The antibody of anti-Ractopamine hydrochloride and application thereofTechnical field
The invention belongs to biological technical field, particularly relate to the antibody of anti-Ractopamine hydrochloride, and detect the ELISA kit of Ractopamine hydrochloride, the application of colloidal gold test paper card in preparation.
Background technology
" clenbuterol hydrochloride " is that a class belongs to suprarenal gland class nervous excitation agent medicine.It can make livestock promote protein synthesis in metabolic process, accelerates the conversion Sum decomposition of fat, then improves the speed of growth of livestock, increases lean ratio; Feed can also be reduced use, meat is gone on the market ahead of time, reduce costs simultaneously." clenbuterol hydrochloride " kind catalogue that office of the food safety council of State Council " " clenbuterol hydrochloride " focus efforts on special areas scheme " (food peace does [2011] No. l4) specifies is: Clenbuterol hydrochloride (ClenbuterolHydrochloride), Ractopamine hydrochloride (Ractopamine, Rac), salbutamol (Salbutamo1) etc.Wherein, Ractopamine hydrochloride (Rac), easily in animal tissues, particularly gathers residual, and enters human body by food chain in internal organ.Then cause Skeletal Muscle Contraction to strengthen, destroy the fusion phenomenon between quick muscle and slow switch fibers, cause muscular tremor, occur the untoward reaction such as flushed face, headache, dizziness, uncomfortable in chest, palpitaition, numbness of the limbs, serious possible threat to life.Ractopamine hydrochloride is all classified as violated thing by many countries, and China has forbidden using Ractopamine hydrochloride with it livestock and poultry.
At present, the common method both at home and abroad for detecting Ractopamine hydrochloride has: enzyme-linked immunosorbent assay (ELISA), colloidal gold immunity chromatography, high performance liquid chromatography, gas chromatography-mass spectrography, chromatography, capillary electrophoresis, immunosensor method etc.Chromatography, capillary electrophoresis, immunosensor method detect sensitive, but required plant and instrument is expensive, sample pre-treatments is complicated, one-time detection sample size is few, detection time is long, and not easily popularizes, and ELISA, colloidal gold immunity chromatography do not need the instrument by costliness, and sample pre-treatments is simple, has the advantages such as high-throughput.But the current existing enzyme linked immunological kit of detection Ractopamine hydrochloride or the detection sensitivity of colloidal gold test paper card or accuracy are not also very good, analyze its reason, be mainly that adopted anti-Anti-ractopamine antibody need to improve.Because Ractopamine hydrochloride is chemical small molecule, its surface site is few, thus for its antibody of preparation brings larger difficulty, especially the antibody that specificity is higher, the specificity of current city antibody is general lower, and half-inhibition concentration (IC50) is higher, cannot meet the demand of application.
Summary of the invention
The present invention aims to provide can the antibody of effective, specific binding Ractopamine hydrochloride (Rac), and detect Ractopamine hydrochloride (Rac) purposes residual in animal body.More particularly:
The present invention's first object is to provide a kind of antibody, and described antibodies specific is in conjunction with Ractopamine hydrochloride (Rac), and described antibody comprises:
Variable region of heavy chain, its aminoacid sequence contains following complementary determining region: the HCDR1 as shown in sequence SEQIDNO:2, the HCDR2 as shown in sequence SEQIDNO:3 and/or the HCDR3 as shown in sequence SEQIDNO:4;
And variable region of light chain, its aminoacid sequence contains following complementary determining region: the LCDR1 as shown in sequence SEQIDNO:6, the LCDR2 as shown in sequence SEQIDNO:7 and/or the LCDR3 as shown in sequence SEQIDNO:8.
Antibody preferably in the present invention contains the variable region of heavy chain of aminoacid sequence as shown in SEQIDNO:1 and the variable region of light chain of aminoacid sequence as shown in SEQIDNO:5.
In preferred the present invention, the encoding gene of antibody is containing, for example the variable region of heavy chain shown in SEQIDNO:9 and the variable region of light chain as shown in SEQIDNO:10.
The present invention's second object is to provide a kind of single-chain antibody, and the aminoacid sequence of described single-chain antibody is as shown in SEQIDNO:11.Preferably, the HIS label be made up of six Histidines is not contained in described single-chain antibody.
The present invention's the 3rd object is to provide a kind of nucleotide sequence of above-mentioned single-chain antibody of encoding, and described nucleotide sequence is as shown in SEQIDNO:12.
The present invention's the 4th object is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
The present invention's the 5th object is to provide a kind of recombinant host cell containing above-mentioned expression vector, and affiliated host cell can be intestinal bacteria, yeast or mammalian cell, is preferably pichia spp.
The present invention's the 6th object is to provide a kind of method of producing above-mentioned antibody, comprising:
1) cultivate above-mentioned recombinant host cell under suitable conditions and express antibody;
2) then from Host Strains purifying, collect antibody.
The present invention's the 7th object is to provide above-mentioned antibody for detecting the residual novelty teabag of Ractopamine hydrochloride (Rac).
The present invention's the 8th object is to provide a kind of colloidal gold test paper card or the ELISA kit that detect Ractopamine hydrochloride, and described colloidal gold test paper card or ELISA kit contain antibody described above.
Preferably, ELISA kit described above, comprising: bag is by enzyme plate, the antibody working fluid containing antibody described above, enzyme mark thing, Ractopamine hydrochloride reference liquid, nitrite ion, stop buffer, the concentrated cleaning solution of the carrier protein couplet thing of antigen Ractopamine hydrochloride.Preferred, described carrier proteins is BSA, OVA, KLH.
Preferably, antibody working fluid described above is obtained by above-mentioned antibody dilution with enzyme diluent, and described enzyme diluent is PBST (pH7.4) damping fluid of 0.01M, and wherein containing mass volume ratio is the Na of 0.335%2hPO412H2o, 0.02%NaH2pO42H2o, 0.8%NaCl, 0.02%KCl, 0.1%Casein, 0.05%BSA.
Preferably, enzyme mark thing described above is anti-His antibody-HRP, anti-His antibody-AP, ProteinL-HRP or ProteinL-AP.
Preferably, nitrite ion described above is TMB nitrite ion or BCIP/NBT nitrite ion.
Preferably, stop buffer described above is the H of 2M2sO4.
Preferably, concentrated cleaning solution described above is the 0.2MPBS containing 0.05%TWEEN-20.
9th object of the present invention is to provide a kind of method detecting Ractopamine hydrochloride, comprises the following steps:
1. number: sample and micropore corresponding to standard substance are numbered according to the order of sequence, each sample and standard substance establish multiple hole parallel;
2. application of sample: every hole adds standard substance, sample, enzyme mark thing and antibody working fluid according to the order of sequence, reacts in room temperature lucifuge;
3. wash plate: after reaction, wash plate and drying treatment;
4. develop the color: add nitrite ion, react in room temperature lucifuge;
5. stop and reading: add stop buffer, measure every hole OD value.
6. result judges:
I). qualitatively judge: judged by the simple contrast of the light absorption ratio of sample well and the light absorption ratio of standard sample wells: the color of sample well than Ractopamine hydrochloride in the then sample of light color of a certain standard sample wells this hole of concentration ratio contained by the concentration of standard substance high, the color of sample well than a certain standard sample wells color deeply then in sample Ractopamine hydrochloride this hole of concentration ratio contained by the concentration of standard substance low;
Ii). quantitative analysis: with the per-cent of the absorbance of the absorbance values of standard substance and first standard (0ppb) for ordinate zou, with the logarithm of the concentration of Ractopamine hydrochloride standard substance (ppb) for X-coordinate, drawing standard graphic representation.The per-cent of the absorbance of the absorbance values of sample and first standard (0ppb) is substituted in typical curve, thus calculates the actual concentrations of Ractopamine hydrochloride in sample.
Of the present invention ten object is to provide a kind of colloidal gold test paper card detecting Ractopamine hydrochloride, comprise sample pad 1, gold mark pad 6, nitrocellulose filter 2, absorption pad 3, liner plate 7, print pad 1, gold mark pad 6, nitrocellulose filter 2, absorption pad 3 is adjacent between two and part is stacked is pasted on successively from left to right on liner plate 7, described nitrocellulose filter 2 there are quality control band 4 (C line) and detection zone 5 (T line), containing the above-mentioned anti-Anti-ractopamine antibody through colloid gold label in described gold mark pad 6.
Preferably, quality control band 4 position is coated with anti-His antibody or ProteinL, and detection zone 5 position is coated with the conjugate of Ractopamine hydrochloride haptens and carrier proteins.
It is more desirable that commercially available prod is compared in sensitivity and the accuracy of the ELISA detection kit prepared of antibody provided by the present invention and colloidal gold test paper card, and specificity is fine.
Accompanying drawing explanation
Fig. 1. antibody K07 heavy chain, chain variable region gene electrophorogram.Band 1 is standard DNA, and band 2 is K07 antibody heavy chain variable region DNA, Lane3 is K07 antibody chain variable region DNA
Fig. 2. antibody K07 structural representation.Vhrepresent weight chain variabl area sequence, Vlrepresent light-chain variable sequence, His label is six Histidines.
Fig. 3. the agarose gel electrophoresis figure of antibody expression PCR primer.
Fig. 4. recombinant pichia yeast strain abduction delivering supernatant nutrient solution qualification figure.Figure a is SDS-PAGE electroresis appraisal figure, figure b is Westernblot qualification figure.
Fig. 5. antibody purification design sketch (SDS-PAGE).The antibody K07 that band 1-10 obtains for different collection tube.
Fig. 6. the WesternBlot qualification figure of antibody K07.Swimming lane 1 is standard protein, swimming lane 2 is Rac-BSA, swimming lane 3 is Rac-OVA, swimming lane 4 is Cle-BSA, swimming lane 5 is Cle-OVA, swimming lane 6 is Sal-BSA, swimming lane 7 is Sal-OVA.
Fig. 7. the typical curve of the ELISA detection kit of Ractopamine hydrochloride of the present invention.Wherein X-coordinate is Ractopamine hydrochloride standard concentration logarithm (lg); Ordinate zou is Ractopamine hydrochloride standard substance percentage light absorption ratio.
Fig. 8. Ractopamine hydrochloride colloidal gold test card structure schematic diagram of the present invention.
1 be sample pad, 2 be wherein nitrocellulose filter, 3 be absorption pad, 4 be nature controlling line (C line), 5 be detection line (T line), 6 for gold mark pad, 7 is for liner plate
Specific embodiment
Definition
" antibody " is also known as immunoglobulin (Ig), the large-scale Y shape protein that a class is secreted by bone-marrow-derived lymphocyte, can by the immunoglobulin molecules of complementary site (antigen bound site) the specific binding target antigen on wherein two bifurcated tops of Y shape, described target antigen is as protein, sugar, polynucleotide, fat, polypeptide, micromolecular compound etc.This term not only comprises complete polyclonal antibody, also comprises its fragment (such as Fab, Fab ', F (ab ')2, Fv), single-chain antibody (singlechainantibodyfragment, scFv), its mutant, comprise antibody moiety fusion rotein and arbitrary comprise antigen recognition site other change the immunoglobulin molecules of configurations.Antibody comprises the antibody of arbitrary class or multiclass, and as IgG, IgA, IgD, IgE or IgM (or its subclass), and antibody does not need for arbitrary certain kinds.
" single-chain antibody " (scFv) refers to the variable region of heavy chain (V of antibodyh) and variable region of light chain (Vl) the single chain fusion protein that is connected to form by 15 ~ 20 amino acid short peptides (linker), be usually rich in glycine and Serine for the linker connected, be beneficial to stability and the snappiness of single-chain antibody.Mode of connection can by Vln end be connected to Vhc-terminal, or on the contrary.Although eliminate constant region and introduce linker, single-chain antibody still remains the specificity of antibody to antigen, and it has that molecular weight is little, penetration power is strong and the feature such as antigenicity is weak.
Complementary determining region (complementarity-determiningregion, CDR), is also called hypervariable region.Taking shape in the amino acid whose end of antibody monomer, is the most critical zone of target antigen and antibodies, and in Artificial Immune Network Theory, the complementary determining region of each antibody is otherwise known as idiotype or genotype.In the application, HCDR refers to the complementary determining region in variable region of heavy chain, and LCDR refers to the complementary determining region in variable region of light chain.
embodiment 1: the preparation of anti-Ractopamine hydrochloride hybridoma cell strain
1, animal immune
With Ractopamine hydrochloride coupling bovine serum albumin (Rac-BSA) conjugate (immunogen) according to general immune programme for children immunity BALB/c female mice (purchased from this laboratory animal company limited of Cavan, Changzhou).First mixed with Rac-BSA conjugate equal-volume by Freund's complete adjuvant/Freund's incomplete adjuvant and repeatedly blow and beat, making immunogen fully emulsified, obtaining concentration is the emulsified immunogen of 0.5mg/mL.Choose the female BAl BIc/c mouse in 6-8 age in week, every mouse adopts subcutaneous multi-point injection mode to inoculate above-mentioned emulsified immunogen, and immunity in every two weeks once.Concrete Immunity sees the following form:
From after second time immunity, orbital venous plexus blood sampling is adopted when one week after immunity, tiring of immune serum (Ractopamine hydrochloride coupling oralbumin conjugate Rac-OVA detects) and half suppression valency is detected with indirect ELISA and Inhibition ELISA, choose serum titer and all the highest mouse of suppression valency, carry out immune mouse spleen cell and murine myeloma cell merges.
2, cytogamy
(1). the preparation of spleen cell
Get the immune mouse that serum titer and suppression valency in step 1 are all the highest, pluck eyeball and get blood, be placed on immersion in the alcohol of 75% (v/v) through disconnected cervical vertebra execution, after 10 minutes, in aseptic operating platform, to get its spleen, be placed in cell screen cloth, abundant grinding cell, cross screen cloth, after aseptic 1640 substratum (purchased from Gibco company) centrifuge washing several, re-suspended cell is to make single cell suspension, and count, for subsequent use.
(2). the preparation of feeder cell
Get the female of 8 ~ 10 week age or male BALB/c mouse one, eyeball is got blood and is prepared negative serum, puts to death to be placed in 75% (v/v) alcohol soak 10 minutes through disconnected cervical vertebra; Asepticly open skin of abdomen, expose peritonaeum, with syringe, about 5mL1640HT substratum (purchased from SIGMA company) is injected mouse peritoneal, gently abdomen massage blow and beat several.The substratum drawn containing scavenger cell injects 20%1640HAT substratum, for subsequent use;
Get the female of 2 ~ 3 week age or male BALB/c mouse one, put to death to be placed in 75% (v/v) alcohol through disconnected cervical vertebra and soak 10 minutes; Aseptic thymus gland of getting is in cell screen cloth, and grinding, crosses screen cloth, makes thymus cell suspension and injects the above-mentioned 20%1640HAT substratum containing scavenger cell, for subsequent use.
(3). cytogamy
Select the mouse myeloma strain SP2/0 being in logarithmic phase, collect and count, for subsequent use.
Get about 108individual above-mentioned spleen cell and 2 × 107individual above-mentioned SP2/0 adds in fusion pipe and mixes, and 1000rpm abandons supernatant (as far as possible abandoning clean) after centrifugal 10 minutes, fusion pipe put on palm and rub gently back and forth to make precipitation loose.Add soon after first slow in 60 seconds PEG1450 (polyoxyethylene glycol 1450) (purchased from the SIGMA company) of 1mL preheating, add 1640HT substratum 30mL to stop, centrifugal 10 minutes of 1000rpm, remove supernatant, friction makes precipitation loose gently, adds in the 20%1640HAT substratum that step (2) obtains.
After fully being mixed by above-mentioned 20%1640HAT substratum, 200 μ L/ holes are divided and are filled in 96 porocyte culture plates, are placed in 37 DEG C, 5%CO2cultivate in cell culture incubator.
After one week, 10%1640HT substratum replaces 20%1640HAT substratum; Change liquid to get supernatant after 10 days and detect.
3, anti-Ractopamine hydrochloride specific lymphocyte hybridoma cell strains screening
(1). the preparation of check-out console
With CB coating buffer dilution Ractopamine hydrochloride coupling oralbumin conjugate (Rac-OVA) to 2 μ g/mL, be added in (100uL/ hole) in 96 hole enzyme plates, 2 ~ 8 DEG C of bags are spent the night, and washing pats dry; 2% casein is closed, and closes 2 hours for 37 DEG C, and PBST washing pats dry, for subsequent use.
(2). the screening of positive colony
Added in above-mentioned check-out console in cells and supernatant 100 μ L/ hole to be checked, under being placed in 37 DEG C of conditions, reaction is washed after 30 minutes and pats dry; Add the IgG antibody of the HRP mark in 100 μ L/ holes, under being placed in 37 DEG C of conditions, reaction is washed after 30 minutes and pats dry; Add the TMB nitrite ion in 100 μ L/ holes, with the 2MH in 50 μ L/ holes after 37 DEG C of lucifuges develop the color 15 minutes2sO4termination reaction, and in OD450 place reading numerical values.Principle is determined in positive hole: OD450 value/negative control value >=2.1.
Choose positive clone strain be at war with ELISA screening, determine its suppression valency: successively by the Rac small molecules of 50 μ L, the sheep anti-mouse igg (being purchased from doctor's moral biological) of the HRP mark of 50 μ L and the positive clone strain cells and supernatant of 50 μ L add in check-out console, and under being placed in 37 DEG C of conditions, reaction is washed after 40 minutes and pats dry; Add the TMB nitrite ion in 100 μ L/ holes, with the 2MH in 50 μ L/ holes after 37 DEG C of lucifuges develop the color 15 minutes2sO4termination reaction, and in OD450 place reading numerical values and calculation of half inhibitory concentration (IC50).Choose the cell strain best with half-inhibition concentration of tiring to reserve seed for planting.
Wherein hybridoma cell strain supernatant detects the >10 that tires5, half inhibiting rate is 2.5ng/mL, called after anti-Ractopamine hydrochloride specific hybrid tumor cell strain C07, is called for short hybridoma cell strain C07.
embodiment 2: the mensuration of anti-Ractopamine hydrochloride hybridoma cell strain antibody variable sequences
Above-mentioned hybridoma cell strain C07 antibody variable sequences is measured.
The extraction of a.RNA: with reference to cell total rna extraction agent box (purchased from Roche company) specification sheets, Total RNAs extraction is carried out to above-mentioned hybridoma cell strain C07 and also carry out reverse transcription immediately;
B.RNA reverse transcription becomes DNA: carry out reverse transcription with reference to ThermoScientificRevertedFirststrandcDNASynthesisKit (purchased from Thermo company) to the total serum IgE extracted in previous step, obtained cDNA, frozen for subsequent use in-20 DEG C;
C. the pcr amplification of variable region sequences and recovery: with gained cDNA in previous step for template, with mouse IgG subclass antibodies variable region sequences universal primer for primer, pcr amplification is carried out to the variable region sequences of heavy chain and light chain, PCR primer is reclaimed test kit (purchased from TIANGEN company) through DNA glue reclaim, see accompanying drawing 1;
D. the clone of variable region sequences and sequencing: according to cloning vector pMD18-Tkit (purchased from Takara company) specification sheets, heavy chain is connected with pMD18-T carrier respectively with chain variable region gene, transformation of E. coli DH5 α, picking positive colony, transfers to InvitrogentMcompany checks order.
The antibody heavy chain variable region gene sequence that order-checking obtains hybridoma cell strain C07 as shown in SEQIDNO:9, chain variable region gene sequence is as shown in SEQIDNO:10.The above-mentioned sequence of Vbase2 database analysis, the aminoacid sequence of each complementary determining region of its variable region of heavy chain respectively: the HCDR1 as shown in sequence SEQIDNO:2, the HCDR2 as shown in sequence SEQIDNO:3 and the HCDR3 as shown in sequence SEQIDNO:4; The aminoacid sequence of each complementary determining region of variable region of light chain is: the LCDR1 as shown in sequence SEQIDNO:6, the LCDR2 as shown in sequence SEQIDNO:7 and the LCDR3 as shown in sequence SEQIDNO:8.
recombinant expressed and the purifying of embodiment 3. antibody
According to sequencing result in embodiment 2, connection peptides (GGGGS) will be added between the heavy chain of antibody of hybridoma cell strain C07 and variable region of light chain3, introduce six Histidines and its full genome carried out the recombinant expressed of codon optimized and antibody according to the preferences of pichia yeast expression system, expressed by the antibody called after antibody K07 that obtains, its structure composition is as shown in Figure 2.The recombinant expressed of above-mentioned antibody has following steps:
A) expression plasmid of antigen-4 fusion protein gene builds
The gene order of the antibody K07 after codon optimized as shown in SEQIDNO:12, aminoacid sequence is as shown in SEQIDNO:11.DNA sequence dna after XhoI sequence in the fragment upstream introducing pPICZ α A carrier that antibody K07 full genome after optimizing is synthesized, XbaI enzyme cutting site is introduced in downstream, be building up in pMD19-TSimpleVector plasmid (purchased from Invitrogen company), obtain one and preserve plasmid for a long time, plasmid is designated as pMD19-K07.Carry out pcr amplification, the primer sequence is as follows:
Upstream primer:
P1:CGCCAGGGTTTTCCCAGTCACGAC
Downstream primer:
P2:AGCGGATAACAATTTCACACAGGA
After Standard PCR program, agarose gel electrophoresis analysis (accompanying drawing 3), display product size is consistent with expection size (790bp).After PCR being obtained gene product recovery purifying, adopt XhoI (#R0146S, purchased from NewEnglandBiolabs company) and XbaI (#R0145V, purchased from NewEnglandBiolabs company) double digestion, be connected in pPICZ α A (V19520, purchased from Invitrogen) plasmid with T4 ligase enzyme, be transformed in DH5 α competent cell, 37 DEG C of overnight incubation in the LB flat board containing Zeocin (R250-01, purchased from Invitrogen company).Screening positive clone bacterium order-checking in second day, comparison, completely the same with expected sequence, namely obtain the expression plasmid of antibody K07, be designated as pPICZ α-K07.
B) antigen-4 fusion protein gene is in the structure of pichia spp host engineering strain, screening and expression
Pichia spp competent cell and relevant YPDS solid medium thereof, BMGY substratum, BMMY substratum are all purchased from Invitrogen company.
By pPICZ α-K07 plasmid, with the linearizing of SacI digestion with restriction enzyme.By linearized vector after alcohol settling, electricity transforms and enters into X-33 competence yeast cell, is applied to the YPDS solid medium containing Zeocin, cultivates 3-5 days, just has positive colony to produce for 30 DEG C.
The mono-clonal of the above-mentioned acquisition of picking is in 5mLBMGY substratum, and 30 DEG C are cultured to OD600when=2.0 ~ 6.0, get 1mL and preserve bacterial classification, and transfer to BMMY Small Amount abduction delivering after resuspended for residue bacterium liquid, adding methyl alcohol to final concentration every 24h is 1% (v/v).After one week, collected by centrifugation bacterium liquid supernatant, by SDS-PAGE gel electrophoresis and Western blot analysis (Westernblot), object observing protein expression situation (accompanying drawing 4).In Westernblot, primary antibodie is anti-HIS-Tag antibody (His-Tag (2A8) MousemAb, M20001 are purchased from Ai Bimate biological medicine (Shanghai) Co., Ltd.).
Be inoculated in BMGY substratum respectively by the K07 recombination fusion protein engineering strain of above-mentioned acquisition, 30 DEG C, 220rpm is cultured to cell density to OD600=2.0 ~ 6.0, adding methyl alcohol to final concentration every 24 hours is 1.0% (v/v).After one week, collect fermentation culture.
C) fusion protein purification
Main employing histidine-tagged affinity column antibody purification K07 fusion rotein, prepackage pillar is chosen as HisTrapHP, and concrete steps are as follows:
(1) the removal of impurities pre-treatment of fermented liquid: above-mentioned expression is obtained antibody K07 fusion rotein fermented liquid supernatant, collected by centrifugation supernatant, and add binding buffer liquid, makes supernatant final concentration be 300mMNaCl, 20mMNaH2pO4, 10mMImidazole, adjusts pH7.5,0.45 μm of membrane filtration.
(2) HisTrapHP affinity column purifying: use fully-automatic intelligent protein purification system (AKTAavant150, purchased from GEhealcare company) affinity purification is carried out to the antibody K07 fusion rotein fermented liquid that pre-treatment obtains, pillar is HisTrapHP (17-5248-02, purchased from GEhealcare company).Binding buffer liquid is 300mMNaCl, 20mMNaH2pO4, 10mMImidazole, pH7.5, elution buffer is 300mMNaCl, 20mMNaH2pO4, 500mMImidazole, pH7.5.Carry out linear elution during wash-out, and collect each elution peak.By SDS-PAGE electroresis appraisal purity, from accompanying drawing 5, the purity of protein after purifying reaches more than 95%; Merge satisfactory collection tube, exchange buffering liquid is PBS solution and ultrafiltration and concentration (1mg/ml), and filtration sterilization saves backup in-20 DEG C.
Those skilled in the art know, connection peptides (GGGGS) 3 can will be added between the heavy chain of antibody of hybridoma cell strain C07 and variable region of light chain, and do not introduce six Histidines, the fusion rotein of recombinant expressed one-tenth not with His label, now the purifying of albumen can adopt ProteinL albumen affinity purification method (concrete with reference to GE company affinity chromatography medium CaptoL).
the Performance Testing of embodiment 4. antibody K07
1. the Westernblot qualification of antibody K07
A. polyacrylamide gel electrophoresis: configure 12% separation gel, 5% concentrated glue, loading standard protein, Ractopamine hydrochloride coupling BSA (Rac-BSA), Ractopamine hydrochloride coupling OVA (Rac-OVA), Clenbuterol hydrochloride coupling BSA (Cle-BSA), Clenbuterol hydrochloride coupling OVA (Cle-OVA), salbutamol coupling BSA (Sal-BSA), salbutamol coupling OVA (Cle-OVA) (above-mentioned antigen is all bought in Shanghai You Long company) respectively, electrophoresis 1 hour under constant voltage;
B. transferring film: transferring film 1 hour under constant current (35mA/ film) condition, moves to the protein transduction on polyacrylamide gel on two nitrocellulose filters.Coomassie brilliant blue G250 dyes to the SDS-PAGE glue completing transferring film, observes the residual condition of albumen;
C. close: containing the TBST buffer blind (confining liquid) of 5% skimmed milk, 4 DEG C are spent the night; After closing, washings (TBST refers to TaKaRa company's T BSTbuffer) washing once, 10 minutes;
D. antigen antibody reaction: confining liquid dilution (by 1:400 volume ratio) horseradish peroxidase-labeled K07 (K07-HRP, 1mg/mL, our company adopts classical Over-voltage protection to mark), add in above-mentioned nitrocellulose filter, room temperature reaction 1 hour; TBST washs 5 times, each 10 minutes;
E. develop the color and take pictures: blotting residual liquid on nitrocellulose filter, nitrocellulose filter adds the mixed solution (buying in Thermo company) of 2mL stable form Peroxidase Solution (1mL) and luminol,3-aminophthalic acid cyclic hydrazide/toughener solution (1mL), the surface of uniform wet nitrocellulose filter, take pictures in gel imaging system (buying in GE company) after room temperature lucifuge reacts one minute, leave and take result.
Experimental result (accompanying drawing 6) shows, K07 antibody and Ractopamine hydrochloride have specific reaction, not with Clenbuterol hydrochloride and salbutamol generation cross reaction.
2. antibody K07 is in the performance evaluation of Ractopamine hydrochloride Radioactive colloidal gold detection platform
Except the parameter in following tableand confining liquid (0.5%Casein+0.05%PEG2000) outward, the preparation process of all the other test cards and parameter are with embodiment 8.Preparation is containing the positive pig urine containing Ractopamine hydrochloride standard substance of 5ppb, 10ppb, 50ppb, 100ppb respectively, detects together with urinating with negative pig.
The anti-Anti-ractopamine antibody of K07 and the detected result of commercialization Anti-ractopamine antibody in same buffer show, antibody prepared by the present invention can be used for the structure of colloidal gold test card, and urine specimen detection sensitivity is suitable with commercially available test card, after optimization, be expected to the level reaching commercially available test card completely; Antibody of the present invention is for building colloidal gold test paper card, and K07 consumption and C line antibody consumption are less, more economical saving.
Remarks: commercialization Anti-ractopamine antibody is bought in Wuhan Sino-American Biotechnology Company
the preparation of embodiment 5. kit for testing lecdopamine ELISA of the present invention
This ELISA kit adopts indirect competitive ELISA method.
1. the preparation of check-out console: it (is the Na of 0.3% containing mass volume ratio that bag is buffered liquid2cO3and the NaHCO of 0.58%3) dilution Ractopamine hydrochloride coupling bovine serum albumin conjugate (Rac-BSA) be diluted to 1 μ g/mL, add in 96 hole enzyme plates with 100 μ L/ holes, 2 ~ 8 DEG C of bags are spent the night; Next day, wash plate one time with the PBST containing 0.05%TWEEN-20; The confining liquid adding 200 μ L/ holes (is 0.3%Na containing mass volume ratio2cO3, 0.58%NaHCO3, 10% sucrose, volume ratio 10% calf serum) in room temperature (25 DEG C) close 2 hours, discard liquid, cryodrying, encapsulate for subsequent use.
Those skilled in the art know, because Ractopamine hydrochloride is micromolecular compound, more difficult direct coated is on enzyme plate, generally can wrap by enzyme plate more further by coupling carrier albumen thereon, therefore, the bovine serum albumin (BSA) of Ractopamine hydrochloride coupling also can use the carrier proteins such as oralbumin (OVA), keyhole limpet hemocyanin (KLH) to substitute completely.
2. enzyme diluent, antibody working fluid, the preparation of enzyme labelled antibody:
1. PBST (pH7.4) damping fluid of enzyme diluent: 0.01M, is the Na of 0.335% containing mass volume ratio2hPO412H2o, 0.02%NaH2pO42H2o, 0.8%NaCl, 0.02%KCl, 0.1%Casein, 0.05%BSA
2. the preparation of antibody working fluid: K07 antibody is diluted with 1/500000 volume ratio with enzyme diluent, vortex mixes, packing (10mL/ bottle);
3. the preparation of enzyme labelled antibody: diluted with 1/2000 volume ratio by mouse-anti His antibody-HRP with enzyme diluent, vortex mixes, packing 7mL/ bottle.
3.TMB nitrite ion, stop buffer and Ractopamine hydrochloride reference liquid:
1. the concentration of TMB nitrite ion: TMB (purchased from SIGMA company) is 0.3g/L, is packed as 14mL/ bottle;
The H of 2. stop buffer: 2M2sO4, be packed as 7mL/ bottle;
3., 4., 5., 6., 7., 8. Ractopamine hydrochloride reference liquid: each 1mL, concentration is respectively 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb.
4.20 times of concentrated cleaning solutions: containing the 0.2MPBS of 0.05%TWEEN-20, are packed as 40mL/ bottle.
5. assemble: all bottled reagent is inserted in the corresponding aperture of box holder, puts into carton together with elisa plate, specification sheets, valve bag, shrouding film, be stored in 2 ~ 8 DEG C, paste seal inspection label after the assay was approved.
In the present embodiment, enzyme labelled antibody can substitute with anti-His antibody-AP, ProteinL-HRP, ProteinL-AP completely according to actual needs, and nitrite ion also can substitute with BCIP/NBT nitrite ion completely.
the using method of embodiment 6. kit for testing lecdopamine ELISA of the present invention
1. Sample pretreatment:
Urine specimen: limpid urine specimen can be directly used in mensuration, first more than 3000g is centrifugal to obtain supernatant for muddy urine palpus; Get 50uL for pattern detection;
Pork or pork liver equal samples: take 2 ± 0.05g homogenate sample in 50mL centrifuge tube, add 4mL wash operating solution, then add 2mL0.1M hydrochloric acid, vortex 30s, centrifugal under room temperature condition (>=3000g, 5 minutes); Get supernatant liquor 1mL, add 30uL0.5MNaOH to sample liquid pH value between 6.5 ~ 8.0, mixing; Get 50uL for pattern detection;
2. required reagent and enzyme plate are taken out from cold storage environment, be placed in room temperature (25 DEG C) balance;
3. with deionized water by 20 times of concentrated cleaning solutions by volume 1:19 carry out dilution and make wash operating solution, for the washing of enzyme plate, prepare before use;
4. number: sample and micropore corresponding to standard substance are numbered according to the order of sequence, each sample and standard substance establish multiple hole parallel;
5. application of sample: add 50 μ L/ hole standard substance or samples in the micropore of correspondence according to the order of sequence respectively, 50 μ L/ hole enzyme labelled antibodies and 50 μ L/ hole antibody working fluids, mixing of vibrating gently, reacts 40 minutes in room temperature (25 DEG C) lucifuge;
6. wash plate: after the reaction times reaches, liquid in hole is dried, washes plate with the wash operating solution in 300 μ L/ holes, fully wash 4 ~ 5 times, every minor tick 10s, dry liquid, pat dry with thieving paper;
7. develop the color: the TMB nitrite ion adding 100 μ L/ holes, mixing of vibrating gently, reacts 15 minutes in room temperature (25 DEG C) lucifuge;
8. stop and reading: the stop buffer adding 50 μ L/ holes, mixing of vibrating gently, setting microplate reader, in 450nm place, measures every hole OD value.
9. result judges:
I). judge roughly: judged by the simple contrast of the light absorption ratio of sample well and the light absorption ratio of standard sample wells: the color of sample well than Ractopamine hydrochloride in the then sample of light color of a certain standard sample wells this hole of concentration ratio contained by the concentration of standard substance high, the color of sample well than a certain standard sample wells color deeply then in sample Ractopamine hydrochloride this hole of concentration ratio contained by the concentration of standard substance low;
Ii). quantitative analysis: with the per-cent of the absorbance of the absorbance values of standard substance and first standard (0ppb) for ordinate zou, with the logarithm of the concentration of Ractopamine hydrochloride standard substance (ppb) for X-coordinate, drawing standard graphic representation.The per-cent of the absorbance of the absorbance values of sample and first standard (0ppb) is substituted in typical curve, thus calculates the actual concentrations of Ractopamine hydrochloride in sample.
the Performance Testing of embodiment 7. kit for testing lecdopamine ELISA of the present invention
Kit for testing lecdopamine ELISA of the present invention and commercially available similar test kit are carried out the comparison of detection perform by the implementation case.
Two kinds of test kits detect 70 parts of fresh negative urine specimens and 70 parts of fresh positive urine fluid samples (adding 1ppb Ractopamine hydrochloride in fresh negative urine specimen) respectively.Kit for testing lecdopamine ELISA of the present invention operates according to above-mentioned detection method; Commercially available similar test kit operates according to reagent specification sheets.Detected result sees the following form, the appearance of two kinds of equal non-false positives of detection kit; During positive sample detects, the rate of recovery scope of kit for testing lecdopamine ELISA of the present invention is narrower compared with commercial reagent box, reflects higher accuracy, possesses and detects advantage more accurately.
the preparation of embodiment 8. Ractopamine hydrochloride colloidal gold test of the present invention card
This test card application Competitive assays immunochromatography, in order to the qualitative detection of Ractopamine hydrochloride in animal body fluid, can be particularly useful for the qualitative detection of Ractopamine hydrochloride in pig urcine, detect and be limited to 3-5ppb.Concrete preparation method is as follows:
The colloid gold label of the anti-Anti-ractopamine antibody of 1.K07:
Use 0.2MK2cO3regulate Radioactive colloidal gold pH value to 8.15, in colloidal gold solution, slowly add the K07 antibody that dilutes with the PBS of 0.02M to 10ug/mL, stirring at low speed 30 minutes;
Add encapsulant: adding BSA to its final concentration is 1% (mass percent), stir that to continue to add PEG2000 to its final concentration after 10 minutes be 0.05% (mass percent);
Radioactive colloidal gold redissolves: stir after 10 minutes and add and the redissolution liquid of Radioactive colloidal gold same volume (1%BSA+5% sucrose+0.1%Tween20+25mMPBS damping fluid (pH7.5)), in 2 ~ 8 DEG C of hold over night.Gained is the K07 antibody of colloid gold label;
2. be sprayed in gold mark pad 6 with the K07 antibody of above-mentioned colloid gold label, mouse-anti His antibody after being diluted by coated antibody diluent (3% methyl alcohol+25mMPBS damping fluid (pH7.5)), Rac-OVA conjugate line C line 4, T line 5 respectively on nitrocellulose filter 2, again by print pad 1 gold medal mark pad 6, nitrocellulose filter 2, absorption pad 3 by adjacent between two shown in accompanying drawing 8 and part is stacked is pasted on successively from left to right on liner plate 7, dress bar.
Those skilled in the art know, because Ractopamine hydrochloride is micromolecular compound, usually be used as bag quilt again after needing coupling macromolecular carrier albumen, the oralbumin (OVA) of coupling Ractopamine hydrochloride used for the present embodiment also can use the carrier proteins such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) to substitute.
3. test card is put into carton together with specification sheets, dropper, be stored in 4 ~ 30 DEG C of shady and cool lucifuge places, paste seal inspection label after the assay was approved.
4. the present invention once attempted multiple test card preparation method, and following table shows the detection perform of several different preparation method (step unlisted in table, parameter are equal to described in the present embodiment) and corresponding reagent box.
the use of embodiment 9. Ractopamine hydrochloride colloidal gold test of the present invention card
1. sample pre-treatments: limpid urine specimen can be directly used in mensuration, muddy urine palpus first centrifugal (>=3000g) is to obtain supernatant;
2. test card is taken out from cold storage environment, be placed in room temperature (25 DEG C) balance;
3. kept flat by plastic clip, draw measuring samples with dropper, vertical dropping 3 (about 75 μ L) is in well;
4. start timing during liquid-flow, in 5 ~ 10 minutes, read result.
5. result judges: if T line is without colour developing, the colour developing of C line, then judges that sample is as the positive, represent in sample containing the Ractopamine hydrochloride higher than detectability; As T line and C line all develop the color, then judge that sample is as feminine gender, to represent in sample without the concentration of Ractopamine hydrochloride or Ractopamine hydrochloride lower than detectability; As C line does not develop the color, then show that operation is incorrect, or this test card used lost efficacy.
the detection perform evaluation of embodiment 10. Ractopamine hydrochloride colloidal gold test of the present invention card
1, sensitivity technique
1.1 reagent and solution:
A) Ractopamine hydrochloride storing solution (1mg/mL): accurately take Ractopamine hydrochloride standard substance 50mg, be diluted to 50mL with after dissolve with methanol, shake up, store for subsequent use in less than-18 DEG C refrigerators, validity period 90 days.
B) Ractopamine hydrochloride working fluid (1 μ g/mL): get Ractopamine hydrochloride storing solution 0.1mL, add methanol constant volume to 100mL, store for subsequent use in 2 DEG C ~ 8 DEG C refrigerators, validity period 7 days.
C) 3ng/mL Ractopamine hydrochloride standard substance: get Ractopamine hydrochloride working fluid 90 μ L, be diluted to 30mL by urine, mixing.
D) 5ng/mL Ractopamine hydrochloride standard substance: get Ractopamine hydrochloride working fluid 100 μ L, be diluted to 20mL by urine, mixing.
E) 10ng/mL Ractopamine hydrochloride standard substance: get Ractopamine hydrochloride working fluid 200 μ L, be diluted to 20mL by urine, mixing.
1.2 detect and result
Get 9, the Rec of the present invention Test paper card of same lot number, detect each 3 times of the Rac standard substance (3ng/mL, 5ng/mL, 10ng/mL) of three kinds of concentration respectively, 3 results of often kind of concentration are and positively just can judge that the result of concentration is for this reason for the positive.When 3ng/ml standard substance detect, in three detected results, being for twice negative, is once positive; When 5ng/ml standard substance detect, three times detected result is the positive.Detected result according to three kinds of concentration judges, the sensitivity of Test paper card of the present invention is 3-5ppb.
2, specificity
2.1 reagent and solution:
A) clenbuterol hydrochloride working fluid 100 μ g/mL, methyl alcohol is as solvent.
B) 500ng/mL clenbuterol hydrochloride standard substance: get clenbuterol hydrochloride working fluid 100 μ L, be diluted to 20mL by urine, mixing.
C) salbutamol working fluid 100 μ g/mL, methyl alcohol is as solvent.
D) 500ng/mL salbutamol standard substance: get salbutamol working fluid 100 μ L, be diluted to 20mL by urine, mixing.
2.2 detect and result
Detect 500ng/mL clenbuterol hydrochloride, 500ng/mL salbutamol standard substance respectively, each repetition 3 times, 3 results of each solution are feminine gender.Detected result shows, Ractopamine hydrochloride Test paper jig of the present invention has good specificity.