CN104844692B - A kind of dodecapeptide and its application with reference to zearalenone - Google Patents

Abstract

The invention belongs to biological technical field, is related to the dodecapeptide that can specifically bind zearalenone (Zearalenone, ZEN) and its application, the amino acid sequence of the peptide molecule is LLNISRRQIKNL.The present invention is by the way of the affine elutriation of magnetic bead liquid phase and combination ZEN antibody competition elutions, it is incubated by optimizing, elution time, the conditions such as temperature, quick elutriation obtains the peptide molecule that can specifically bind ZEN, the peptide molecule can substitute traditional ZEN antibody and applied in the immune analysis system of ZEN, its linear detection range is broad, the acquisition of the peptide molecule is without immunologic process, prepare simple, cost is low, cycle is short, the peptide molecule both can largely be expanded by way of biological culture, can also largely it be prepared by the method for chemical synthesis or genetic engineering, new path is provided for the preparation of ZEN antibody, with preferable application value.

Description

A kind of dodecapeptide and its application with reference to zearalenone

Technical field

The invention belongs to biological technical field, and in particular to a kind of dodecapeptide with reference to zearalenone and its should With.

Background technology

Zearalenone (Zearalenone, ZEN) is a kind of oestrogen-like hormone sample mycotoxin produced by sickle-like bacteria, Entitled 6- (- 6 epoxides of 10- hydroxyls-undecenyl) β of its chemistry-thunder lock acid lactone, be widely present in the corn to go mouldy, sorghum, In the cereal crops such as wheat, oat, barley and milk.Since ZEN pollutes generally in grain, the work of food chain can be passed through Toxic action is produced with to poultry and health, therefore the monitoring to ZEN is just particularly important.

The method of detection ZEN more commonly used at present mainly includes：High performance liquid chromatography (High performance Liquid chromatography, HPLC), gas chromatography (Gaschromatography, GC), thin layer chromatography (Thin Layer chromatography, TLC), tablets by HPLC-MS (High performance liquid Chromatography-mass sepectrum, HPLC-MS), microbiological method and immunodetection etc., wherein immunology Analysis method is with its high sensitivity, easy to operate and the advantages that can realize scale selection, in the field of fast detection of ZEN It is widely used.

The core of immunological analysis method is specific recognition and combination between Ag-Ab, therefore prepares specificity Just become the premise and core of development immunological analysis method with reference to the antibody of antigen.The research of antibody develops rapidly in recent years, From traditional polyclonal, monoclonal antibody, it is developed to single-chain antibody, phage displaying antibody, single domain heavy chain antibody, anti- Idiotype antibody etc. is the genetic engineering antibody field of representative.Novel antibodies have molecular weight it is small, it is simple in structure, can self into Change, prepare the characteristics of quick and development trend.For example, monoclonal antibody, single-chain antibody, the molecular weight of single domain heavy chain antibody are gradual Reducing, be respectively 150,30-50,15-20kDa, the antibody analog using Lipocalin as skelemin is 18-20kDa, and Aptamer is only then a bit of nucleotide sequence.

With developing rapidly for phage-displayed polypeptides storehouse technology, by the elutriation of phage display peptide library, can quickly, letter The peptide molecule combined with specific target body molecular specificity singly is obtained, therefore, peptide molecule can become potential novel antibodies Molecule.Being mainly characterized by of phage display peptide library technology can effectively filter out the phage display technology with target target body specific bond Show polypeptide, the technology is in the ligand explored the binding site that interacts between acceptor and ligand, seek high-affinity bioactivity Molecule, explore agnoprotein matter space structure epitope, the development etc. of new generation vaccine is widely used, but above-mentioned phage display technology Show that the application of polypeptide techniques is confined to carry out affine elutriation by target body molecule of macro-molecular protein mostly.Due to protein surface Epitope with multiple copies, therefore it is very easy to obtain the corresponding peptide molecule combined therewith.Current peptide molecule is most Mimic epitope (antigen substitute) as antigen is applied in immune analysis, and resists peptide molecule as small-molecule substance The report that the substitute of body is applied to immune analysis is less, the reason is that small-molecule substance volume is too small, and surface does not have Abundant amino, carboxylic group, it is difficult to combined with peptide molecule, so as to cause elutriation difficult.

The present invention using the affine elutriation of magnetic bead liquid phase and combines ZEN antibody competitions by phage-displayed polypeptides storehouse technology Property elution mode, by optimize be incubated, the condition such as elution time, temperature, quickly screened from phage-displayed polypeptides storehouse The peptide molecule that can be specifically bound with ZEN, the peptide molecule have similar to traditional ZEN monoclonal antibody molecules be immunized Detection characteristic is learned, the immune analysis system of ZEN can be applied to as the substitute of tradition ZEN antibody.This method avoid system The flows such as immunologic process, cell fusion, the monoclonal screening required for standby traditional monoclonal antibody molecule by cycle length, tool Have without immunologic process, screen the features such as convenient, the cycle is short, manufacturing cost is low.

The content of the invention

The present invention with ZEN holoantigens (the conjugate ZEN-BSA of ZEN and bovine serum albumin(BSA)) for target molecule, by target molecule It is attached on magnetic bead, input phage random displaying dodecapeptide storehouse, carries out the affine elutriation of magnetic bead liquid phase, with reference to ZEN antibody competitions Property elution mode, obtain a kind of peptide molecule for specifically binding ZEN, its amino acid sequence is：

LLNISRRQIKNL。

In aforementioned polypeptides molecular structure, capitalization English letter represents known natural L-form amino acid residue or its D- type respectively One kind of isomers, i.e. L represent leucine residue, and N represents asparagine acid residue, and I represents isoleucine residues, and R represents essence Histidine residue, S represent serine residue, and Q represents glutamine residue, and K represents lysine residue.

The invention further relates to the nucleotide of encoding such polypeptides molecule amino acid sequence, its sequence is:

CTT CTT AAT ATT AGT CGT CGT CAG ATT AAG AAT CTT

The present invention refer to peptide molecule can Phage amplification, chemical synthesis or genetic engineering recombination expression by way of into Row is a large amount of to be prepared.Phage amplification refers to show the bacteriophage for having peptide molecule, by way of biology expands, amount reproduction Production displaying has the bacteriophage particles of peptide molecule.Chemical synthesis refers to the amino acid sequence according to the mimic epitope announced, and leads to The mode for crossing chemically synthesized polypeptide carries out Peptide systhesis.The mode of genetic engineering recombination expression refers to the base of coded polypeptide molecule Cause, by being cloned into expression vector, carries out a large amount of preparations of peptide molecule in the form of polypeptide-fusion protein.

The invention further relates to application of the peptide molecule in immunology detection analysis.The type of immunology detection includes The immune analysis detection type based on Ag-Ab specific reaction such as MBP enzyme linked immuno-adsorbent assay.

The beneficial effects of the invention are as follows：The alternative traditional ZEN antibody molecules of peptide molecule mentioned by the present invention, as The binding molecule of ZEN directly applies to immunology detection analysis, indirect competitive ELISA standard curve its line based on peptide molecule Property have a wide reach, the peptide molecule have the characteristics that without immunologic process, obtain easily and fast, the cycle it is short.

Brief description of the drawings

ZEN indirect competitive ELISA standard curves of the Fig. 1 based on 12 polypeptides.

Embodiment

Embodiment 1. and the affine elutriation of magnetic bead liquid phase and its identification of ZEN specific binding polypeptide molecules

1) affine elutriation and the specific method of ZEN specific binding polypeptide molecules are：Can be special with ZEN molecules efficiently to obtain The peptide molecule that the opposite sex combines, employs the affine elutriation of magnetic bead liquid phase and combines the mode of ZEN antibody competitions elution, specific side Method is：The magnetic bead molecule (diameter 400nm) of 1.0mg carboxylated is taken, is washed 3 times with PBST.Add the displaying of 10 μ L phage randoms 12 peptide libraries (2 × 10¹¹Pfu, NEB company), it is rear to add 900 μ L PBST, mixed with magnetic bead, jog concussion is anti-at room temperature 60min, after Magneto separate (purpose of this step combined in phage display peptide library is removed with magnetic bead surfaces modification group it is more Peptide molecule, the absorption of this kind of peptide molecule are got rid of in magnetic bead surfaces and by Magneto separate), careful Aspirate supernatant (not with magnetic The peptide molecule that bead surface modification group combines) to another new centrifuge tube, add the magnetic bead (diameter that 1.0mg ZEN-BSA are coupled The coupling amount of 400nm, ZEN-BSA are 1.0mg, ZEN:The coupling ratio of BSA is 15:1, the preparation method of conjugate is referring to Burkin Ea al., 2000, Applied biochemistry and microbiology, 36,282-288)), concussion reaction at room temperature 45min.After Magneto separate, washed 6 times with TBST, wash away uncombined bacteriophage.Add 1mL 150ng/mL ZEN antibody (PBS System), jog shakes 60min at room temperature.After Magneto separate, supernatant is carefully drawn, the first round affine elutriation product is obtained, stays 10 μ L Titre is surveyed, ER2738 of the remaining bacteriophage input in logarithm early period is expanded, then carries out the 2nd wheel and the 3rd wheel successively Elutriation, panning step are substantially the same with the first round, and the phagocytosis scale of construction added every time is 2 × 10¹¹Pfu, difference are：The 2nd, the incubation time of the phage-displayed polypeptides and ZEN-BSA coupled beads of 3 wheel screenings is respectively 30min, 15min, and competition is washed The concentration of de- liquid (ZEN antibody) is respectively 75ng/mL, 50ng/mL.

2) identification of positive phage clones：Random picking 80 in the tablet of phage titre is measured after third round elutriation A bacteriophage spot, carries out the amplification of bacteriophage, and positive phage clones are carried out using Immunofluorescent antibody detection method Identification, specific method are：First, with 10mM PBS (pH 7.4) dilute ZEN-BSA antigens, 2 μ g/mL be coated with 96 hole elisa Plates, 4 DEG C be incubated overnight.After second day is washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), with containing 3% degreasing The PBS of milk powder is closed, when 37 DEG C of incubations 1 are small；Put into 100 μ l bacteriophages spots amplification liquid (1.0 × 10¹²Pfu), bitten with original Thalline peptide storehouse as negative control, 37 DEG C be incubated 1 it is small when；Add 1：5000 times of diluted HRP mark anti-M13 bacteriophages secondary antibody 100 μ l, when 37 DEG C of incubations 1 are small；100 μ l tmb substrate liquid, lucifuge colour developing 5min are added, microplate reader reads the absorption at 450nm Value.Choose OD₄₅₀Phage clone of 2 times more than negative control is positive colony.

3) identification of ZEN peptide molecules is specifically bound：Carried out and ZEN specificity using the method for indirect competitive ELISA With reference to the identification of phage-displayed polypeptides, specific method is：ZEN-BSA, 2 μ g/mL coatings are diluted with 10mM PBS (pH 7.4) 100 μ L of ELISA Plate, 4 DEG C of overnight incubations；After second day is washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), Closed with the PBS containing 3% skimmed milk power, when 37 DEG C of incubations 1 are small；Put into 50 μ l and be accredited as the positive through indirect ELISA Phage clone (1.0 × 10¹¹Pfu) and 50 μ l ZEN standard items (concentration range 0-1000ng/mL), 37 DEG C be incubated 1 it is small when； Add 1：The 100 μ l of anti-M13 bacteriophages secondary antibody of 5000 dilution HRP marks, when 37 DEG C of incubations 1 are small；Add 100 μ l tmb substrates Liquid, lucifuge colour developing 5min, reads OD₄₅₀.If the peptide molecule of phage display can specifically bind ZEN, ZEN is added The OD values in the hole of standard items will be less than control wells (not adding ZEN standard items).

The sequencing of the peptide molecule encoding gene of embodiment 2. and ZEN specific bindings and its determining for amino acid sequence

The bacteriophage for there are specific binding ZEN peptide molecules by indirect competitive ELISA identification displaying is expanded, is carried Take the DNA sequencing template of bacteriophage.Simplified process is as follows：Phage amplification is carried out, after first step centrifugation, 800 μ l is contained and are bitten Thalline supernatant is transferred to a new centrifuge tube.Add 200 μ l PEG/NaCl precipitating phages.Precipitation is resuspended in 100 μ l iodine after centrifugation Compound buffer solution (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), adds 250 μ l absolute ethyl alcohols precipitation DNA, from After the heart precipitation (DNA sequencing template) is washed with 70% ethanol again.Precipitation is finally resuspended in 20 μ l aqua sterilisas, takes 2 μ l to carry out agar Sugared gel electrophoresis analysis；5 μ L bacteriophages templates are taken to carry out DNA sequencing, its -96gIII sequencing primer is：5’-^HOCCC TCA TAG TTA GCG TAA CG-3’.The nucleotide sequence of its amino acid encoding of DNA sequencing the results show：CTT CTT AAT ATT AGT CGT CGT CAG ATT AAG AAT CTT, amino acid sequence are：LLNISRRQIKNL.

Embodiment 3. specifically binds application of the ZEN peptide molecules in ELISA

(1) sample extraction

5g samples to be tested are weighed, add methanol-PBS solution of 25ml 60%, 200rpm vibrates 5 minutes；Extracting solution is used After No. 1 filter paper of whatman is filtered, 1ml filtrates are taken to add 4ml PBS (phosphate buffer, pH=7.2)

It is sample extracting solution after mixing, it is stand-by.

(2) it is coated with and closes

With 10mM PBS (pH 7.4) diluted ZEN-BSA (2 μ g/ml), coated elisa plate, 4 DEG C of overnight incubations.Second day After being washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), closed with the PBS containing 3% skimmed milk power, 37 DEG C be incubated 1 it is small when after, it is stand-by with PBST board-washings 6 times.

(3) foundation of standard curve

The lath handled well through step (2) is taken out, putting into 50 μ l displayings respectively per hole there are specific binding ZEN peptide molecules Bacteriophage (1.0 × 10¹²Pfu) and a series of various concentrations 50 μ l ZEN standard items (0-1000ng/ml), 37 DEG C incubation 20min.Add 1:The anti-M13 bacteriophages secondary antibody of 5000 dilution HRP marks, when 37 DEG C of incubations 1 are small.Then developed the color with tmb substrate, Read OD₄₅₀.Using ZEN log concentrations as abscissa, Percentage bound (adds the OD in the hole of ZEN₄₅₀/ do not add ZEN hole OD₄₅₀× 100%) it is ordinate, establishes indirect competition standard curve.

(4) detection of sample

The lath handled well through step (2) is taken out, putting into 50 μ l displayings respectively per hole there are specific binding ZEN peptide molecules Bacteriophage (1.0 × 10¹²Pfu) and sample to be tested extracting solution, 37 DEG C be incubated 1 it is small when.Add 1:5000 dilution HRP marks resist M13 bacteriophage secondary antibodies, when 37 DEG C of incubations 1 are small.Then developed the color with tmb substrate, read OD₄₅₀, calculations incorporated rate, and according to standard Curve, retrodicts out the content of ZEN in sample.

Embodiment 4 and a large amount of preparations of ZEN specific binding polypeptide molecules

1) in a manner of Phage amplification

It is inoculated with being added with the bacteriophage that ZEN is specifically bound to 20ml in the culture of ER 2738,37 degree of 220rpm Shaken cultivation 4.5h.Culture is transferred in another centrifuge tube, 4 DEG C of 10000rpm centrifuge 10min, by 80% turn of the top of supernatant Enter in a fresh tube, add the PEG/NaCl of 1/6 volume, 120min is stood at 4 DEG C.4 DEG C of 10000rpm centrifugations PEG/NaCL are quiet Put solution 15min.Supernatant is abandoned, residual supernatant is sucked after of short duration centrifugation.Add 1mL TBS to be resuspended, be that bacteriophage is expanded Increase liquid.

2) prepared in a manner of polypeptide-fusion protein

A.PCR expands the external source encoding gene of peptide molecule

PCR reaction systems：(50μL)

PCR reaction conditions：

Using PCR product QIAquick Gel Extraction Kit purified pcr product, trace dna quantitative instrument quantifies.The coding base of peptide molecule Because sequence is：CTT CTT AAT ATT AGT CGT CGT CAG ATT AAG AAT CTT.

B. the double digestion of external source encoding gene and expression vector

The external source code gene of ACC65I and Eag I enzymes and expression vector is respectively adopted, and (pMAl-pIII, NEB company, can Express MBP fusion proteins) carry out double digestion.

C. after digestion product connection and conversion

Plasmid pMal-PIII and purpose fragment are mixed with 1: 20 (molar ratio), 12h is connected in 20 DEG C of water-baths, takes 15 μ L Connection product is added in 100 μ L competent cell DH5 α, is fully mixed.After ice bath 30min, 42 DEG C of water-bath heat shock 40s, ice immediately 800 μ L LB liquid mediums are added after bath 3min, 37 DEG C, 200rpm culture 1h, 8000rpm centrifugation 10min, suck supernatant and stay About 300 μ L are taken, are coated in LB-A solids (Amp) culture medium, 37 DEG C are incubated overnight, and obtain subclone positive colony.

D. Cloning Transformation

The subclone that above-mentioned steps obtain Tiangeng kit is extracted into purpose plasmid, takes 10 μ L thin to 100 μ L competence In born of the same parents TB1, fully mix.After ice bath 30min, 45 DEG C of water-bath heat shock 50s, add the training of 800 μ L LB liquid immediately after ice bath 5min Nutrient solution, 37 DEG C, 200rpm culture 45min, 7000rpm centrifugation 10min, suck supernatant and leave and take about 300 μ L, be coated on LB-A solids (Amp) in culture medium, 37 DEG C are incubated overnight, and obtain positive colony.

E. the expression of polypeptide-MBP fusion proteins

By the positive clone molecule of above-mentioned acquisition, a single bacterium colony is chosen from tablet and is inoculated in 5mL LB-A, in 0.5% sucrose, 37 DEG C, 220r/min, shaken cultivation is overnight, and overnight culture is inoculated in the LB-A of 50mL by 1% inoculum concentration (v/v), and 0.2% In sucrose culture medium, 3 bottles of inoculation respectively, 37 DEG C, 220r/min shaken cultivations, when culture bacterial concentration OD600 reaches 0.6 When, IPTG to final concentration of 0.5mmol/L is added into three bottles of cultures, 120r/min shaken cultivations, (IPEG is molten by inducer Liquid) in 4 DEG C, 5000g, centrifugation 15min collects bacterial sediment, abandons supernatant.Cell is resuspended in 400mL 30mM Tris-HCl, 25% sucrose, pH 8.0 (80mL/g wet cell weights), adds EDTA to 1mM, shakes 5-10min, 8000g, 4 DEG C of centrifugations at room temperature 15min, abandons supernatant, and precipitation is resuspended in the 5mM MgSO4 of 400ml precoolings, shakes 15min, 8000g, 4 DEG C on ice, centrifuges 20min, retains supernatant, and 8mL 1M Tris-HCl, pH 7.4 is added into supernatant, obtains polypeptide-MBP fusion proteins.

Embodiment 5 and the immunology detection stability experiment of ZEN specific binding polypeptide molecules

1) resistance to acid and alkali is tested

Chemical synthesis can be used into pH=4.0 respectively with the ZEN dodecapeptides specifically bound and ZEN monoclonal antibodies, 5.0,6.0,7.4,8.0 buffer solution is diluted to working solution, carries out indirect competitive ELISA measure respectively, and specific method is：With 10mM PBS (pH 7.4) diluted ZEN-BSA (2 μ g/ml), coated elisa plate, 4 DEG C of overnight incubations.Use PBST within second day After (10mM PBS, 0.05%Tween-20 (v/v)) is washed 3 times, closed with the PBS containing 3% skimmed milk power, 37 DEG C incubate Educate 1 it is small when after, it is stand-by with PBST board-washings 6 times.The lath handled well is taken out, ZEN can be specifically bound by putting into 50 μ l respectively per hole Peptide molecule/ZEN monoclonal antibodies (20 μ g/ml) and a series of various concentrations 50 μ l ZEN standard items (0-1000ng/ Ml), 37 DEG C of incubation 20min.Add 1:The sheep anti-mouse igg two of anti-M13 bacteriophages secondary antibody/HRP marks of 5000 dilution HRP marks It is anti-, when 37 DEG C of incubations 1 are small.Then developed the color with tmb substrate, read OD₄₅₀.Using ZEN log concentrations as abscissa, Percentage bound (adds The OD in the hole of ZEN₄₅₀/ do not add ZEN hole OD₄₅₀× 100%) it is ordinate, establishes indirect competition standard curve.

Experimental result is shown：Indirect competitive ELISA using peptide molecule as ZEN recognition components, in pH=4.0,5.0, When 6.0,7.4,8.0, its IC₅₀Respectively 60,55,53,63,58ng/ml；Using ZEN monoclonal antibodies as the indirect of recognition component Competitive ELISA, does not there is good blocking effect in pH=4.0,5.0,6.0, and smoothed curve, the immune analysis of antibody is presented Performance is significantly affected, in pH=7.4,8.0, its IC₅₀Respectively 46,39ng/ml, surface is mono- compared to traditional ZEN Clonal antibody, the peptide molecule of ZEN antibody substitutes have more preferable soda acid patience.

2) high temperature resistance is tested

By being respectively placed in temperature with the ZEN peptide molecules specifically bound and ZEN monoclonal antibodies and be for chemical synthesis 37 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 30min is incubated in 70 DEG C of water-bath, carries out indirect ELISA experiment after taking-up respectively, specific side Method is：With 10mM PBS (pH 7.4) diluted ZEN-BSA (2 μ g/ml), coated elisa plate, 4 DEG C of overnight incubations.Second day use After PBST (10mM PBS, 0.05%Tween-20 (v/v)) is washed 3 times, closed with the PBS containing 3% skimmed milk power, 37 DEG C be incubated 1 it is small when after, it is stand-by with PBST board-washings 6 times.The lath handled well is taken out, puts into per hole and is incubated by different temperatures respectively The 50 μ l crossed can specifically bind peptide molecule/ZEN monoclonal antibodies (20 μ g/ml) of ZEN, 37 DEG C of incubation 20min.Add 1: The sheep anti-mouse igg secondary antibody of anti-M13 bacteriophages secondary antibody/HRP marks of 5000 dilution HRP marks, when 37 DEG C of incubations 1 are small.Then use Tmb substrate develops the color, and reads OD₄₅₀, OD values during using 37 DEG C calculate OD under different temperatures as 100%₄₅₀The stability % of value.

Experimental result is shown:Indirect ELISA using peptide molecule as ZEN recognition components, temperature be 37 DEG C, 45 DEG C, At 50 DEG C, 60 DEG C, 70 DEG C, its temperature degree is respectively 100%, 98%, 95%, 88%, 86%；Using ZEN monoclonal antibodies as The indirect ELISA of ZEN recognition components, when temperature is 37 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, its temperature degree is respectively 100%, 75%, 30%, 25%, 16%, show compared to traditional ZEN monoclonal antibodies, the polypeptide point of ZEN antibody substitutes Son has more preferable high temperature resistance.

SEQUENCE LISTING

<110>University Of Nanchang

<120>A kind of dodecapeptide and its application with reference to zearalenone

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213>Artificial sequence

<400> 1

Leu Leu Asn Ile Ser Arg Arg Gln Ile Lys Asn Leu

1 5 10

Claims (4)

1. the dodecapeptide of zearalenone can be combined, it is characterised in that its amino acid sequence is:

LLNISRRQIKNL。

2. encode the nucleotide for the dodecapeptide amino acid sequence that zearalenone can be combined described in claim 1.

3. nucleotide as claimed in claim 2, its sequence are:

CTT CTT AAT ATT AGT CGT CGT CAG ATT AAG AAT CTT。

4. the preparation method of the dodecapeptide of zearalenone can be combined as claimed in claim 1, it is characterised in that pass through phagocytosis The mode of body amplification, chemical synthesis or genetic engineering recombination expression is largely prepared；The Phage amplification refers to show The bacteriophage of the dodecapeptide, by way of biology expands, amount reproduction production；The chemical synthesis refers to according to described 12 The amino acid sequence of peptide, carries out Peptide systhesis by way of chemically synthesized polypeptide；The mode of the genetic engineering recombination expression Refer to that the gene of the dodecapeptide will be encoded, by being cloned into expression vector, largely made in the form of polypeptide-fusion protein It is standby.