CN106916790A - The anti-general monoclonal antibody of free gossypol and its application - Google Patents
The anti-general monoclonal antibody of free gossypol and its application Download PDFInfo
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- CN106916790A CN106916790A CN201710033126.2A CN201710033126A CN106916790A CN 106916790 A CN106916790 A CN 106916790A CN 201710033126 A CN201710033126 A CN 201710033126A CN 106916790 A CN106916790 A CN 106916790A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses the general monoclonal antibody of anti-free gossypol and its application, belong to immunology detection analysis technical field.The invention provides the hybridoma cell strain 2D4 of the general monoclonal antibody of anti-free gossypol, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, deposit number is CGMCC No.10872.Present invention also offers the monoclonal antibody secreted by above-mentioned hybridoma cell strain.The anti-general monoclonal antibody of free gossypol of the present invention, has preferably specificity, detection sensitivity IC to free gossypol50It is 100ng/mL to be worth, and can be used for the specific detection of free gossypol in cottonseed and its product.
Description
Technical field
The present invention relates to immunology detection analysis technical field, more particularly, to hybridoma cell strain 2D4 and its generation
The general monoclonal antibody of anti-free gossypol and application.
Background technology
Gossypol (gossypol) be present in malvaceae plant belong to pigment gland generation polyphenol naphthalene derivatives, be present in leaf and
In seed.The structure of gossypol is 1,1 ' 6,6 ' 7,7 '-hexahydroxy -3,3 '-dimethyl -5,5 '-diisopropyl -2,2 '-dinaphthalene -8,
8 '-dialdehyde.Gossypol can be divided into free gossypol and the class of bound gossoypol two by its existence form.Active group (aldehyde in free gossypol
Base and hydroxyl) can be combined with protein, enzyme activity is destroyed, protein digestibility is reduced, cause slow growth of animal, poisoning and dead
Die.
Gossypol is partially converted into during free gossypol remains in cotton cake dregs after cottonseed oil expression, and average content is 700-
1920mg/kg.Cottonseed meal content is high, and nutritive value close to dregs of beans, under protein raw material situation in short supply, make by cotton cake dregs
Applied increasingly extensively in Chinese Production of Livestock and Poultry for good protein raw material, but contain free gossypol in cotton cake dregs, if
Improper use in daily ration, easily causes animal gossypol poisoning, if while the edible livestock products containing gossypol of the mankind there is also safety wind
Danger.Therefore, feed, feedstuff and gossypol content limitation in human food have been formulated in the countries and regions such as European Union, China, U.S.
Standard.Wherein, China's current forage health standard (GB13078-2001) is defined as cotton for free gossypol allowance in feed
Seedcake dregs of rice raw material≤1200mg/kg.Gossypol is important endogenous toxin mass parameter in cottonseed, sets up trip quick, efficiently, easy
From gossypol detection method can in acquisition process quality calibration provide safeguard.
Detecting the method for free gossypol in cotton cake dregs and cottonseed oil at present mainly includes high performance liquid chromatography, efficient hair
Cons electrophoresis method, fluorimetric HPLC method, AAS (aniline process), immunochemistry analytic approach etc..But, efficient liquid
Although the method sensitivity of the utilization instrument such as phase chromatography, high performance capillary electrophoresis, fluorimetric HPLC method is higher, behaviour
Make simple, but, testing cost high to instrument and equipment requirement is high, it is difficult to promote the use of；Although AAS is universal, the method
Have that sensitivity is low, toxicity big, operating cost when the shortcomings of.And immunoassay method has low cost, high flux, highly sensitive, right
The low feature of technical staff's relative requirement, it is adaptable to the rapid screening of a large amount of samples.Therefore it provides having to free gossypol higher
The monoclonal antibody of affinity and detection sensitivity, has extremely important for free gossypol in detection cotton cake dregs and cottonseed oil
Meaning.
The content of the invention
It is an object of the present invention to provide the general monoclonal antibody hybridoma cell line of anti-free gossypol；
It is a further object to provide the monoclonal antibody for having above-mentioned hybridoma cell strain secretion, the antibody is to trip
There is preferable affinity and detection sensitivity from gossypol, can be used to set up free gossypol total amount in measure cottonseed and its product
Immunological detection method.
To reach above-mentioned purpose, the present invention uses following technical proposals：
The hybridoma cell strain 2D4 of the anti-general monoclonal antibody of free gossypol of the present invention is in the preservation of May 19 in 2015
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, (abbreviation CGMCC, address is:Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.10872, and Classification And Nomenclature is single
Clonal cell line.
It by preserving number is the mouse monoclonal of CGMCC No.10872 that the anti-general monoclonal antibody of free gossypol of the present invention is
Secreted by antibody hybridoma cell strain 2D4.
The hybridoma cell strain of the above-mentioned general monoclonal antibody of anti-free gossypol and the monoclonal antibody secreted by it belong to
In protection scope of the present invention.
Hybridoma cell strain of the present invention or monoclonal antibody can be applied to detect the free cotton in cottonseed and its product
Phenol.
Further, hybridoma cell strain of the present invention or monoclonal antibody can be used to prepare for detecting cottonseed and its system
In product in the medicament or kit of free gossypol.
The preparation basic step of the hybridoma cell strain 2D4 of the general monoclonal antibody of anti-free gossypol that the present invention is provided
For：
(1) preparation of immunogene and identification：Gossypol acetate is taken to be dissolved in methyl alcohol, it is mixed with BSA (bovine serum albumin(BSA)) solution
Close, sodium cyanoborohydride (NaBH is added afterwards3CN), lucifuge stirring at room temperature.Dialysed with PBS solution afterwards, packing is preserved；
(2) mouse is immune：It is immune by nape part multi-point injection by antigen and equivalent after not formula adjuvant is well mixed
BALB/c mouse.Immunologic process six times altogether：Every 100 μ L, are spaced 21 days first, and two exempt to five to exempt from every 50ul afterwards, most
Afterwards once for abdominal cavity spurt is immune, every 25ul；
(3) cell fusion is set up with cell line：By polyethylene glycol (PEG4000) method by mouse boosting cell and mouse bone marrow cells
Oncocyte is merged, and by HAT medium cultures, positive cell hole is detected using indirect ELISA, and further with indirect competition
ELISA method determines the inhibition of positive cell hole, by limiting dilution assay to there is the positive cell hole for preferably suppressing to carry out three
Secondary subclone, final screening obtains hybridoma cell strain 2D4；
(4) identification of hybridoma cell strain property：It is set with ELIAS secondary antibody using mouse monoclonal Ig classes/subgroup identification and is surveyed
It is fixed；IC50 values are determined by ELISA method.
Wherein, in step (1), after the amino on the aldehyde radical and albumen of gossypol acetate occurs condensation reaction, Schiff is formed
Alkali, but because carbon-to-nitrogen double bon is unstable, therefore carbon-to-nitrogen double bon is reduced for singly-bound further through sodium cyanoborohydride is added, produce coupling
Thing can be with stable existence.
Beneficial effects of the present invention are as follows：
(1) sodium cyanoborohydride used in the present invention reduces the carbon-to-nitrogen double bon between albumen and small molecule, makes to exempt from
Epidemic focus is more stablized, and is more beneficial for the preservation of immunogene；
(2) the anti-free gossypol cell strain of monoclonal antibody that the present invention is obtained, has to free gossypol and preferably detects sensitive
Degree and affinity (IC50It is 100ug/kg to be worth)；
(3) immunoassay method that cell line application of the invention is produced is low to instrument and equipment requirement, simple to operate, is easy to
Quick detection and promote the use of.
Brief description of the drawings
Specific embodiment of the invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is comlete antigen ultra-violet absorption spectrum phenogram；Wherein, GOS-BSA30, GOS-BSA60, GOS-BSA90 point
Not Biao Shi small molecule and albumen ingredient proportion be 30:1、60:1、90:1；
Fig. 2 produces standard suppression curve of the monoclonal antibody to free gossypol by cell line 2D4.
Specific embodiment
In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings
It is bright.Similar part is indicated with identical reference in accompanying drawing.It will be appreciated by those skilled in the art that institute is specific below
The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
The preparation of the hybridoma cell strain 2D4 of embodiment 1
1st, the synthesis of comlete antigen
Take gossypol acetate (5mg) to be dissolved in the methyl alcohol of 2ml, BSA (bovine serum albumin(BSA)) solution with 15ml is (dilute with PBS
Release 50mg BSA) mixing, 60mg sodium cyanoborohydrides (NaBH is added afterwards3CN), lucifuge is stirred 12 hours at room temperature.Exist afterwards
4 DEG C are dialysed 48 hours with PBS solution, and -20 DEG C of packing are preserved.
After there is condensation reaction because of the amino on the aldehyde radical and albumen of gossypol acetate, schiff bases is formed, but because carbon nitrogen is double
Key is unstable, therefore reduces carbon-to-nitrogen double bon for singly-bound further through sodium cyanoborohydride is added, and makes the coupled product can be with stable existence.
Comlete antigen and the small haptens not being coupled are separated by being dialysed 48 hours with PBS solution at 4 DEG C, and by UV absorption
Whether scan method identification comlete antigen is coupled successfully, as shown in figure 1, the characteristic peak of BSA is at 280nm, small molecular protein is anti-
Ying Hou, the ultraviolet figure of conjugate has obvious projection at small molecule characteristic peak 240nm, and it has the characteristic peak of BSA simultaneously,
Therefore judgement is coupled successfully.
2nd, animal immune
The Balb/C mouse of 6~8 week old of health are selected to be immunized.Take gossypol acetate comlete antigen (1mg/mL) with etc.
After amount Freund's adjuvant emulsification is uniform, BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection.First immunisation is using not
Family name's Freund's complete adjuvant, booster immunization uses incomplete Freund's adjuvant, and immunizing dose is the one of a preceding immunizing dose when spurt is immune
Half, intraperitoneal injection is directly carried out after being well mixed with physiological saline；Each time immunization interval is 21 days.After third time is immune, interval
Blood sampling detection serum titer and suppression in one week；Selection suppresses best mouse, and spurt in 21 days is immune after exempting from five, and assistant is not used
Agent, intraperitoneal injection prepares fusion.
3rd, cell fusion
After immune three days of impact, according to conventional PEG (polyethylene glycol, molecular weight is that 4000) method carries out cell fusion,
Comprise the following steps that：(1) it is aseptic to take mouse spleen, grind and obtain splenocyte suspension by 200 mesh cell screen clothes, and carry out thin
Born of the same parents count；(2) SP2/0 cells are collected, is suspended in RPMI-1640 basic culture solutions, carry out cell count；(3) by splenocyte
With SP2/0 cells according to 1：10 ratio mixing, is merged, time 1min after centrifugation with 50%PEG, afterwards according to from slowly to fast,
RPMI-1640 basic culture solutions are added, is suspended in after centrifugation containing 20% hyclone, the RPMI-1640 of 2% 50 × HAT is sieved
Select in nutrient solution, be added to 96 porocyte culture plates, be placed in 37 DEG C, 5%CO2Incubator in cultivate.4th, cell screening and cell
Strain is set up
Liquid is partly changed in the RPMI-1640 screening and culturing liquid that carried out to fused cell for the 3rd day of cell fusion, is used within the 5th day
Containing 20% hyclone, the RPMI-1640 transition nutrient solutions of 1% 100 × HT are changed liquid entirely, and cell conditioned medium was taken at the 7th day
Screened.
Screening is in two steps：The first step first filters out positive cell hole with indirect ELISA, and second step is standard from gossypol
Product, inhibition measure is carried out with indirect competitive ELISA to positive cell.The concrete operations of indirect competitive ELISA method are as follows：
(1) wrapper sheet：Envelope antigen is diluted into 1000 times of addition ELISA Plates with coating buffer solution (CBS), per the μ l of hole 100,37
DEG C be incubated 2h；
(2) board-washing：ELISA Plate is washed with cleaning solution (PBST) 3 times, each 3min, blotting paper is patted dry；
(3) close：PBSs of the 200 μ l containing 0.2% gelatin, 37 DEG C of incubation 2h are added per hole；
(4) board-washing：With (2)；
(5) gossypol standard liquid and cell conditioned medium are added；The gossypol standard liquid that concentration is 100 μ g/ml is added per hole, is put down
Row sets positive control, negative control；
(6) board-washing：With (2)；
(7) ELIAS secondary antibody is added：100 μ l are added per hole through 1:Horseradish peroxidase-the goat-anti of 3000 times of PBST dilutions is small
Mouse IgG, 37 DEG C of incubation 0.5h；
(8) board-washing：With (2)；
(9) develop the color：100 μ l TMB nitrite ions, 37 DEG C of incubation 0.5h are added per hole；
(10) terminate：The sulfuric acid solution of 50 μ l 2mol/L is added per hole；
(11) absorbance measurement：Each hole light absorption value at 450nm wavelength is determined with ELIASA；
The positive hole part occurred in experimental result can have preferably suppression to free gossypol, and another part is to the cotton that dissociates
Phenol does not suppress or inhibition is poor, and selection has the cell hole of preferable suppression to gossypol, and Ya Ke is carried out using limiting dilution assay
It is grand, detected with same method.In triplicate, cell line 2D4 is obtained.5th, the preparation of monoclonal antibody and identification
Take 8-10 week old BALB/c mouses, every mouse peritoneal injection paraffin oil 1mL；Every mouse peritoneal injection 1 after 7 days
×106Hybridoma, collected ascites since the 7th day, and ascites is purified by caprylic acid-ammonium, and the monoclonal antibody of acquisition is put
In -20 DEG C of preservations.
The hypotype for determining monoclonal antibody is set with ELIAS secondary antibody using mouse monoclonal Ig classes/subgroup identification, can from table 1
To find out：Its hypotype is IgG2a types.
The hypotype identification of the hybridoma cell strain 2D4 monoclonal antibodies of table 1
By indirect competitive ELISA, monoclonal antibody is drawn to free gossypol standard suppression curve (such as Fig. 2), by mark
Directrix curve, it is 100ug/kg that can calculate the monoclonal antibody to the IC50 of free gossypol, wherein, indirect competitive ELISA method sensitivity
It is 0.03ug/kg, disclosure satisfy that the measure of free gossypol in cottonseed and its product.
Specification Curve of Increasing process is as follows：
(1) wrapper sheet：Envelope antigen is diluted into 1000 times of addition ELISA Plates with coating buffer solution (CBS), per the μ l of hole 100,37
DEG C be incubated 2h；
(2) board-washing：ELISA Plate is washed with cleaning solution (PBST) 3 times, each 3min, blotting paper is patted dry；
(3) close：PBSs of the 200 μ l containing 0.2% gelatin, 37 DEG C of incubation 2h are added per hole；
(4) board-washing：With (2)；
(5) gossypol standard liquid and antibody are added；0 is respectively configured with the phosphate buffer (PBS) containing 30% methyl alcohol,
The free gossypol standard liquid of 0.01,0.02,0.05,0.1,0.2,0.5,1 μ g/mL.Standard liquid is added separately to
In the ELISA Plate closed, per the μ L of hole 50, the standard liquid of each concentration repeats 3 holes, then 50 μ L 1 are added per hole:16000
The anti-free gossypol monoclonal antibody of dilution, 37 DEG C of incubation half an hour；
(6) board-washing：With (2)；
(7) ELIAS secondary antibody is added：100 μ l are added per hole through 1:Horseradish peroxidase-the goat-anti of 3000 times of PBST dilutions is small
Mouse IgG, 37 DEG C of incubation 0.5h；
(8) board-washing：With (2)；
(9) develop the color：100 μ l TMB nitrite ions, 37 DEG C of incubation 0.5h are added per hole；
(10) terminate：The sulfuric acid solution of 50 μ l 2mol/L is added per hole；
(11) absorbance measurement：Each hole light absorption value at 450nm wavelength is determined with ELIASA；
(12) Specification Curve of Increasing：Standard suppression curve is drawn with origin 8.5.
The anti-free gossypol monoclonal antibody specific application of embodiment 2
1st, the configuration of solution：
Carbonate buffer solution (CBS)：Weigh Na2CO31.59g, NaHCO32.93g, mixes after a small amount of distilled water is dissolved in respectively
Close, plus distilled water is mixed to about 800mL, adjusts pH value to 9.6, plus distilled water to be settled to 1000mL, 4 DEG C of storages are standby；
Phosphate buffer (PBS)：8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9gNa2HPO4·12H2O, it is molten
In 800mL pure water, adjust pH to 7.2~7.4 with NaOH or HCl, be settled to 1000mL；
PBST：PBS containing 0.05% polysorbas20；
TMB nitrite ions：A liquid：Na2HPO4 12H2O 18.43g, citric acid 9.33g, pure water is settled to 1000mL；B liquid：
60mg TMB are dissolved in 100mL ethylene glycol.A, B liquid press 1：5 mixing are TMB nitrite ions, are now mixed with existing；
Isopropanol-n-hexane mixed solvent：6:4(V/V)；
Solvent orange 2 A：Measure that about 500m L isopropyls are liquor-saturated, n-hexane mixed solvent, 2mL 3- amino -1- propyl alcohol, 8m L glacial acetic acids
With 50mL water in the volumetric flask of 1000mL, then scale is settled to isopropanol-n-hexane mixed solvent.
2nd, hybridoma cell strain 2D4 is applied into free gossypol ELISA by monoclonal antibody prepared by internal ascites to add
Add-back acceptance test：
(1) the 1.5 μ g/mLAFB1-BSA that carbonate buffer solution (CBS) has diluted are used as the coating hole enzyme mark of primordial covering 96
Plate, per the μ L of hole 100, after 37 DEG C of coating 2h, with PBST washing lotions board-washing three times, every time per hole 250 μ L, each 3min, pats dry；
(2) closed with the CBS containing 0.01% gelatin, per hole 200 μ L, 37 DEG C of closing 2h, with PBST washing lotions board-washing three
It is secondary, every time per hole 250 μ L, each 3min, pat dry；
(3) 0,0.01,0.02,0.05,0.1,0.2 is respectively configured with the phosphate buffer (PBS) containing 30% methyl alcohol,
The free gossypol B1 standard liquids of 0.5,1 μ g/L.By standard liquid and detected sample extract solution, it is added separately to seal
In the ELISA Plate for closing, per the μ L of hole 50, each sample repeats 3 holes, then 50 μ L 1 are added per hole：The anti-of 16000 dilutions dissociates
Gossypol monoclonal antibody, after 37 DEG C of reaction half an hour, board-washing is patted dry；
(4) PBSs 1 of the 100 μ L containing 0.01% gelatin is added per hole：The sheep anti-mouse igg two of the HRP marks of 3000 dilutions
Anti-, after 37 DEG C of reaction half an hour, board-washing is patted dry.100 μ L TMB nitrite ions are added per hole, after 37 DEG C of colour developing 15min, is added per hole
50μL 2M H2SO4Terminate liquid, 450nm surveys light absorption value；
(5) addition is reclaimed and sample pre-treatments：The cottonseed sample for weighing 5g crushing is inserted in 250mL conical flask with stopper, point
Not Tian Jia 10ng, 50ng and 100ng free gossypol, add 20 beades, 50mL solvent orange 2 As are accurately added with pipette, stopper
Bottle stopper, is put into vibration 1h (120 times per minute or so) in oscillator.The filtering of pier filter paper is determined with dry, during filtering on funnel
Add a cover a watch crystal to reduce solvent volatilization, discard initial a few drop filtrates, collection filtrate is in 100mL tool plug conical flasks.Filter
After liquid dilutes 4 times with the PBS containing 0.01% gelatin, as ELISA sample extracting solutions, it is added using indirect competitive ELISA
Recovery test, its rate of recovery is respectively 72%, 85%, 93%.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not right
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all of implementation method cannot be exhaustive here, it is every to belong to this hair
Obvious change that bright technical scheme is extended out changes row still in protection scope of the present invention.
Claims (4)
1. the hybridoma cell strain of the general monoclonal antibody of anti-free gossypol, it is characterised in that：Its biological deposits numbering is CGMCC
No.10872。
2. the general monoclonal antibody of anti-free gossypol, it is characterised in that：Produced as secreted by the hybridoma described in claim 1
It is raw.
3. the monoclonal antibody described in the hybridoma cell strain or claim 2 described in claim 1 is in detection cottonseed and its system
The application in free gossypol in product.
4. the monoclonal antibody described in the hybridoma cell strain or claim 2 described in claim 1 is being prepared for detecting cotton
Application in seed and its product in the medicament or kit of free gossypol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111912988A (en) * | 2020-07-17 | 2020-11-10 | 国家粮食和物资储备局科学研究院 | Double-signal colloidal gold immunochromatographic test strip for detecting free gossypol as well as preparation method and application thereof |
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| CN105567645A (en) * | 2016-01-28 | 2016-05-11 | 江南大学 | Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof |
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2017
- 2017-01-18 CN CN201710033126.2A patent/CN106916790A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567645A (en) * | 2016-01-28 | 2016-05-11 | 江南大学 | Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| WANG X等: "Monoclonal Antibodies for the Analysis of Gossypol in Cottonseed Products", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 刘萍等: "单克隆抗体研究进展", 《中国畜牧兽医》 * |
| 张积仁等: "醋酸棉酚对人宫颈癌细胞增殖的影响及其机制初探", 《解放军医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111912988A (en) * | 2020-07-17 | 2020-11-10 | 国家粮食和物资储备局科学研究院 | Double-signal colloidal gold immunochromatographic test strip for detecting free gossypol as well as preparation method and application thereof |
| CN111912988B (en) * | 2020-07-17 | 2022-07-15 | 国家粮食和物资储备局科学研究院 | Double-signal colloidal gold immunochromatographic test strip for detecting free gossypol as well as preparation method and application thereof |
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