, 2 500mL beakers, 2 50mL centrifuge tubes, 3 15mL centrifuge tubes.

5, culture medium

IMDM culture medium, fetal calf serum are purchased from HyCLone company, are configured to IMDM complete medium (containing 15% serum)； PEG1500 is purchased from Roche, article No.: 78364；HAT culture solution, HT culture solution are purchased from Sigma, and article No. is respectively H0262- 10VL and H0137-10VL.Hybridoma supematant ELISA detection: coating buffer: sodium carbonate-bicarbonate buffer, pH9.6；0.1M PBS buffer solution pH7.4；Confining liquid: 2% skim milk；Washing lotion: PBST (0.05% tween, PBS)；Developing solution soluble T MB； Terminate liquid: 2M sulfuric acid.

The prokaryotic expression of 1 Der p10 albumen of embodiment and purifying

1. design of primers

It (is logged according to tropomyosin (tropomyosin) gene order of tyrophagus putrescentiae listed in Genbank Number: AAT40866.1), PCR amplification primer is designed, sequence is as follows: 5 '-ATGAATCACAAAGTGCATCATCATC of upstream primer ATCATCATGGC- 3 ', 5 '-CCG of downstream primerTCTAGATTAATAACCGGTCAGTTCGGCAAA-3 ', underscore are digestion position Point.

2. the extraction and reverse transcription of tyrophagus putrescentiae total serum IgE

Total serum IgE is extracted from tyrophagus putrescentiae using Trizol method as template, carries out reverse transcription synthesis with random primer cDNA。

Trizol method extracts RNA step: picking tyrophagus putrescentiae adult mite 30 under microscope, and it is mixed that 0.5mL Trizol is added It is even, it is stored at room temperature 5min, 0.1mL chloroform is added, firmly shakes 15s, is incubated at room temperature 2-3min, 12000g, 4 DEG C of centrifugation 15min, It is careful to take out upper layer colourless liquid, the isopropanol being pre-chilled in equal volume is added, be incubated at room temperature after mixing 10min, 12000g, 4 DEG C from Heart 10min, abandons supernatant, and the ethyl alcohol (DEPC water is prepared) of 0.5mL75%, 15s, 7500g, 4 DEG C of centrifugations of gently shaking are added in precipitating 5min, careful to abandon supernatant to the greatest extent, precipitating is air-dried at room temperature 3-5min, and the dissolution of 20-30 μ L DEPC water, -20 DEG C of preservations are added.

Reverse transcription synthesis cDNA is carried out with the total serum IgE of extraction, system is as follows:

In reaction process, first by template ribonucleic acid solution and random primer in 95 DEG C of incubation 10min, the cooling 5min of ice bath, then Remaining reagent is added, mixes, is placed at room temperature for 10min, 42 DEG C of incubations 60min cool down 2min in ice.

3.Der p10 gene magnification and purifying

The cDNA obtained using reverse transcription, using designed PCR amplification primer, expands Der p10 gene as template.

PCR reaction system (50 μ L) is as follows:

Reaction condition is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 3min, 30 are followed Ring；72 DEG C of extension 10min.Whole pcr amplification products are carried out agarose gel electrophoresis by the recycling of PCR product glue, and in ultraviolet lamp Under cut the blob of viscose containing target gene, recycle PCR according to the precious biological Co., Ltd's plastic recovery kit specification in Dalian later The Der p10 gene amplified.

The building of 4.Der p10 albumen pronucleus expression recombinant vector

Der p10 gene and prokaryotic expression carrier pCzn1 that glue recycles are attached respectively, linked system Are as follows:

Condition of contact: 16 DEG C of connections are overnight.

5. converting and choosing bacterium

Connection product obtained in 4 is all transferred in Ecoli BL21 competent cell, ice bath 30min, 42 DEG C later Water-bath thermostimulation 90s, then rapid ice bath 2min.250 μ L LB liquid mediums are added in Xiang Guanzhong, shake culture in 37 DEG C of shaking tables 100 μ L bacterium solutions are coated on the LB plate containing ampicillin (100 μ g/mL), 37 DEG C of overnight incubations by 1h.From plate with Machine picking single bacterium colony is inoculated into 3mL LB (Amp respectively⁺, 100 μ g/mL) and 37 DEG C of shaken cultivations in fluid nutrient medium.

6. the PCR identification and sequencing of recombinant plasmid

1) the small amount plasmid extraction agent box for utilizing AXYGEN company is cultivated from 5 according to kit specification operation Plasmid is extracted in bacterium solution, and extracted plasmid is subjected to PCR identification and is sequenced.

2) PCR identification is carried out to doubtful recombinant plasmid

PCR reaction system (50 μ L) is as follows:

Reaction condition is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 3min, 30 are followed Ring；72 DEG C of extension 10min.

Plasmid (as shown in Figure 1) Jing Guo the PCR result positive is delivered into Nanjing Qing Ke Bioisystech Co., Ltd and carries out sequence Column measurement, will identify that correct recombinant plasmid is named as pCzn1-TM-T by sequencing.

The prokaryotic expression of 7.Der p10 gene and purifying

Recombinant plasmid pCzn1-TM-T is transformed into prokaryotic expression BL21 competent cell according to 5 method for transformation, It then takes out 100 μ L bacterium solutions to be coated on the LB plate containing ampicillin (100 μ g/mL), 37 DEG C of overnight incubations, picking LB White colony on plating medium is inoculated in 3mL and contains in the LB liquid medium of ampicillin (100 μ g/mL) 37 DEG C Overnight incubation.Induced expression concrete operations are as follows: 1mL recombinant bacterium bacterium solution being taken to be added to 37 DEG C of concussions in the LB culture medium of 100mL Cultivating to OD600nm is about 0.6-0.8 (about 2h)；Add IPTG to final concentration of 1mmol/L, 37 DEG C of induction 4h.By expression product SDS-PAGE electrophoresis is carried out, is purified by the affinity chromatography method of HIS label, expression and purification result are shown in Fig. 2.Affine layer Analysis method is as follows:

1, it will be centrifuged by the bacterium solution of IPTG induction, 7000rpm, 10min.Somatic cells are collected, are rinsed twice with PBS, The bacterium solution ultrasonication that will be gathered, until clarification, 13000rpm are centrifuged 10min, take precipitating.The loading used with purifying again Buffer rinses one time, is finally resuspended with sample-loading buffer, 13000rpm, is centrifuged 10min, supernatant is taken, with 0.45 μm of filter mistake Filter.

2, chromatographic column is added after mixing filler, is stored at room temperature 10 minutes, after gel and solution layering, going out for bottom Liquid mouth is opened, and ethyl alcohol is allowed slowly to flow out by gravity.

3, supernatant is loaded into upper prop, flow velocity is 10 times of column volume/hours, and collection flows through liquid.Use 15 times of column volumes Binding Buffer rinses pillar, washes away foreign protein.It is eluted using 5mlElution Buffer, collects eluting peak.It will purifying Product carries out SDS-PAGE electrophoresis detection afterwards.

The preparation of 2 monoclonal antibody of embodiment

1. mouse immune

It is small with 46 week old female SPF BALB/c females of recombination Der p10 protein immunization of the prokaryotic expression of affinity purification Mouse is immunized 3 times, each immunization interval two weeks altogether, and immunizing dose is 30 μ g/, and immunization route is peritoneal immunity.

Respectively two exempt from three exempt from it is latter week to mouse carry out eye socket blood sampling, separation serum (4 DEG C, 10000rpm, 20min), antibody level is detected with indirect ELISA, it is best to the results are shown in Table 1, No. 3 Mouse titers, and cell fusion selects No. 3 mouse. 3 days before cell fusion, booster immunization, every mouse peritoneal note are carried out again to No. 3 SPF BALB/c mouses of good immune effect Penetrate 50 μ g immunizing antigens.

Indirect ELISA method: with recombination Der p10 proteantigen after purification, 2ug/ml, the hole 100uL/, 4 DEG C were coated with Night；It is washed 3 times with washing lotion afterwards；The closing of 2% skimmed milk confining liquid, the hole 200uL/, 37 DEG C of incubators, 2h；It is washed 3 times with washing lotion afterwards； Serum 2 times of gradient dilutions since 200 times, blank control (blank) are PBS, and negative control (negative) is negative serum 200 times of dilutions.It is the hole 100uL/, 37 DEG C of incubators, 1h；It is washed 3 times with washing lotion afterwards；The secondary antibody that PBS dilutes 20000 times is added (sheep anti-mouse igg of HRP label), the hole 100uL/, 37 DEG C of incubators, 1h；It is washed 3 times after taking-up with washing lotion；Colour developing, developing solution The hole 100uL/, developing time 5min；Every hole is added 50uL terminate liquid and terminates；OD value is surveyed at 450nm, record saves data.

Table 1 recombinates Der p10 protein ELISA potency result

2. cell fusion

First 1 day preparation feeder cells are merged, SPF BALB/c mouse peritoneal macrophage is conventionally taken to spread 96 It is stand-by in porocyte culture plates.Disconnected neck puts to death the mouse of spleen to be taken, sterile to take spleen and separating Morr. cell, by splenocyte and SP2/0 The ratio of myeloma cell 3:1 carries out cell fusion with PEG, and fused cell is laid on ready feeder cells.

3. screening and the clone of positive hybridoma cell strain

It is thin that indirect ELISA detection method screening positive hybridoma is established using prokaryotic expression Der p10 albumen after purification Born of the same parents' strain expands culture to the hybridoma of reacting positive, while carrying out the Ya Ke of positive hybridoma cell with limiting dilution assay It is grand, it is at least subcloned 3 times, the positive hybridoma cell being subcloned is frozen in time.Final one plant of acquisition can be with stably excreting The hybridoma cell strain of anti-Der p10 allergen protein monoclonal antibody, and the monoclonal antibody secreted is named as AntiTput-13。

4. the ascites preparation and purification of monoclonal antibody

To 10 week old or so healthy SPF BALB/c mouse be injected intraperitoneally incomplete Freund's adjuvant, 0.5mL/ only, after 1 week Intraperitoneal injection 1 × 10⁶A hybridoma extracts ascites when mouse web portion extreme expansion after 7~10d, taken out every 2d it is primary, will The ascites 10000r/min of extraction is centrifuged 10min, removes upper layer grease and precipitating, and supernatant packing is stored in -20 DEG C.

Mouse ascites antibody uses G-protein affinity purification.Method is as follows: filler being loaded into chromatographic column, with caching liquid Ascites is diluted about 5 times by (20mM PBS, 300mM NaCl, pH7.4-8.5), and upper prop is purified.After loading, equalizing and buffering is used Liquid (20mM PBS, 300mM NaCl, pH7.4-8.5) removes the unbonded part of albumen；Then elution buffer (0.1M is added Sodium citrate, pH4.0), binding protein is eluted, finally carries out PH with neutralization buffer (1M tris-HCl, pH9.0) Value adjustment, antibody concentration and purity are shown in Table 2.Antibody after purification carries out SDS-PAGE detection, as a result sees Fig. 3.

Table 2: AntiTput-13 purity, concentration and potency after purification

The identification of 3 monoclonal antibody of embodiment

1. the subgroup identification of monoclonal antibody

According to SBA Clonotyping^TMSystem-HRP (Southern Biotech) Subclass of antibody identification kit behaviour It explains book and subgroup identification is carried out to the obtained monoclonal antibody of embodiment 2.

The heavy chain of monoclonal antibody AntiTput-DP10 of the present invention is IgG1 as the result is shown, and light chain is Kappa chain, identification It the results are shown in Table 3.

3 monoclonal cell subgroup identification of table

2.Western blot qualification test

SDS-PAGE electrophoresis is carried out after centrifugation processing after 5 tyrophagus putrescentiae adult mite polypide pestles are ground, then through electricity By on protein delivery to nitrocellulose filter, it is 18V voltage 30min that electricity, which turns condition, for transfer, will be turned with 5% skimmed milk power confining liquid 4 DEG C of nitrocellulose filter closings after print are overnight；Monoclonal antibody supernatant is added and is incubated at room temperature 1h, (is spat containing 0.5mL/L with PBST The PBS buffer solution of the pH7.4 of temperature -20) it washs three times, then the mountain with 4000 times of diluted horseradish peroxidases (HRP) labels Sheep anti-mouse igg antibody is incubated at room temperature 1h, after PBST is washed 3 times, is developed the color with DAB colour reagent box, scanning record.

Test result confirms that monoclonal antibody AntiTput-DP10 prepared by the present invention can occur with tyrophagus putrescentiae Specific reaction, and do not react with (oyster cap fungus mycelia) is compareed, as a result see Fig. 4, thus it is speculated that AntiTput-13 was identified Der p10 protein B cell epitope is a linear epitope.

Embodiment 4 establishes detection dust mite mistake using tyrophagus putrescentiae allergen Der p10 monoclonal antibody AntiTput-DP10 The sandwich ELISA method in quick source

Double antibody sandwich method, the antigen or antibody for being usually incorporated in surface of solid phase carriers still keep its immunologic competence, The antigen or antibody of enzyme label had not only retained its immunologic competence, but also retained the activity of enzyme.By inspection sample and surface of solid phase carriers Antigen or antibody react.Make other objects in the antigen antibody complex and liquid that are formed on solid phase carrier with the method for washing Matter separates.The antigen or antibody of enzyme label is added, is also integrated on solid phase carrier by reaction.After the substrate of enzyme reaction is added, Substrate becomes color products by enzymatic, and the amount of product is directly related with the amount of tested substance in sample, according to the depth of colour generation Carry out qualitative or quantitative analysis.The catalytic efficiency of enzyme is very high, be exaggerated indirectly immune response as a result, reaching measuring method Very high susceptibility.This research prepares the positive polyvalent antibody of tyrophagus putrescentiae, as coated antibody；Monoclonal antibody AntiTput-DP10 marks horseradish peroxidase as colour developing antibody, and detectable substance is doubtful tyrophagus putrescentiae culture.This method Can using with mushroom acarid hazard detection, have very strong practicability.

1, the determination of standard Der p10 albumen difference dilution standard curve

A. the determination of antibody working concentration: coated antibody (the positive polyvalent antibody of tyrophagus putrescentiae) and enzyme mark are marked anti- Body (monoclonal antibody AntiTput-DP10) carries out gradient dilution, determines its working concentration using Checkerboard titration method, experiment shows Coated antibody concentration is 100 μ g/ml, and when enzyme labelled antibody dilution 1:2000, effect is best, and the positive is than negative value highest.

The determination of B double antibody sandwich method operating procedure

1, be coated with: positive No. I of tyrophagus putrescentiae and coating buffer are diluted to 100 holes μ g/ml, 100ul/, 37 DEG C of insulating box 1.5h, Board-washing machine cleans 1 time.

2 closings: 10% skimmed milk+3%BSA is closed, every hole 250ul, and board-washing machine cleans 4 times for 24 hours for 4 DEG C of closings.

3 sample 100ul are diluted using coating buffer (1% skimmed milk), and 37 DEG C of insulating box 1.5h board-washing machines clean 3 times.

4 secondary antibody 1:2000 dilute 100 μ l, PBS (1% skimmed milk), 37 DEG C of 1h, and board-washing machine cleans 2 times.

5 colour developings: 100 μ l room temperature 3min of TMB.50 μ l of 2M H2SO4 terminate liquid.

Sequence table

<110>Jiangsu Province Agriculture Science Institute

<120>hybridoma cell strain AntiTput-13 and its AntiTput-DP10 monoclonal antibody of secretion

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 295

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Met Asn His Lys Val His His His His His His Met Glu Ala Ile Lys

1 5 10 15

Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp Asn Ala Ile Asp Arg

20 25 30

Ala Glu Ile Ala Glu Gln Lys Ala Arg Asp Ala Asn Leu Lys Ser Glu

35 40 45

Lys Thr Glu Glu Glu Val Arg Ala Leu Gln Lys Lys Ile Gln Gln Ile

50 55 60

Glu Asn Glu Leu Asp Gln Val Gln Glu Asn Leu Thr Gln Ala Thr Thr

65 70 75 80

Lys Leu Glu Glu Lys Glu Lys Ala Leu Gln Thr Ala Glu Ala Asp Val

85 90 95

Ala Ala Leu Asn Arg Arg Ile Gln Leu Ile Glu Glu Asp Leu Glu Arg

100 105 110

Ser Glu Glu Arg Leu Lys Val Ala Thr Ala Lys Leu Glu Glu Ala Ser

115 120 125

His Ser Ala Asp Glu Ser Glu Arg Met Arg Lys Met Leu Glu His Arg

130 135 140

Ser Ile Thr Asp Glu Glu Arg Met Asp Gly Leu Glu Ser Gln Leu Lys

145 150 155 160

Glu Ala Arg Leu Met Ala Glu Asp Ala Asp Arg Lys Tyr Asp Glu Val

165 170 175

Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu

180 185 190

Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu

195 200 205

Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys

210 215 220

Ala Gln Gln Arg Glu Glu Ala Tyr Glu Gln Gln Ile Arg Ile Met Thr

225 230 235 240

Ser Lys Leu Lys Glu Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser

245 250 255

Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val

260 265 270

His Glu Lys Glu Lys Tyr Glu Ser Ile Ser Asp Glu Leu Asp Gln Thr

275 280 285

Phe Ala Glu Leu Thr Gly Tyr

290 295

Claims (3)

1. a strain of hybridoma strain AntiTput-13, the monoclonal of the anti-tyrophagus putrescentiae allergen Der p10 of secretion that can be stable Antibody, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.16298, preservation address are as follows: BeiChen West Road, Chaoyang District, BeiJing City institute number, Institute of Microorganism, Academia Sinica.

2. a kind of monoclonal antibody antiTPUT-DP10 of anti-tyrophagus putrescentiae allergen Der p10, which is characterized in that by right It is required that hybridoma cell strain AntiTput-13 described in 1 secretes, sequence is as shown in SEQ ID NO:1.

3. the monoclonal antibody antiTPUT-DP10 of anti-tyrophagus putrescentiae allergen Der p10 described in claim 2 is preparing environment The reagent of middle tyrophagus putrescentiae Der p10 diagnosis or the purposes in detection kit.