1、Biochemical Engineering Journal 43(2009)5863Contents lists available at ScienceDirectBiochemical Engineering Journaljournal homepage: highly sensitive enzyme-linked immunosorbent assay for quantificationof adipocytokines secreted by mouse adipocytesMinori Mimura1,Risako Nabeshima,Miwako Maeda,Naofum
2、i ShiomiDepartment of Biosphere Sciences,Kobe College,4-1 Okadayama,Nishinomiya 662-8050,Japana r t i c l ei n f oArticle history:Received 3 June 2008Received in revised form 24 July 2008Accepted 15 August 2008Keywords:ELISATNFLeptinAdipocytesa b s t r a c tThe mouse preadipocyte 3T3-L1 line is the
3、most useful cell line for the study of adipocytes.Adipocytessecrete adipocytokines,and abnormal adipocytokine production can cause the metabolic syndrome.Although it is important to understand the characteristics of mouse adipocytokine secretion,it is dif-ficult to quantify these products because th
4、ey are produced in low concentrations.Here,we developed ahighly sensitive enzyme-linked immunosorbent assay(ELISA)for detecting the concentrations of mouseadipocytokines,such as TNF?and leptin.In this method,the antigen was sandwiched by using goat-and rabbit-derived polyclonal antibodies,and the fl
5、uorescence intensity produced in the reaction with4-methylumbelliferyl-?-galacto-sidase pyranosidase(MUG)was measured.TNF?and leptin could bemeasured at concentrations as low as approximately 1pg/ml.By using our ELISA method,we alsomeasured the concentrations of TNF?and leptin in mouse 3T3-L1 preadi
6、pocytes and adipocytes.The dif-ferentiation of preadipocytes into adipocytes enhanced TNF?production and secretion and reduced theleptin production.The amount of TNF?produced in the adipocytes was 3.0ng/mg-protein;this amountwas considerably higher than that of leptin.2008 Elsevier B.V.All rights re
7、served.1.IntroductionThe metabolic syndrome,a diseases manifested in someobesity-induced conditions,has been remarked at a standpoint ofpreservation of a lifestyle-related disease such as diabetes.Whiteadipose tissue(WAT)was initially considered to function merelyas an energy-storage tissue that acc
8、umulates triglycerides.How-ever,since the discovery of adipocytokines in adipocyte producedby adipocytes in the 1990s,WAT has been recognized to play anadditionalimportantroleasanendocrinetissue1.Abnormalpro-duction of the adipocytokines,such as tumor necrosis factor alpha(TNF?),leptin and adiponect
9、in,accompanied by the accumulationof lipids in adipocytes,leads to insulin resistance and a decrease intheglucoseconsumption;theseunfavorableconditionsmayinducethe metabolic syndrome 2.The regulation of adipocytokine production is a key factorin the management of obesity-related diseases.Leptin is o
10、neof the adipocytokines and inhibits an appetite by working to aneuron 3,4.The administration of leptin has been shown topositively affect insulin resistance in patients with lipoatrophicCorresponding author.Tel.:+81 798 51 8421;fax:+81 798 51 8421.E-mail address:n-shiomimail.kobe-c.ac.jp(N.Shiomi).
11、1Present address:Oriental Yeast Co.Ltd.,Japan.diabetes;however,adequate improvement in the condition is notachieved solely by leptin treatment 5.The relationship betweenadiponectin,which is produced in large amounts in adipocytes7,and insulin resistance 68 was also investigated in detail.The normal
12、adiponectin concentration in the blood is 510?g/ml;however,thisconcentrationdecreasesinpatientswithtype2glyco-genesis and ischemic heart disease.Effective treatment strategieshave been described for increasing the blood adiponectin con-centration in these patients 9.TNF?causes insulin resistanceby i
13、nhibiting the activity of tyrosine kinasean insulin acceptor10.The level of macrophage migration inhibitory factor(MIF),whichisalsoassociatedwithinsulinresistance,increasewithTNF?levels 11.Activated macrophages in WAT secrete TNF?and arethus involved in the development of insulin resistance 12.Thus,
14、insulin resistance is considered to be caused by the induction ofTNF?and the suppression of leptin and adiponection in WAT.Themechanism underlying insulin resistance,however,is complicated,and the involvement of cross-talk among cells such as adipocytes,macrophages and liver cells should be investig
15、ated in this regard.Aquantitative and kinetic analysis of the adipocytokines is requiredfor elucidating this complicated mechanism and for preventing themetabolic syndrome.The mouse preadipocyte line 3T3-L1,which was originally gen-erated from the Swiss albino mouse fibroblast cell line 3T3 byGreen
16、and Kehinde 13,is one of the most useful cell lines1369-703X/$see front matter 2008 Elsevier B.V.All rights reserved.doi:10.1016/j.bej.2008.08.008M.Mimura et al./Biochemical Engineering Journal 43(2009)586359Fig.1.Schemes of ELISAs performed using ABTS and MUG.for investing the characteristics of ad
17、ipocytes and their secretedadipocytokines.This is because these preadipocytes can easilybe induced to differentiate into adipocytes 14.Some previ-ous studies have used the reverse transcription-polymerase chainreaction(RT-PCR)and western blotting methods to investigatemRNA and protein expression of
18、adipocytokines 1517.How-ever,since all adipocytokines,except adiponectin,are producedin very low concentration in mouse cells and since it is diffi-cult to obtain monoclonal antibodies from these cells,very fewquantitative studies have been conducted for TNF?and leptinusing the enzyme-linked immunos
19、orbent assay(ELISA)method.Thus,in this study,we developed a highly sensitive sandwichELISA technique involving the use of 4-methylumvelliferyl-?-d-galactopyranosidase(MUG)and polyclonal antibodies,which areless expensive than monoclonal ones.TNF?and leptin concen-trations as low as approximately 1pg
20、/ml could be detected usingthis method:thus,our method was sufficiently efficient for detect-ing the low concentration of these factors in 3T3-L1 cells.Inaddition,we investigated production and secretion characteris-tics of TNF?and leptin in adipocytes using our novel ELISAmethod.2.Materials and met
21、hods2.1.Chemicals used for ELISA,strains and mediaRabbit and goat anti-mouse TNF?polyclonal antibodies(Cat.No.AB410-NA,R&D Systems Inc.and Cat.No.P-350,PieceBiotechnology Inc.,Rockford,USA),respectively,rabbit and goatanti-mouse leptin polyclonal antibodies(Cat.No.AF498,R&DSystems Inc.and Cat.No.500
22、-P68,Perro Tech EC,London,UK),and horseradish peroxide(HRP)-conjugated and?-galactosidase-conjugatedgoatanti-rabbitimmunoglobulin(IgG;Cat.No.A102GT,American Qualex Manufacturers,California,USA)were used asantibodies in the ELISA.Mouse TNF?(Cat.No.3410,TecheneTechnol.Corp.,Minneapolis,USA)and leptin(
23、Cat.No.429700,Calbiochem-Novabiochem Corp.,California,USA),were used as theantigen.2,2-Azino-di(3-ethylbenzthiazoline)sulfuric acid(ABTS;Zymed Laboratories Inc.,CA,USA)and 4-methylumvelliferyl-?-d-galactopyranosidase(MUG)were used as the substrates.Casein(Hammerstein grade)and Block One(Nacalai Tesq
24、ue Inc.,Kyoto,Japan)were used for blocking.Cells of preadipocyte line 3T3-L1 were cultured in Dulbeccosmodified Eagles medium(DMEM)containing sodium bicarbon-ate(MP Biomedicals Inc.,Illkirch,France).The medium wasenriched with human insulin,dexamethasone and 3-isobutyl-1-methylxanthine(IBMX)to facil
25、itate cell differentiation.Fetalbovine serum(FBS)was used after treatment at 56C for 30min.2.2.ELISA methodFig.1 shows a schematic representation of the ELISA methodused in our study.A sandwich ELISA assay was carried out usingABTS as the substrate.First,100?l of the goat anti-mouse TNF?polyclonal a
26、ntibody(10?g/ml)dissolved in phosphate-bufferedsaline(PBS)was added into each well of a 96-well flat-bottomELISA plate(Coster 3590;Data Packing Co.,MA,USA),and the platewas incubated at 4C for 12h to enable adsorption.The plate waswashed four times with PBS following which 400?l of 5%caseinsolution
27、dissolved in PBS was added into the wells for blocking andthe plate incubated for 2h.Further,100?l of sample solution con-tainingTNF?wasaddedintothewells,andtheplatewasincubatedfor 1h and subsequently washed six times with PBS.Next,100?l ofthe rabbit anti-TNF?polyclonal antibody,diluted to 4?g/ml wi
28、tha blocking solution(Block One:PBS=1:9),was added to the wells,and the plate was incubated for 1h.It was washed six times withPBS,and 100?l of HRP-conjugated goat anti-rabbit IgG(4?g/ml)diluted with the blocking solution was then added into the wells.Theplateswasincubatedfor1handsubsequentlywasheds
29、ixtimeswith PBS.Further,200?l of a reaction buffer(3mg/ml ABTS,50?l;30%H2O2,5?l;250mM citrate buffer(pH 4.0),2.5ml;distilledwater,2.5ml)wasaddedtothewells,andtheabsorbancewasmea-sured at 525nm using a microplate reader(Model 680;Bio-RadLaboratories,Tokyo,Japan).In the ELISA assay for leptin,the goat
30、andrabbitanti-leptinpolyclonalantibodiesdilutedto4?g/mlwithblocking solution were used.The next ELISA involving the use of MUG was performed in a96-well flat-bottom FIA plate(Greiner Bio-One 655077;GreinerJapan,Tokyo,Japan).Here,?-galactosidase-conjugated goat anti-rabbit IgG diluted(1:200)with the
31、blocking solution was added tothe wells;the plate was incubated for 1h to permit adsorption andwas subsequently washed six times with PBS.Further,200?l ofreaction buffer(1mM MgCl2,10mM NaCl,100mM sodium phos-phate buffer(pH 7.0),0.1g/l bovine serum albumin(fraction V),60M.Mimura et al./Biochemical E
32、ngineering Journal 43(2009)5863Fig.2.Calibration curves constructed for TNF?and leptin using sandwich ELISAwith ABTS.(?and?)TNF?and leptin dissolved in PBS;(and?)TNF?and leptindissolved in a 1:1 mixture of PBS and DMEM containing 10%FBS.The values ofabsorbance in case of lacking TNF?and leptin(negat
33、ive control),0.10 and 0.11.and 0.1mg/l MUG)was added to the wells and the fluorescenceintensity was measured at an excitation wavelength of 355nm andan emission wavelength 460nm by using a fluorescent microplatereader(Fluoroskan Ascent FL;Dainippon Sumitomo Pharma Co.Ltd.,Osaka,Japan).2.3.TNF and le
34、ptin productions in mouse adipocyteDMEM containing 10%(v/v)FBS,0.5?M IBMX and 4?M dex-amethasone(induction medium)was used to induce adipocytedifferentiation.The 3T3-L1 preadipocytes(2106cells)were cul-tured in 15ml of DMEM containing 10%FBS in a 75cm2flask;theflask was incubated at 37C in the prese
35、nce of CO2.Two cultureflasks were prepared and maintained under two different condi-tions.In the first flask,3T3-L1 preadipocytes were cultured for 6days,and in the second flask,they were cultured for 2 days,fol-lowing which the growth medium was replaced with an inductionmedium,and the cells were c
36、ultured for four additional days.Afterthe culture,the culture broths were discarded from the flask,andthecellsthatadheredtotheflaskwerewashedwithPBScontaining0.02mM of EDTA,harvested and suspended in 0.1ml of PBS.Thesuspended cells were disrupted by using an ultrasonic disrupter(Tomy Seiko Co.Ltd.,T
37、okyo,Japan).To measure the total proteins,10?lofthesolutionwasused,andtheremainingsolutionwasusedto quantify TNF?(or leptin).The protein concentrations were mea-sured using a protein assay kit(Bio-Rad Laboratories).Oil dropletsin the cells were stained red with oil-red stain using an Adipo-genesis A
38、ssay Kit(Chemicon International Inc.,California 92590,USA).2.4.Detection of mRNAs by RT-PCR methodRT-PCR was used to detect the mRNA expression levels of per-oxisome proliferator activated receptor(PPAR)?,CCAT/enhancerbinding protein(C/EBP)?,C/EBP?and C/EBP?.The cells,which hadbeenculturedin8mlofame
39、diumina25-cm2flask,werecollectedin a 2ml of microtube and washed twice with PBS.After the totalmRNAs in the cell pellet were purified by using an RNeasy Lipid Kit,cDNAs were synthesized using Omniscript Reverse Transcriptase.PCR was carried out in the following manner,using a Hot Start PCRkit:50?l o
40、f the reaction mixture containing 0.5?l of Hot Start Taqpolymerase,3?l of cDNA and 8?l each of the forward and reverseprimers at a concentration of 10?M,was subjected to 35 cycles oftreatmentat95Cfor30s,55Cfor30sand72Cfor1min.ThekitsusedforRT-PCRwereobtainedfromQiagenK.K.(Tokyo,Japan).Theprimer sequ
41、ences used have been described previously(1517).Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)mRNA wasusedasastandardsample.TheamplifiedDNAfragmentsofC/EBP?,C/EBP?,C/EBP?and PPAR?were separated by performing agarosegel electrophoresis using a 3%NuSieve 3:1 agarose gel(Takara BioInc.,Shiga,Japan),an
42、d were stained with ethidium bromide solu-tion.3.Results3.1.Sandwich ELISA for TNF and leptin with ABTS as the substrateGoat-and rabbit-derived polyclonal antibodies,which are usedin direct ELISA,were used to identify the antigen,and a combi-nation of HRP and ABTS was used for detection.Fig.2 shows
43、thecalibration curve constructed for various concentrations of TNF?dissolved in PBS,which were measured by ELISA using ABTS asthe substrate.TNF?could be detected in the concentration rangeof 1100ng/ml.To investigate the effect of reaction inhibition bythe serum contained in the medium,TNF?dissolved
44、in DMEMcontaining 10%of FBS,which was diluted with PBS(volume ratio,1:1),was also quantified.We considered that the reaction was notinhibited because the values obtained for TNF?dissolved in DMEMwere almost identical to those obtained for the samples dissolvedinPBS.Fig.2alsoshowsthecalibrationcurvec
45、onstructedforleptin.The detectable concentration range for leptin dissolved in PBS was1001000ng/ml,which was lower than that for TNF?.In case ofFig.3.Enzyme stability in the ELISA reactions.(A)Absorbance measured for TNF?by performing ELISA with ABTS along the time course.(B)Fluorescence intensity s
46、ignalsobtained for TNF?by performing ELISA with MUG along the time course.M.Mimura et al./Biochemical Engineering Journal 43(2009)586361Fig.4.Calibration curves constructed for TNF?and leptin by using sandwich ELISAwith MUG.(?)TNF?and(?)leptin.The values of fluorescence intensity in case oflacking T
47、NF?and leptin(negative control),30 and 200.leptin dissolved in a medium containing FBS,the absorbance val-ues were almost identical.The results suggest that the associationconstants of the anti-leptin antibodies were worse than those ofthe anti-TNF?antibodies.3.2.Highly sensitive measurement by perf
48、orming ELISA withMUGA high-sensitivity method(0.01ng/ml)was required forthe detection of TNF?and leptin produced in mouse 3T3-L1preadipocytes.Antigen concentrations lower than 1ng/ml couldnot be detected during the 30-min of reaction time by using themethod involving ABTS;this was because the measur
49、ed valuediffered marginally from the negative control value(approxi-mately 0.1).Therefore,we attempted to increase this differenceby extending the reaction time.Fig.3A shows the absorbancevalues determined for TNF?over the time course.However,thereaction almost arrested within 2h due to the inactiva
50、tion ofHRP by H2O2,and the difference between the measured valuesand the negative control values remained small.Thus,differ-ent substrate-enzyme combinations were tested to realize highlysensitive measurement system.The results revealed that the com-bination of MUG and?-galactosidase produced optima
