金黄色葡萄球菌肠毒素A检测方法的建立及其应用（英文）
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摘要
目的建立一种敏感、特异、便捷的方法,用于检测食源性金黄色葡萄球菌肠毒素A(SEA)。方法采用经典的细胞融合技术制备抗SEA的单克隆抗体,亲和层析纯化抗体蛋白,ELISA方法测定抗体滴度,免疫层析分析抗体亚型。以抗SEA单抗为一抗,建立Western印迹法,检测不同食物样品中的SEA。结果筛选出3株抗SEA的单抗,命名为SEA-7,SEA-18和SEA-86。纯化后的3株单抗纯度均>90%,抗体滴度均在1∶32 000,经鉴定均为IgG1亚型。利用3株单抗建立检测SEA的Western印迹法,SEA-7的检测灵敏度最高,可达1.56 ng。以SEA-7为一抗,可检测出不同食物中污染的SEA,如污染了1.56 ng SEA的水,污染了5 ng SEA的粥或火腿肠匀浆。结论建立的检测SEA方法可靠灵敏,可用于监测食源性金黄色葡萄球菌污染。
OBJECTIVE To establish a sensitive, specific and easy-to-use method to detect staphylococcal enterotoxin A(SEA) in the food poisoning. METHODS Anti-SEA monoclonal antibodies(McA bs)were established using mouse hybridoma method. The McA bs were purified by immunoaffinity chromatography and confirmed by SDS-PAGE. The titers of the McA bs were detected by ELISA. Their isotypes were analyzed by immunochromatography strips. With the McAbs as the primary antibody, the SEAWestern blotting was developed. SEA from a variety of food samples was also successfully assayed by this protocol. RESULTS Three hybridoma cell lines secreting McAbs against SEA were established.Three McA bs(SEA-7, SEA-18 and SEA-86) were purified and confirmed. The purity of the McA bs was above 90%, and the titer of McAbs was above 1∶32 000. Their isotypes were identified and the results showed they all belonged to IgG1 subclass. McAb SEA-7 showed the highest sensitivity when used to detect SEA by Western blotting. The detection limit was 1.56 ng of SEA. SEA from a variety of food samples was also successfully assayed by Western blotting using McAb SEA-7 as the primary antibody, such as water spiked with 1.56 ng SEA, ham or porridge spiked with 5 ng SEA. CONCLUSION A reliable and sensitive method to detect SEA is established, which can be used in the detection of food staphylococcal pollution.
OBJECTIVE To establish a sensitive, specific and easy-to-use method to detect staphylococcal enterotoxin A(SEA) in the food poisoning. METHODS Anti-SEA monoclonal antibodies(McA bs)were established using mouse hybridoma method. The McA bs were purified by immunoaffinity chromatography and confirmed by SDS-PAGE. The titers of the McA bs were detected by ELISA. Their isotypes were analyzed by immunochromatography strips. With the McAbs as the primary antibody, the SEAWestern blotting was developed. SEA from a variety of food samples was also successfully assayed by this protocol. RESULTS Three hybridoma cell lines secreting McAbs against SEA were established.Three McA bs(SEA-7, SEA-18 and SEA-86) were purified and confirmed. The purity of the McA bs was above 90%, and the titer of McAbs was above 1∶32 000. Their isotypes were identified and the results showed they all belonged to IgG1 subclass. McAb SEA-7 showed the highest sensitivity when used to detect SEA by Western blotting. The detection limit was 1.56 ng of SEA. SEA from a variety of food samples was also successfully assayed by Western blotting using McAb SEA-7 as the primary antibody, such as water spiked with 1.56 ng SEA, ham or porridge spiked with 5 ng SEA. CONCLUSION A reliable and sensitive method to detect SEA is established, which can be used in the detection of food staphylococcal pollution.
引文
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[6]Park CE,Akhtar M,Rayman MK.Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit(TECRA)[J].Appl Environ Microbiol,1993,59(7):2210-2213.
[7]Park CE,Warburton D,Laffey PJ.A collaborative study on the detection of staphylococcal enterotoxins in foods by an enzyme immunoassay kit(RIDAS-CREEN)[J].Int J Food Microbiol,1996,29(2-3):281-295.
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[10]Sospedra I,Soler C,Ma?es J,Soriano JM.Analysis of staphylococcal enterotoxin A in milk by matrixassisted laser desorption/ionization-time of flight mass spectrometry[J].Anal Bioanal Chem,2011,400(5):1525-1531.
[11]Zhang Y,Wang Y,Cai R,Shi L,Li C,Yan H.Prevalence of enterotoxin genes in Staphylococcus aureus isolates from pork production[J].Foodborne Pathog Dis,2018,15(7):437-443.
[12]Atanassova V,Meindl A,Ring C.Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham-a comparison of classical culturing detection and RFLP-PCR[J].Int J Food Microbiol,2001,68(1-2):105-113.
[13]Rasooly A,Rasooly RS.Detection and analysis of staphylococcal enterotoxin a in food by Western immunoblotting[J].Int J Food Microbiol,1998,41(3):205-212.
[14]Ahmadvand D,Rahbarizadeh F,Vishteh VK.Highexpression of monoclonal nanobodies used in the preparation of HRP-conjugated second antibody[J].Hybridoma(Larchmt),2008,27(4):269-276.
[15]Chen D,Song Q,Xu Z,Zhang D.Characterization of enterotoxin A-producing Staphylococcus aureus[J].Infect Drug Resist,2018,11:531-538.
[16]Sedighian H,Halabian R,Amani J,Heiat M,Taheri RA,Imani Fooladi AA.Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA[J].Anal Biochem,2018,548:69-77.
[17]Singh M,Agrawal RK,Singh BR,Mendiratta SK,Agarwal RK,Singh MK,et al.Development and evaluation of simple dot-blot assays for rapid detection of staphylococcal enterotoxin-A in food[J].Indian J Microbiol,2017,57(4):507-511.
[18]Ben Haddada M,Hu D,Salmain M,Zhang L,Peng C,Wang Y,et al.Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A(SEA)detection[J].Anal Bioanal Chem,2017,409(26):6227-6234.
[19]Nouri A,Ahari H,Shahbazzadeh D.Designing a direct ELISA kit for the detection of Staphylococcus aureus enterotoxin A in raw milk samples[J].Int JBiol Macromol,2018,107(Pt B):1732-1737.
[2]Kroning IS,Iglesias MA,Mendon?a KS,Lopes GV,Silva WP.Presence of classical enterotoxin genes,agr typing,antimicrobial resistance,and genetic diversity of Staphylococcus aureus from milk of cows with mastitis in southern Brazil[J].JFood Prot,2018,81(5):738-742.
[3]Ercoli L,Gallina S,Nia Y,Auvray F,Primavilla S,Guidi F,et al.Investigation of a staphylococcal food poisoning outbreak from a chantilly cream dessert,in Umbria(Italy)[J].Foodborne Pathog Dis,2017,14(7):407-413.
[4]Chiang YC,Liao WW,Fan CM,Pai WY,Chiou CS,Tsen HY.PCR detection of staphylococcal enterotoxins(SEs)N,O,P,Q,R,U,and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan[J].Int J Food Microbiol,2008,121(1):66-73.
[5]Huang LM,Hu LX,Yu H,Chen SC,Huang CP,Liu H.Field epidemiological investigation on a foodborne outbreak caused by Staphylococcus aureus enterotoxin,in Hangzhou,2014[J].Chin J Epidemiol(中华流行病学杂志),2017,38(12):1642-1644.
[6]Park CE,Akhtar M,Rayman MK.Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit(TECRA)[J].Appl Environ Microbiol,1993,59(7):2210-2213.
[7]Park CE,Warburton D,Laffey PJ.A collaborative study on the detection of staphylococcal enterotoxins in foods by an enzyme immunoassay kit(RIDAS-CREEN)[J].Int J Food Microbiol,1996,29(2-3):281-295.
[8]Tsai WC,Li IC.SPR-Based immunosensor for determining staphylococcal enterotoxin A[J].Sensors Actuators B,2009,136:8-12.
[9]Salmain M,Ghasemi M,Boujday S,Spadavecchia J,Técher C,Val F,et al.Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A(SEA)at the ng level[J].Biosens Bioelectron,2011,29(1):140-144.
[10]Sospedra I,Soler C,Ma?es J,Soriano JM.Analysis of staphylococcal enterotoxin A in milk by matrixassisted laser desorption/ionization-time of flight mass spectrometry[J].Anal Bioanal Chem,2011,400(5):1525-1531.
[11]Zhang Y,Wang Y,Cai R,Shi L,Li C,Yan H.Prevalence of enterotoxin genes in Staphylococcus aureus isolates from pork production[J].Foodborne Pathog Dis,2018,15(7):437-443.
[12]Atanassova V,Meindl A,Ring C.Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham-a comparison of classical culturing detection and RFLP-PCR[J].Int J Food Microbiol,2001,68(1-2):105-113.
[13]Rasooly A,Rasooly RS.Detection and analysis of staphylococcal enterotoxin a in food by Western immunoblotting[J].Int J Food Microbiol,1998,41(3):205-212.
[14]Ahmadvand D,Rahbarizadeh F,Vishteh VK.Highexpression of monoclonal nanobodies used in the preparation of HRP-conjugated second antibody[J].Hybridoma(Larchmt),2008,27(4):269-276.
[15]Chen D,Song Q,Xu Z,Zhang D.Characterization of enterotoxin A-producing Staphylococcus aureus[J].Infect Drug Resist,2018,11:531-538.
[16]Sedighian H,Halabian R,Amani J,Heiat M,Taheri RA,Imani Fooladi AA.Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA[J].Anal Biochem,2018,548:69-77.
[17]Singh M,Agrawal RK,Singh BR,Mendiratta SK,Agarwal RK,Singh MK,et al.Development and evaluation of simple dot-blot assays for rapid detection of staphylococcal enterotoxin-A in food[J].Indian J Microbiol,2017,57(4):507-511.
[18]Ben Haddada M,Hu D,Salmain M,Zhang L,Peng C,Wang Y,et al.Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A(SEA)detection[J].Anal Bioanal Chem,2017,409(26):6227-6234.
[19]Nouri A,Ahari H,Shahbazzadeh D.Designing a direct ELISA kit for the detection of Staphylococcus aureus enterotoxin A in raw milk samples[J].Int JBiol Macromol,2018,107(Pt B):1732-1737.