CN105440137B - The antibody of anti-Ractopamine and its application - Google Patents
The antibody of anti-Ractopamine and its application Download PDFInfo
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- CN105440137B CN105440137B CN201510048100.6A CN201510048100A CN105440137B CN 105440137 B CN105440137 B CN 105440137B CN 201510048100 A CN201510048100 A CN 201510048100A CN 105440137 B CN105440137 B CN 105440137B
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- ractopamine
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Abstract
The present invention provides a kind of Anti-ractopamine antibody and its applications, belong to field of immunological detection.The present invention by Ractopamine antigen-immunized animal and screening obtain hybridoma cell strain, and further carry out sequencing and recombinant expression obtain the antibody with high specificity.The present invention also provides the enzyme linked immunological kits and colloidal gold strip of the application Anti-ractopamine antibody, and its preparation and detection method.The Anti-ractopamine antibody specificity that the present invention is prepared is high, it is at low cost, Ractopamine enzyme linked immunological kit and colloidal gold test paper card the detection Ractopamine being prepared with this have the characteristics that easy to operate, testing cost is low, specific height, high sensitivity, accuracy are high, can quantitatively or the Rct opamine residue in the samples such as qualitative detection animals urine, blood sample, tissue and internal organ.
Description
Technical field
The invention belongs to biotechnology more particularly to the antibody of anti-Ractopamine, and its detect Lake preparing
The ELISA kit of dopamine, the application of colloidal gold test paper card.
Background technology
" clenbuterol hydrochloride " is that one kind belongs to adrenal gland class CNS stimulant drug.It can make livestock promote egg in metabolic process
White matter synthesizes, and accelerates the conversion and decomposition of fat, then improves the speed of growth of livestock, increases lean meat percentage；It can also reduce simultaneously
Feed uses, meat is made to list ahead of time, reduce cost.Office of the food security committee of State Council《" clenbuterol hydrochloride " focus efforts on special areas
Scheme》" clenbuterol hydrochloride " kind catalogue is as defined in (food peace does [2011] No. l4)：Clenobuterol hydrochloride (Clenbuterol
Hydrochloride), Ractopamine (Ractopamine, Rac), salbutamol (Salbutamo1) etc..Wherein, Lake is more
Bar amine (Rac) gathers residual easily in animal tissue, especially internal organ, and enters human body by food chain.Then cause bone
Flesh shrinks enhancing, destroys the fusion phenomenon between quick muscle and slow switch fibers, causes muscular tremor, occur flushed face, headache,
The adverse reactions such as dizzy, uncomfortable in chest, palpitaition, numb limb, serious possible threat to life.Many countries all arrange Ractopamine
For violated object, China has forbidden using Ractopamine with livestock and poultry.
Currently, the common method for detecting Ractopamine has both at home and abroad：Enzyme-linked immunosorbent assay (ELISA),
Colloidal gold immunity chromatography, high performance liquid chromatography, gas chromatography-mass spectrography, chromatography, capillary electrophoresis, immune biography
Sensor method etc..The sensitive but required instrument and equipment of chromatography, capillary electrophoresis, the detection of immunosensor method is expensive, before sample
Processing is complicated, and one-time detection sample size is few, and detection time is long, and is not easy to popularize, and ELISA, colloidal gold immunity chromatography are not required to
Will be by expensive instrument, and sample pre-treatments are simple, have many advantages, such as high throughput.But current existing detection Ractopamine
Enzyme linked immunological kit or colloidal gold test paper card detection sensitivity or accuracy be not also very ideal, analyze its reason,
Anti- Anti-ractopamine antibody need to be improved used by essentially consisting in.Since Ractopamine is chemical small molecule, surface
Site is few, thus brings bigger difficulty to prepare its antibody, especially specific higher antibody, at present city's antibody
Specific universal relatively low, half-inhibition concentration (IC50) is higher, cannot be satisfied the demand of application.
Invention content
The present invention is intended to provide energy is effective, the antibody of specific binding Ractopamine (Rac), and its detection Lake is more
Bar amine (Rac) remaining purposes in animal body.More specifically：
First purpose of the invention is to provide a kind of antibody, and the antibody specificity combination Ractopamine (Rac) is described
Antibody includes：
Heavy chain variable region, amino acid sequence contain complementary determining region below：Such as sequence SEQ ID NO：Shown in 2
HCDR1, such as sequence SEQ ID NO：HCDR2 shown in 3 and/or such as sequence SEQ ID NO：HCDR3 shown in 4；
And light chain variable region, amino acid sequence contain complementary determining region below：Such as sequence SEQ ID NO：Shown in 6
LCDR1, such as sequence SEQ ID NO：LCDR2 shown in 7 and/or such as sequence SEQ ID NO：LCDR3 shown in 8.
Antibody preferably in the present invention contains amino acid sequence such as SEQ ID NO:Heavy chain variable region and ammonia shown in 1
Base acid sequence such as SEQ ID NO:Light chain variable region shown in 5.
The encoding gene of antibody contains such as SEQ ID NO in the preferred present invention:Heavy chain variable region shown in 9 and such as
SEQ ID NO:Light chain variable region shown in 10.
Second purpose of the invention is to provide a kind of single-chain antibody, the amino acid sequence such as SEQ ID of the single-chain antibody
NO:Shown in 11.Preferably, without the HIS labels being made of six histidines in the single-chain antibody.
Third purpose of the present invention is to provide a kind of nucleotide sequence of the above-mentioned single-chain antibody of coding, the nucleotide sequence
Such as SEQ ID NO:Shown in 12.
4th purpose of the invention is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
5th purpose of the invention is to provide a kind of recombinant host cell containing above-mentioned expression vector, affiliated host cell
Can be Escherichia coli, yeast or mammalian cell, preferably Pichia pastoris.
6th purpose of the invention is to provide a kind of method producing above-mentioned antibody, including：
1) above-mentioned recombinant host cell expression antibody is cultivated under suitable conditions；
2) it and then from host strain purifies, collect antibody.
7th purpose of the invention is to provide above-mentioned antibody for detecting Ractopamine (Rac) remaining new application.
The present invention the 8th is designed to provide a kind of colloidal gold test paper card or ELISA reagents of detection Ractopamine
Box, the colloidal gold test paper card or ELISA kit contain antibody described above.
Preferably, ELISA kit described above, including：It has been coated with the carrier protein couplet object of antigen Ractopamine
ELISA Plate, the antibody working solution containing antibody described above, enzyme mark object, Ractopamine titer, developing solution, terminate liquid,
Concentrated cleaning solution.It is furthermore preferred that the carrier protein is BSA, OVA, KLH.
Preferably, antibody working solution described above is to dilute to obtain by above-mentioned antibody with enzyme dilution, the enzyme dilution
It is PBST (pH7.4) buffer solution of 0.01M, wherein the Na for being 0.335% containing mass volume ratio2HPO4·12H2O, 0.02%
NaH2PO4·2H2O, 0.8%NaCl, 0.02%KCl, 0.1%Casein, 0.05%BSA.
Preferably, enzyme mark object described above be anti-His antibody-HRP, anti-His antibody-AP, Protein L-HRP or
Protein L-AP。
Preferably, developing solution described above is TMB developing solutions or BCIP/NBT developing solutions.
Preferably, terminate liquid described above is the H of 2M2SO4。
Preferably, concentrated cleaning solution described above is the 0.2M PBS containing 0.05%TWEEN-20.
The 9th of the present invention is designed to provide a kind of method of detection Ractopamine, includes the following steps：
1. number：Sample and the corresponding micropore of standard items are sequentially numbered, it is parallel that each sample and standard items set multiple holes；
2. sample-adding：Standard items, sample, enzyme mark object and antibody working solution are sequentially added per hole, is protected from light in room temperature；
3. board-washing：After reaction, board-washing is simultaneously dried；
4. colour developing：Developing solution is added, is protected from light in room temperature；
5. terminating and reading：Terminate liquid is added, measures per hole OD values.
6. result judgement：
I) is qualitatively judged：It is compared with the simple of the absorptance of standard sample wells to judge by the absorptance of sample well：Sample
Concentration of the color in hole than standard items contained by the concentration ratio of Ractopamine hole in the then sample of light color of a certain standard sample wells
Height, standard items contained by the concentration ratio of the Ractopamine hole in the color of sample well then sample deeper than the color of a certain standard sample wells
Concentration it is low；
Ii) quantitative analyses：With the percentage of the absorbance values of standard items and the absorbance value of first standard (0ppb)
Than drawing canonical plotting using the logarithm of the concentration (ppb) of Ractopamine standard items as abscissa for ordinate.By sample
Absorbance values and first standard (0ppb) absorbance value percentage substitute into standard curve in, to calculate sample
The actual concentrations of Ractopamine in this.
The tenth of the present invention is designed to provide a kind of colloidal gold test paper card of detection Ractopamine, including sample pad
1, gold-labelled pad 6, nitrocellulose filter 2, absorption pad 3, liner plate 7, print pad 1, gold-labelled pad 6, nitrocellulose filter 2,3 liang of absorption pad
Two it is adjacent and part be laminated be pasted on successively on liner plate 7 from left to right, have on the nitrocellulose filter 2 quality control band 4 (C lines) and
Band 5 (T lines) is detected, contains the above-mentioned anti-Anti-ractopamine antibody through colloid gold label in the gold-labelled pad 6.
Preferably, 4 position of quality control band is coated with anti-His antibody or Protein L, and it is more that 5 position of detection band is coated with Lake
The conjugate of bar amine haptens and carrier protein.
The ELISA detection kit of Antibody preparation provided by the present invention and the sensitivity of colloidal gold test paper card and accuracy phase
It is more ideal than commercial product, and specificity is very well.
Description of the drawings
Fig. 1 antibody K07 heavy chains, chain variable region gene electrophoretogram.Band 1 is standard DNA, and band 2 is K07 antibody weights
Chain variable region DNA, Lane3 are K07 antibody light chains variable region DNA
Fig. 2 antibody K07 structural schematic diagrams.VHIndicate weight chain variabl area sequence, VLIndicate light-chain variable sequence, His marks
Label are six histidines.
The agarose gel electrophoresis figure of Fig. 3 antibody expression PCR products.
Fig. 4 recombinant pichia yeast strain induced expression supernatant culture solution qualification figures.Figure a be SDS-PAGE electroresis appraisals figure,
Figure b is Western blot qualification figures.
Fig. 5 antibody purifications design sketch (SDS-PAGE).The antibody K07 that band 1-10 is obtained by different collecting pipes.
The Western Blot qualification figures of Fig. 6 antibody K07.Swimming lane 1 is standard protein, swimming lane 2 is Rac-BSA, swimming lane
3 be 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.
The standard curve of the ELISA detection kit of Fig. 7 Ractopamines of the present invention.Wherein abscissa is Lake DOPA
Amine standard concentration logarithm (lg)；Ordinate is Ractopamine standard items percentage absorptance.
Fig. 8 Ractopamine colloidal gold test card structure schematic diagrames of the present invention.
Wherein 1 it is sample pad, 2 be nitrocellulose filter, 3 be absorption pad, 4 be nature controlling line (C lines), 5 is detection line (T
Line), 6 be gold-labelled pad, 7 be liner plate
Specific embodiment
Definition
" antibody " is also known as immunoglobulin, is a kind of large-scale Y shape protein secreted by bone-marrow-derived lymphocyte, can pass through Y shape
Two of which bifurcated top complementary site (antigen binding position) specific binding target antigen immunoglobulin molecules, it is described
Target antigen such as protein, sugar, polynucleotides, fat, polypeptide, micromolecular compound etc..The term includes not only complete polyclonal
Antibody, also include its segment (such as Fab, Fab ', F (ab ')2, Fv), single-chain antibody (single chain antibody
Fragment, scFv), its mutant, the fusion protein comprising antibody moiety and any other comprising antigen recognition site change
The immunoglobulin molecules of allosteric type.Antibody includes any sort or the antibody of multiclass, as IgG, IgA, IgD, IgE or IgM (or its
Subclass), and antibody need not be any certain kinds.
" single-chain antibody " (scFv) refers to the heavy chain variable region (V of antibodyH) and light chain variable region (VL) pass through 15~20
The single chain fusion protein that amino acid short peptide (linker) connection is formed, the linker for connection are generally rich in glycine and silk
Propylhomoserin, in favor of the stability and flexibility of single-chain antibody.Connection type can be by VLN-terminal be connected to VHC-terminal, Huo Zhexiang
Instead.Although eliminating constant region and introducing linker, single-chain antibody still remains specificity of the antibody to antigen, and it has
Molecular weight is small, penetration power is strong and it is antigenic weak the features such as.
Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Molding
It is the most critical zone that target antigen is combined with antibody in the end of antibody monomer amino acid, in Artificial Immune Network Theory, Mei Gekang
The complementary determining region of body is otherwise known as idiotype or genotype.HCDR refers to the complementary decision in heavy chain variable region in the application
Area, LCDR refer to the complementary determining region in light chain variable region.
Embodiment 1:The preparation of anti-Ractopamine hybridoma cell strain
1, animal immune
Exempted from according to general immune programme with Ractopamine coupling bovine serum albumin(BSA) (Rac-BSA) conjugate (immunogene)
Epidemic disease BALB/c female mices (are purchased from this experimental animal Co., Ltd of Changzhou Cavan).It is first that Freund's complete adjuvant/Freund is endless
Full adjuvant is mixed with Rac-BSA conjugates and is blown and beaten repeatedly in equal volume, is kept immunogene fully emulsified, is obtained a concentration of 0.5mg/mL
Emulsified immunogen.Female BAl BIc/c mouse of 6-8 week old are chosen, every mouse is inoculated with above-mentioned using subcutaneous multi-point injection mode
Emulsified immunogen is immunized primary every two weeks.Specific Immunity see the table below：
Since after second immune, taken a blood sample using orbital venous plexus at immune latter week, with indirect ELISA and competition
The potency and half of ELISA method detection immune serum (Ractopamine is coupled oralbumin conjugate Rac-OVA detections)
Number inhibition valence chooses serum titer and inhibits all highest mouse of valence, carries out immune mouse spleen cell and murine myeloma cell
It is merged.
2, cell fusion
(1) preparation of spleen cells
It takes serum titer in step 1 and inhibits all highest immune mouse of valence, pluck eyeball and take blood, postposition is put to death through disconnected cervical vertebra
It is impregnated after ten minutes in the alcohol of 75% (v/v), its spleen is taken in aseptic operating platform, is placed in cell screen clothes, is fully ground
Levigate born of the same parents cross sieve, with sterile 1640 culture medium (be purchased from Gibco companies) centrifuge washing for several times after, cell is resuspended so that list is made
Cell suspension, and count, it is spare.
(2) preparation of feeder cells
The female or male BALB/c mouse one of 8~10 week old is taken, eyeball takes blood to prepare negative serum, at disconnected cervical vertebra
It is after death placed in 75% (v/v) alcohol and impregnates 10 minutes；Sterile to open skin of abdomen, exposure peritonaeum will about 5mL with syringe
1640HT culture mediums (be purchased from SIGMA companies) injection mouse peritoneal gently abdomen massage and is blown and beaten for several times.It draws thin containing macrophage
It is spare in the culture medium injection 20%1640HAT culture mediums of born of the same parents；
The female or male BALB/c mouse one for taking 2~3 week old is placed on 75% (v/v) alcohol through disconnected cervical vertebra execution
It is middle to impregnate 10 minutes；Sterile to take thymus gland in cell screen clothes, grinding crosses sieve, thymus cell suspension is made and injects above-mentioned contain
Have in the 20%1640HAT culture mediums of macrophage, it is spare.
(3) cell fusions
Mouse myeloma strain SP2/0 of the selection in exponential phase, collects and counts, spare.
Take about 108A above-mentioned spleen cell and 2 × 107A above-mentioned SP2/0, which is added in fusion pipe, to be mixed, 1000rpm centrifugations 10
Supernatant (abandoning as possible net) is abandoned after minute, and fusion pipe is set and is gently rubbed back and forth on palm so that precipitation is loose.After elder generation is slow in 60 seconds
The PEG1450 (polyethylene glycol 1450) (being purchased from SIGMA companies) of 1mL preheatings is added soon, it is whole that 1640HT culture mediums 30mL is added
Only, 1000rpm is centrifuged 10 minutes, removes supernatant, and gently friction keeps precipitation loose, and the 20%1640HAT that step (2) is obtained is added
In culture medium.
After above-mentioned 20%1640HAT culture mediums are mixed well, 200 holes μ L/ are dispensed into 96 porocyte culture plates, are placed in
37 DEG C, 5%CO2It is cultivated in cell incubator.
After a week, 10%1640HT culture mediums replace 20%1640HAT culture mediums；Supernatant is taken to be examined after changing liquid 10 days
It surveys.
3, anti-Ractopamine specific lymphocyte hybridoma cell strains screening
(1) preparation of detection plates
With CB coating buffers dilution Ractopamine coupling oralbumin conjugate (Rac-OVA) to 2 μ g/mL, it is added extremely
In 96 hole elisa Plates (holes 100uL/), overnight, washing pats dry 2~8 DEG C of coatings；2% casein is closed, and 37 DEG C are closed 2 hours,
PBST washings pat dry, spare.
(2) screening of positive colonies
100 holes μ L/ of cells and supernatant to be checked are added in above-mentioned detection plate, after being reacted 30 minutes under the conditions of being placed in 37 DEG C
It washs and pats dry；The IgG antibody of the HRP labels in 100 holes μ L/ is added, washs and claps after being reacted 30 minutes under the conditions of being placed in 37 DEG C
It is dry；The TMB developing solutions in 100 holes μ L/ are added, with the 2M H in 50 holes μ L/ after being protected from light and develop the color 15 minutes in 37 DEG C2SO4Reaction is terminated,
And the reading numerical values at OD450.Positive hole determines principle：OD450 values/negative control value >=2.1.
Choose positive clone strain be at war with ELISA screening, determine its inhibition valence：Successively by the Rac small molecules of 50 μ L, 50
Inspection is added in the sheep anti-mouse igg (being purchased from doctor's moral biology) and the positive clone strain cells and supernatant of 50 μ L of the HRP labels of μ L
In drafting board, is reacted under the conditions of being placed in 37 DEG C and wash and pat dry after forty minutes；The TMB developing solutions in 100 holes μ L/ are added, are kept away in 37 DEG C
Light develop the color 15 minutes after with the 2M H in 50 holes μ L/2SO4Terminate reaction, and in OD450 at reading numerical values and calculate half inhibition it is dense
It spends (IC50).It chooses potency and the best cell strain of half-inhibition concentration is reserved seed for planting.
Wherein hybridoma cell strain supernatant detects potency>105, half inhibiting rate is 2.5ng/mL, and it is more to be named as anti-Lake
Bar amine specific hybrid tumor cell strain C07, abbreviation hybridoma cell strain C07.
Embodiment 2:The measurement of anti-Ractopamine hybridoma cell strain antibody variable sequences
The antibody variable sequences above-mentioned hybridoma cell strain C07 are measured.
The extraction of a.RNA：Reference cell total serum IgE extraction agent box (being purchased from Roche companies) specification is to above-mentioned hybridoma
Cell strain C07 carries out Total RNAs extraction and carries out reverse transcription immediately；
B.RNA reverse transcriptions become DNA：With reference to Thermo Scientific Reverted First strand cDNA
Synthesis Kit (being purchased from Thermo companies) carry out reverse transcription to the total serum IgE extracted in previous step, and cDNA is made, freezes
Be stored in -20 DEG C it is spare；
C. the PCR amplification of variable region sequences and recycling：It is anti-with mouse IgG hypotypes as template using gained cDNA in previous step
Body variable region sequences universal primer is primer, PCR amplification is carried out to the variable region sequences of heavy chain and light chain, by PCR product through DNA
Plastic recovery kit (being purchased from TIANGEN companies) is recycled, and sees attached drawing 1；
D. the clone of variable region sequences and sequencing：According to cloning vector pMD18-T kit (being purchased from Takara companies)
Heavy chain and chain variable region gene are attached with pMD18-T carriers by specification respectively, convert bacillus coli DH 5 alpha, picking
Positive colony transfers to InvitrogenTMCompany is sequenced.
Sequencing obtains the antibody heavy chain variable region gene sequence such as SEQ ID NO of hybridoma cell strain C07:9 shown, light chains
Variable region gene sequence such as SEQ ID NO：Shown in 10.The above-mentioned sequence of Vbase2 database analysis, heavy chain variable region it is each mutually
It mends and determines that the amino acid sequence in area is respectively：Such as sequence SEQ ID NO：HCDR1 shown in 2, such as sequence SEQ ID NO：Shown in 3
HCDR2 and such as sequence SEQ ID NO：HCDR3 shown in 4；The amino acid sequence of each complementary determining region of light chain variable region is：
Such as sequence SEQ ID NO：LCDR1 shown in 6, such as sequence SEQ ID NO：LCDR2 shown in 7 and such as sequence SEQ ID NO：8
Shown in LCDR3.
The recombinant expression of 3. antibody of embodiment and purifying
According to sequencing result in embodiment 2, will be added between the heavy chain of antibody and light chain variable region of hybridoma cell strain C07
Connect peptide (GGGGS)3, introduce six histidines and its full genome be subjected to password according to the preferences of pichia yeast expression system
The recombinant expression of son optimization and antibody, expressed obtained antibody are named as antibody K07, and structure composition is as shown in Fig. 2.On
The recombinant expression for stating antibody has following steps：
A) the expression plasmid structure of antigen-4 fusion protein gene
The gene order of antibody K07 after codon optimization such as SEQ ID NO:12 shown, amino acid sequence such as SEQ ID
NO:Shown in 11.The segment upstream of antibody K07 full genomes synthesis after optimization is introduced into pPICZ α A carriers DNA after XhoI sequences
Sequence, downstream introduce XbaI enzyme cutting site, are building up to pMD19-T Simple Vector plasmids (being purchased from Invitrogen companies)
In, a kind of long-term preservation plasmid is obtained, plasmid is denoted as pMD19-K07.PCR amplification is carried out, the primer sequence is as follows：
Sense primer：
P1:CGCCAGGGTTTTCCCAGTCACGAC
Downstream primer：
P2:AGCGGATAACAATTTCACACAGGA
After Standard PCR program, agarose gel electrophoresis analyzes (attached drawing 3), display primer size and expected size (790bp)
Unanimously.After PCR is obtained gene outcome recovery purifying, using XhoI (#R0146S, purchased from New England Biolabs public affairs
Department) and XbaI (#R0145V is purchased from New England Biolabs companies) double digestion, it is connected to pPICZ α A with T4 ligases
It in (V19520 is purchased from Invitrogen) plasmid, is transformed into DH5 α competent cells, is containing Zeocin (R250-01, purchase
From Invitrogen companies) LB tablets in 37 DEG C of overnight incubations.Screening positive clone bacterium sequencing in second day, compares, with expection
Sequence is completely the same to get to the expression plasmid of antibody K07, is denoted as pPICZ α-K07.
B) antigen-4 fusion protein gene is in the structure of Pichia pastoris host's engineered strain, screening and expression
Pichia pastoris competent cell and its relevant YPDS solid mediums, BMGY culture mediums, BMMY culture mediums are purchased
From Invitrogen companies.
By pPICZ α-K07 plasmids, linearized with SacI digestion with restriction enzyme.By linearized vector after ethanol precipitation,
Electrotransformation enters X-33 competence yeast cells, is applied to the YPDS solid mediums containing Zeocin, 30 DEG C of culture 3-5
It, just there is positive colony generation.
The monoclonal of the above-mentioned acquisition of picking is in 5mL BMGY culture mediums, 30 DEG C of cultures to OD600When=2.0~6.0, take
1mL preserves strain, and will be transferred to BMMY Small Amount induced expressions after the resuspension of remaining bacterium solution, dense to end every adding methanol for 24 hours
Degree is 1% (v/v).After a week, supernatant of bacteria solution is collected by centrifugation, passes through PAGE gel electrophoresis and Western blot analysis
(Western blot), object observing protein expression situation (attached drawing 4).Primary antibody is anti-HIS-Tag antibody in Western blot
(His-Tag (2A8) Mouse mAb, M20001, be purchased from Ai Bimate biological medicines (Shanghai) Co., Ltd.).
The K07 recombination fusion proteins engineering strain of above-mentioned acquisition is inoculated in respectively in BMGY culture mediums, 30 DEG C,
220rpm is cultivated to cell density to OD600=2.0~6.0, methanol was added every 24 hours to final concentration of 1.0% (v/v).
After a week, fermentation culture is collected.
C) fusion protein purification
Histidine tag affinity column antibody purification K07 fusion proteins, prepackage pillar is mainly used to be selected as HisTrap HP,
It is as follows：
(1) the removal of impurities pretreatment of zymotic fluid：Above-mentioned expression is obtained into antibody K07 fusion protein fermented liquid supernatants, centrifugation is received
Collect supernatant, and combination buffer is added so that supernatant final concentration of 300mM NaCl, 20mM NaH2PO4, 10mM
Imidazole adjusts pH7.5,0.45 μm of membrane filtration.
(2) the affine column purifications of HisTrap HP：With fully-automatic intelligent protein purification system, (AKTA avant150, are purchased from
GE healcare companies) affinity purification, pillar HisTrap are carried out to the antibody K07 fusion protein zymotic fluids that pretreatment obtains
HP (17-5248-02 is purchased from GE healcare companies).Combination buffer is 300mM NaCl, 20mM NaH2PO4, 10mM
Imidazole, pH7.5, elution buffer are 300mM NaCl, 20mM NaH2PO4, 500mM Imidazole, pH7.5.Elution
Shi Jinhang linear elutions, and collect each eluting peak.By SDS-PAGE electroresis appraisal purity, by attached drawing 5 it is found that after purification
Purity of protein reaches 95% or more；Merge satisfactory collecting pipe, replaces buffer solution and be PBS solution and (1mg/ is concentrated by ultrafiltration
Ml), filtration sterilization is saved backup in -20 DEG C.
Those skilled in the art know, can will be added between the heavy chain of antibody and light chain variable region of hybridoma cell strain C07
Peptide (GGGGS) 3 is connected, without introducing six histidines, recombinant expression is at the fusion protein without His labels, albumen at this time
Protein L albumen affinity purification method can be used in purifying (referring in particular to GE companies affinity chromatography medium Capto L).
The Performance Testing of 4. antibody K07 of embodiment
1. the Western blot identifications of antibody K07
A. polyacrylamide gel electrophoresis：12% separation gel, 5% concentration glue are configured, respectively loading standard protein, Lake
DOPA amine coupling BSA (Rac-BSA), Ractopamine coupling OVA (Rac-OVA), clenobuterol hydrochloride coupling BSA (Cle-
BSA), clenobuterol hydrochloride coupling OVA (Cle-OVA), salbutamol coupling BSA (Sal-BSA), salbutamol are coupled OVA
(Cle-OVA) (above-mentioned antigen is bought in Shanghai You Long companies), electrophoresis 1 hour under constant pressure；
B. transferring film：Transferring film 1 hour under the conditions of constant current (35mA/ films), extremely by the Protein transfer on polyacrylamide gel
On two nitrocellulose filters.Coomassie brilliant blue G250 dyes the SDS-PAGE glue for completing transferring film, observes the residual of albumen
Show mercy condition；
C. it closes：TBST buffer blinds (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning solution after closing (TBST,
Refer to TaKaRa company's T BST buffer) it washed once, 10 minutes；
D. antigen-antibody reaction：Confining liquid dilution (presses 1:400 volume ratios) horseradish peroxidase-labeled K07 (K07-
HRP, 1mg/mL, our company are marked using classical Over-voltage protection), it is added in above-mentioned nitrocellulose filter, room temperature reaction 1 is small
When；TBST is washed 5 times, every time 10 minutes；
E. it develops the color and takes pictures：Residual liquid on nitrocellulose filter is blotted, 2mL stable type peroxides are added in nitrocellulose filter
The mixed liquor (purchase is in Thermo companies) of compound enzyme solutions (1mL) and luminol/enhancing agent solution (1mL), uniform wet nitre
The surface of acid cellulose film, room temperature are taken pictures after being protected from light one minute in gel imaging system (purchase is in GE companies), and knot is left and taken
Fruit.
Experimental result (attached drawing 6) shows that K07 antibody and Ractopamine have specific reaction, not with hydrochloric acid Ke Lunte
Cross reaction occurs for sieve and salbutamol.
2. antibody K07 is in the performance evaluation of Ractopamine colloidal gold detection platform
Except the parameter in following tableAnd confining liquid (0.5%Casein+0.05%PEG2000) is outside, the preparation of remaining test card
Step and parameter are the same as embodiment 8.It is prepared respectively containing 5ppb, 10ppb, 50ppb, the sun of the standard items containing Ractopamine of 100ppb
Property pig urine, with negative pig urine be detected together.
Testing result table of the anti-Anti-ractopamine antibodies of K07 with commercialization Anti-ractopamine antibody in same buffer
Bright, antibody prepared by the present invention can be used for the structure of colloidal gold test card, and urine specimen detection sensitivity and commercially available examination
Paper card is suitable, is expected to that the level of commercially available test card is fully achieved after optimization；Antibody of the present invention is used to build colloidal gold test paper card,
K07 dosages and C line antibody dosages are less, more economical saving.
Remarks：Anti-ractopamine antibody purchase is commercialized in Wuhan Sino-American Biotechnology Company
The preparation of 5. kit for testing lecdopamine ELISA of the present invention of embodiment
This ELISA kit uses indirect competitive ELISA method.
1. the preparation of detection plate：It is coated with the buffer solution (Na for being 0.3% containing mass volume ratio2CO3And 0.58% NaHCO3)
Dilution Ractopamine coupling bovine serum albumin(BSA) conjugate (Rac-BSA) is diluted to 1 μ g/mL, and 96 holes are added with 100 holes μ L/
In ELISA Plate, 2~8 DEG C of coatings are overnight；Next day, with the PBST board-washings one time containing 0.05%TWEEN-20；200 holes μ L/ are added
Confining liquid (is 0.3%Na containing mass volume ratio2CO3, 0.58%NaHCO3, 10% sucrose, 10% calf serum of volume ratio) in room
(25 DEG C) of temperature is closed 2 hours, and liquid is discarded, and low temperature drying encapsulates spare.
Those skilled in the art know, since Ractopamine is micromolecular compound, it is more difficult to direct coated to ELISA Plate
On, it coupling carrier albumen further can generally be coated on ELISA Plate on it, therefore, the cow's serum of Ractopamine coupling
Albumin (BSA) also can use the carrier proteins such as oralbumin (OVA), keyhole limpet hemocyanin (KLH) to substitute completely.
2. enzyme dilution, antibody working solution, the preparation of enzyme labelled antibody：
1. enzyme dilution：PBST (pH7.4) buffer solution of 0.01M, the Na for being 0.335% containing mass volume ratio2HPO4·
12H2O, 0.02%NaH2PO4·2H2O, 0.8%NaCl, 0.02%KCl, 0.1%Casein, 0.05%BSA
2. the preparation of antibody working solution：K07 antibody is diluted with 1/500000 volume ratio with enzyme dilution, vortex mixing,
It dispenses (10mL/ bottles)；
3. the preparation of enzyme labelled antibody：The anti-His antibody-HRP of mouse are diluted with 1/2000 volume ratio with enzyme dilution, are vortexed mixed
It is even, dispense 7mL/ bottles.
3.TMB developing solutions, terminate liquid and Ractopamine titer：
1. TMB developing solutions：The concentration of TMB (being purchased from SIGMA companies) is 0.3g/L, is packed as 14mL/ bottles；
2. terminate liquid：The H of 2M2SO4, it is packed as 7mL/ bottles；
3., 4., 5., 6., 7., 8. Ractopamine titer：Each 1mL, concentration are respectively 0ppb, 0.1ppb, 0.3ppb,
0.9ppb, 2.7ppb, 8.1ppb.
4.20 concentrated cleaning solution again：0.2M PBS containing 0.05%TWEEN-20, are packed as 40mL/ bottles.
5. assembling：All bottled reagents are inserted in the corresponding aperture of box support, with elisa plate, specification, valve bag, sealing plate film
It is put into togerther carton, is stored in 2~8 DEG C, pastes seal inspection label after the assay was approved.
According to actual needs in the present embodiment enzyme labelled antibody completely can with anti-His antibody-AP, Protein L-HRP,
ProteinL-AP is substituted, and developing solution also can use BCIP/NBT developing solutions to substitute completely.
The application method of 6. kit for testing lecdopamine ELISA of the present invention of embodiment
1. Sample pretreatment：
Urine specimen：Limpid urine specimen can be directly used for measuring, and muddy urine palpus elder generation 3000g or more is centrifuged on to obtain
Clearly；Take 50uL for pattern detection；
Pork or pork liver equal samples：It weighs in 2 ± 0.05g homogenate samples to 50mL centrifuge tubes, 4mL is added and washs work
Liquid, adds 2mL 0.1M hydrochloric acid, and vortex 30s centrifuges (>=3000g, 5 minutes) under room temperature；Supernatant 1mL is taken, is added
30uL 0.5M NaOH to sample liquid pH value between 6.5~8.0, mixing；Take 50uL for pattern detection；
2. required reagent and ELISA Plate are taken out from cold storage environment, it is placed in (25 DEG C) balances of room temperature；
3. with deionized water by 20 times of concentrated cleaning solutions by volume 1:19 are diluted wash operating solution are made, and are used for enzyme
The washing of target, is prepared before use；
4. number：Sample and the corresponding micropore of standard items are sequentially numbered, it is parallel that each sample and standard items set multiple holes；
5. sample-adding：50 μ L/ hole standard items or sample, 50 holes μ L/ enzyme labelled antibodies are sequentially separately added into corresponding micropore
And 50 holes μ L/ antibody working solutions, mixing is gently vibrated, is protected from light 40 minutes in room temperature (25 DEG C)；
6. board-washing：After reaction time reaches, liquid in hole is dried, with the wash operating solution board-washing in 300 holes μ L/, fully
Washing 4~5 times dries liquid, is patted dry with blotting paper per minor tick 10s；
7. colour developing：The TMB developing solutions in 100 holes μ L/ are added, gently vibrate mixing, 15 points are protected from light in room temperature (25 DEG C)
Clock；
8. terminating and reading：The terminate liquid in 50 holes μ L/ is added, gently vibrates mixing, setting microplate reader is surveyed at 450nm
Fixed every hole OD values.
9. result judgement：
I) judges roughly：It is compared with the simple of the absorptance of standard sample wells to judge by the absorptance of sample well：Sample
Concentration of the color in hole than standard items contained by the concentration ratio of Ractopamine hole in the then sample of light color of a certain standard sample wells
Height, standard items contained by the concentration ratio of the Ractopamine hole in the color of sample well then sample deeper than the color of a certain standard sample wells
Concentration it is low；
Ii) quantitative analyses：With the percentage of the absorbance values of standard items and the absorbance value of first standard (0ppb)
Than drawing canonical plotting using the logarithm of the concentration (ppb) of Ractopamine standard items as abscissa for ordinate.By sample
Absorbance values and first standard (0ppb) absorbance value percentage substitute into standard curve in, to calculate sample
The actual concentrations of Ractopamine in this.
The Performance Testing of 7. kit for testing lecdopamine ELISA of the present invention of embodiment
The implementation case is by kit for testing lecdopamine ELISA of the present invention and commercially available similar being detected property of kit
The comparison of energy.
Two kinds of kits detect 70 parts of fresh negative urine specimens and 70 parts of fresh positive urine specimen (fresh feminine genders respectively
1ppb Ractopamines are added in urine specimen).Kit for testing lecdopamine ELISA of the present invention is according to above-mentioned detection method
It is operated；Commercially available similar kit is operated according to reagent specification.Testing result see the table below, and two kinds of detection kits are equal
The appearance of non-false positive；In positive sample detection, the rate of recovery range of kit for testing lecdopamine ELISA of the present invention is compared with city
It is narrower to sell kit, reflects higher accuracy, has the more accurate advantage of detection.
The preparation of 8. Ractopamine colloidal gold test card of the present invention of embodiment
This test card applies Competitive assays immunochromatography, can be used to the qualitative detection of Ractopamine in animal body fluid, especially
It is suitable for the qualitative detection of Ractopamine in pig urcine, detection is limited to 3-5ppb.Specific preparation method is as follows：
The colloid gold label of the anti-Anti-ractopamine antibodies of 1.K07：
With 0.2M K2CO3Colloidal gold pH value is adjusted to 8.15, is slowly added into colloidal gold solution dilute with the PBS of 0.02M
The K07 antibody released is to 10ug/mL, stirring at low speed 30 minutes；
Sealer is added：BSA is added to its final concentration of 1% (mass percent), stirring continuously adds after ten minutes
PEG2000 is to its final concentration of 0.05% (mass percent)；
Colloidal gold redissolves：Stirring be added after ten minutes with the redissolution liquid of colloidal gold same volume (1%BSA+5% sucrose+
0.1%Tween20+25mM PBS buffer solution (pH7.5)), it is stood overnight in 2~8 DEG C.Gained is the K07 of colloid gold label
Antibody；
2. be sprayed in gold-labelled pad 6 with the K07 antibody of above-mentioned colloid gold label, by coated antibody dilution (3% methanol+
25mM PBS buffer solution (pH7.5)) the anti-His antibody of mouse after dilution, Rac-OVA conjugates are respectively on nitrocellulose filter 2
Line C lines 4, T lines 5, then by print pad 1 gold-labelled pad 6, nitrocellulose filter 2, absorption pad 3 by shown in attached drawing 8 two-by-two it is adjacent simultaneously
Part stacking is pasted on liner plate 7 successively from left to right, fills item.
Those skilled in the art know, since Ractopamine is micromolecular compound, need to usually be coupled macromolecular carrier
It is further used as being coated with after albumen, the oralbumin (OVA) of the used coupling Ractopamine of the present embodiment can also use cow's serum
The carrier proteins such as albumin (BSA), keyhole limpet hemocyanin (KLH) substitute.
3. test card and specification, dropper are put into togerther carton, it are stored in 4~30 DEG C of cool places and are protected from light place, it is qualified to examine
Seal inspection label are pasted afterwards.
4. the present invention once attempted a variety of test card preparation methods, several different preparation methods of following table displaying are (unlisted in table
The step of, parameter is equal described in the present embodiment) and corresponding reagent box detection performance.
The use of 9. Ractopamine colloidal gold test card of the present invention of embodiment
1. sample pre-treatments：Limpid urine specimen can be directly used for measuring, and muddy urine must first centrifuge (>=3000g) to obtain
Obtain supernatant；
2. test card is taken out from cold storage environment, it is placed in (25 DEG C) balances of room temperature；
3. plastic clip is kept flat, measuring samples are drawn with dropper, vertical 3 drops (about 75 μ L) that are added dropwise are in well；
4. liquid starts timing when flowing, result is read in 5~10 minutes.
5. result judgement：If T lines are without colour developing, the colour developing of C lines, then sample is judged for the positive, is indicated in sample containing higher than inspection
Survey the Ractopamine of limit；If T lines and C lines all develop the color, then sample is judged for feminine gender, is indicated in sample without Ractopamine or Lay
The concentration of gram dopamine is limited less than detection；If C lines do not develop the color, then incorrect or this test card for using is shown to operate
Failure.
The detection performance of 10. Ractopamine colloidal gold test card of the present invention of embodiment is evaluated
1, sensitivity technique
1.1 reagents and solution：
A) Ractopamine storing solution (1mg/mL)：Ractopamine standard items 50mg accurately is weighed, after being dissolved with methanol
It is diluted to 50mL, is shaken up, spare, the term of validity 90 days is stored in -18 DEG C or less refrigerators.
B) Ractopamine working solution (1 μ g/mL)：Ractopamine storing solution 0.1mL is taken, adds methanol constant volume to 100mL,
Spare, the term of validity 7 days is stored in 2 DEG C~8 DEG C refrigerators.
C) 3ng/mL Ractopamines standard items：90 μ L of Ractopamine working solution are taken, 30mL is diluted to urine, are mixed
It is even.
D) 5ng/mL Ractopamines standard items：100 μ L of Ractopamine working solution are taken, 20mL is diluted to urine, are mixed
It is even.
E) 10ng/mL Ractopamines standard items：200 μ L of Ractopamine working solution are taken, 20mL is diluted to urine, are mixed
It is even.
1.2 detections and result
9, the Lake Test paper card of the present invention of same lot number is taken, detects the Rac standard items (3ng/ of three kinds of concentration respectively
ML, 5ng/mL, 10ng/mL) it is 3 times each, it is sun that 3 results of each concentration, which are the positive result that just can determine whether concentration thus,
Property.It is twice feminine gender three times in testing result when 3ng/ml standard items detect, it is primary for the positive；5ng/ml standard items detect
When, testing result is the positive three times.Judged according to the testing result of three kinds of concentration, Test paper card of the present invention it is sensitive
Degree is 3-5ppb.
2, specific
2.1 reagents and solution：
A) 100 μ g/mL of Clenizole Hydrochloride working solution, methanol is as solvent.
B) 500ng/mL Clenizole Hydrochlorides standard items：100 μ L of Clenizole Hydrochloride working solution are taken, are diluted to urine
20mL, mixing.
C) 100 μ g/mL of salbutamol working solution, methanol is as solvent.
D) 500ng/mL salbutamols standard items：100 μ L of salbutamol working solution are taken, 20mL, mixing are diluted to urine.
2.2 detections and result
500ng/mL Clenizole Hydrochlorides, 500ng/mL salbutamol standard items are detected respectively, are respectively repeated 3 times,
3 results of each solution are feminine gender.It is preferable special that testing result shows that Ractopamine Test paper card of the present invention has
Property.
Claims (9)
1. a kind of anti-Anti-ractopamine antibody, which is characterized in that including
Heavy chain variable region, amino acid sequence contain complementary determining region below：Such as sequence SEQIDNO：HCDR1 shown in 2, such as
Sequence SEQIDNO：HCDR2 shown in 3 and such as sequence SEQIDNO：HCDR3 shown in 4；
And its light chain variable region, amino acid sequence contain complementary determining region below：Such as sequence SEQIDNO：Shown in 6
LCDR1, such as sequence SEQIDNO：LCDR2 shown in 7 and such as sequence SEQIDNO：LCDR3 shown in 8.
2. anti-Anti-ractopamine antibody according to claim 1, which is characterized in that contain amino acid sequence such as SEQID
NO:Heavy chain variable region and amino acid sequence such as SEQIDNO shown in 1:Light chain variable region shown in 5.
3. a kind of single-chain antibody of anti-Ractopamine, the amino acid sequence such as SEQIDNO of the single-chain antibody:Shown in 11.
4. a kind of nucleotide sequence of coding antibody as claimed in claim 3, the nucleotide sequence such as SEQIDNO:Shown in 12.
5. a kind of expression vector containing nucleotide sequence as claimed in claim 4.
6. a kind of recombinant host cell containing expression vector as claimed in claim 5.
7. recombinant host cell as claimed in claim 6, which is characterized in that the recombinant host cell is Pichia pastoris.
8. a kind of method producing single-chain antibody as claimed in claim 3, including：
1) recombinant host cell expression antibody as claimed in claims 6 or 7 is cultivated under suitable conditions；
2) it and then from host cell purifies, collect antibody.
9. claims 1 to 3 any one of them antibody is in the application of detection Rct opamine residue.
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| CN103012592A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of ractopamine monoclonal antibody |
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| CN103012592A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of ractopamine monoclonal antibody |
| CN103163296A (en) * | 2011-12-09 | 2013-06-19 | 大连普瑞康生物技术有限公司 | Immune colloidal gold test strip for detection of ractopamine residue in swine urine |
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