LI Qian,WU Jun-hua,GAO Shan,JIA Pei-yuan,WEI Wen-qing,WANG Yu-xia

(1.The Seventh Medical Center of PLA General Hospital,Beijing 100700,China;2.Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;3.Beijing Center for Disease Prevention and Control,Beijing 100031,China)

Abstract:OBJECTIVE To establish a sensitive,specific and easy-to-use method to detect staphy?lococcal enterotoxin A(SEA)in the food poisoning.METHODS Anti-SEA monoclonal antibodies(McAbs)were established using mouse hybridoma method.The McAbs were purified by immunoaffinity chroma?tography and confirmed by SDS-PAGE.The titers of the McAbs 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 McAbs(SEA-7,SEA-18 and SEA-86)were purified and confirmed.The purity of the McAbs 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 IgG1subclass.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 anti?body,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.

Key words:staphylococcal enterotoxin A;monoclonal antibody;toxin detection

Staphylococcus aureus(S.aureus)is a ubiq?uitous gram-positive coccus and considered to be the second or third most common pathogen that causes outbreaks of food poisoning in many countries[1-3].The symptoms of food poisoning exposed to enterotoxins produced byS.aureusinclude nausea,violentvomiting,abdominal cramps and diarrhea.Staphylococcal enterotoxins(SEs)have been divided into five major serological types(SEA to SEE)[4-5].SEA,a 27 ku monomeric protein,is the most common toxin associated with staphylococcal-borne food poisoning.SEA is heat stable,resistant to gut proteases and stable over a wide range of pH.It is reported that SEA retains both immunological and biological activi?ties after exposure to pasteurization.And even if the bacteria have been sterilized,the biological activity of the toxin remains unchanged[3].

Several methods for detecting SEA have been developed[6-7],such as biosensors[8-9],poly?merase chain reaction(PCR)and matrix-assisted laser desorption/ionization-time of flight mass spec?trometry[10].In recent years,PCR methods were used for identification and quantitative detection of SE gene[11-12].These methods require specific materials and devices,thus too troublesome for routine laboratories.

Western blotting is a routine method for protein analysis.Rasooly Aet al[13]have applied this method to SEA detection,which is reported more reliable and simple for the specific SEA detection among a small number of samples.However,the antibody used by Rasooly Aet alwas a polyclonal antibody,which would have cross-reaction with unrelated antigens in food samples.The purpose of this study is to prepare anti-SEA monoclonal antibodies(McAbs)and estab?lish a Western blotting protocol for detecting SEA in various foods.

1 MATERIALS AND METHODS

1.1 Reagents and main instruments

Purified SEA and SEB(Beijing Institute of Microbiology and Epidemiology).Protein G-Sepha?rose 4 Fast Flow Column(Pharmacia Biotech,Sweden).Horseradish peroxidase(HRP)(Sigma,USA).Bovine serum albumin(BSA)(YHSM Biotech?nology Institutes,Beijing,China).Polyethylene glycol 4000(PEG 4000)(Gibco,UK).Freund′s adjuvant(Sigma,USA).Goat anti-mouse IgG/HRP antibody(Zhongshan Goldenbridge Biotech?nology Co.,Ltd.,Beijing,China).Protein marker(10-170 ku)(Real Time Biotechnology Corpora?tion,Beijing,China).Pro-light HRP chemilumi?nescence detection reagent(Biotech Co.,Ltd,Beijing,China).IsoQuick Kit(Envirologix,Port?land,USA).Ham was purchased from Auchan supermarket.Porridge was obtained from the cafe?teria of Academy of Military Medical Sciences.Mini PROTEAN@Tetra Cell(Bio-Rad Company,USA).Pure Nitrocellulose Blotting Membrane(Pall Corporation,Pensacola,Mexico),DYCP-40C electrophoresis chamber(Beijing Liu Yi Appa?ratus Factory,Beijing,China)and Varioskan Flash(Thermo Scientific,Finland).UV 250 Spec?trophotometer(Shimadzu,Japan).ECL(Applygen Technologies Inc.,Beijing,China).

1.2 Animals

Female BALB/c mice(Beijing Laboratory Animal Research Center).They were housed in a controlled environment 19-21℃,50%-60%of humidity,12 h dark/light cycle with light provided between 6∶00 am and 6∶00 pm.Food and water were givenad libitum.All procedures were followed in accordance with the regulations regarding the"Protection of Animals Used for Experimental and Other Scientific Purposes"from the relevant Direc?tives of GLP.The study protocols were approved by the Ethical Committee of the Seventh Medical Center of PLA General Hospital.

1.3 Preparation of monoclonal antibodies

Five female BALB/c mice(6 weeks old)were immunized subcutaneously with 100 μg/mouse of SEA emulsified in complete Freund′s adjuvant.At 14 d intervals,the mice received three booster immunizations in incomplete Freund′s adjuvant of 100 μg/mouse of SEA.Three days before fusion,mice were boosted intraperitoneally with 100 μg/mouse SEA without adjuvant.Spleen cells from immunized mice were fused with the hypoxanthine-guanine-phosphoribosyltransferasedeficient mouse myeloma cell line(SP2/0)using 50%polyethylene glycol 4000(PEG 4000).Super?natants from hybridoma cultures were screened for anti-SEA McAbs by ELISA.The positive hybrids were cloned and subcloned by limiting dilution.

1.4 Preparation of mouse ascites and purifi?cation of antibodies

Female BALB/c mice were injected intraperi?toneally with hybridoma cells(3×106in 0.5 mL of sterilized 0.9%NaCl)after the mice had been sensitized with liquid paraffin.Ascites fluid was collected and centrifugated at 12 000×gfor 10 min.The supernatant was diluted with 0.02 mol·L-1sodium phosphate buffer(pH 7.0)and loaded on a protein G-Sepharose 4 Fast Flow Column.The antibody bond on the sepharose was eluted with glycine-HCl 0.1 mol·L-1,pH 2.7 and immediately neutralized to pH 7.4 with Tris-HCl(pH 9.0)1 mol·L-1.The eluted antibody was dialyzed against 0.02 mol·L-1sodium phosphate buffer(pH 7.4).The concentration of purified McAbs was calculated from absorbance at 280 nm and 260 nm.The purity of antibody was analyzed by SDS-PAGE with Coomassie blue staining.

1.5 Labeling of anti-SEA McAb by HRP

The purified anti-SEA antibodies were labeled with HRP according to the reported method[14].ThetitersofHRP labeledanti-SEA McAbs(anti-SEA McAbsHRP)were detected by ELISA.

1.6 Determination of the subclass of anti-SEA McAbs

The subclasses of purified McAbs were detected using IsoQuick Strips.

1.7 Analysis of SEA by Western blotting

SEA stock solution(1 g·L-1)was diluted to various concentrations(5.0,2.5,1.25,0.625,0.312,0.156 and 0 μg·L-1)with deionized water.The diluted SEA solution was mixed with an equal volume of 2 × loading buffer(Tris-HCl 50 mmol·L-1,pH 6.8,DTT 50 mmol·L-1,2%SDS,0.1%bromo?phenol blue and 10% glycerol).After being boiled,the supernatant was loaded onto 12%SDS-PAGE and blotted onto nitrocellulose membrane with transfer buffer(Tris 25 mmol·L-1,glycine 190 mmol·L-1,20%MeOH and 0.05%SDS,pH 8.3)through a semi-dry transfer cell.The membrane was blocked with 5%skim milk in TBST(Tris 10 mmol·L-1,NaCl 100 mmol·L-1and 0.1%Tween 20,pH 7.5)for 1 h and incubated with each kind of anti-SEA McAbs diluted 1∶1000(V/V)in TBST at 37℃for 1 h.Then,the membrane was washed,incubated with HRP conjugated goat anti-mouse IgG antibody diluted 1∶1000(V/V)in TBST at 37℃for 1 h.The membrane blotted with SEA could be incubated with each anti-SEA McAbHRPdirectly after being blocked.After being washed with TBST for 6 times,the membrane was detected by ECL.

1.8 Analysis of SEA spiked in food samples by Western blotting

Ham was homogenized with a 10-fold amount of deionized water(W/V)while porridge was homogenized with a two-fold amount of deion?ized water(W/V).Then,the homogenate was centrifugated at 1400×gfor 10 min.The superna?tants were added with SEA to the final concentra?tion of 0.5 mg·L-1.SEA 0.5 mg·L-1in water was used as positive control and the food samples free of SEA as negative control.After being boiled with loading buffer(Tris-HCl 50 mmol·L-1pH 6.8,DTT 50 mmol·L-1,2%SDS,0.1%bromophenol blue and 10%glycerol),the samples were loaded onto a 12%SDS-PAGE gel and then blotted onto nitrocellulose membrane.Following the proce?dure of Western blotting,McAb SEA-7 diluted 1∶1000(V/V)in TBST was added to recognize SEA,and HRP conjugated goat anti-mouse IgG antibody was used to bind with McAb SEA-7.

1.9 Statistical analysis

The statistical analysis was performed with SPSS 13.0 software.The data was expressed asx±s,all experiments was performed in quadru?plicate.

2 RESULTS

2.1 Production of mouse anti-SEA McAbs

Three strains of hybridoma cells(named SEA-7,SEA-18 and SEA-86),which could stably secrete anti-SEA McAbs,were ip injected into BALB/c mice sensitized with liquid paraffin to prepare ascites.Three anti-SEA McAbs were successfully purified by Protein G affinity chromatography.The concentrations of purified antibodies,calculated from its absorbance at 280 and 260 nm,were 4.42,3.28 and 2.15 g·L-1for SEA-7,SEA-18 and SEA-86,respectively.Three McAbs were analyzed by SDS-PAGE with Coomassie blue staining(Fig.1).The result of SDS-PAGE showed that the three purified McAbs had high purities(above 90%)and two bands,representing heavy chains(50 ku)and light chains(25 ku)of antibody,respectively(Fig.1).

2.2 The titer of anti-SEA McAbs

Purified McAbs were diluted with PBST and their titers were determined by ELISA.The results indicated that three McAbs could specifically recog?nize SEA and had high titers(at least 1∶32 000).When the plates were coated with SEB,very low absorbance values were obtained(Fig.2).

2.3 Subclass of anti-SEA McAbs

Following the instruction of IsoQuick Strips,three diluted McAbs were analyzed.The results showed that all the three McAbs developed an IgG1pink line(Fig.3A),while on IgA/IgM strip only appeared the control line(Fig.3B).The results demonstrated that anti-SEA-7,anti-SEA-18 and anti-SEA-86 McAbs all belonged to IgG1subclass.

Fig.2 Titers of anti-SEA McAbs determined by ELISA.Antibodies were serially diluted with PBST(from 1∶250 to 1∶32 000 by two-fold serial dilution).The 96 well microtiter plates were respectively coated with SEA and SEB at the concentra?tion of 3 mg·L-1,100 μL/well.±s,n=4.

2.4 Titer of anti-SEA McAbsHRP

The titers of McAbs(SEA-7HRP,SEA-18HRP,SEA-86HRP)were determined by ELISA.To deter?mine the specificity of the antibodies,SEA and SEB were respectively coated on the 96 well microtiter plate and their reactivity with three McAbs was determined by ELISA.The results indicated that the titers of McAbs SEA-7HRP,SEA-18HRPand SEA-86HRPwere 1∶32 000(Fig.4).No positive signals were obtained when the plate was coated with SEB,demonstrating that three McAbsHRPcould still recog?nize SEA specifically.

Fig.3 Subclass of anti-SEA McAbs.IsoQuick Strips were used to determine the subclass of antibody.M：marker.SEA-7,SEA-18 and SEA-86 were diluted in phosphate buffered saline to 2 mg·L-1.A:IgG strip;B:IgA/IgM strip.

Fig.4 Titers of anti-SEA McAbsHRPdetermined by ELISA.McAbs were serially diluted with PBST(from 1∶500 to 1∶2 048 000 by four-fold serial dilution).The 96 well microtiter plate was coated with SEA or SEB at the concentration of 3 mg·L-1,100 μL/well.±s,n=4.

2.5 Detection of SEA in aqueous solution by Western blotting

The results indicated that detection limits of SEA-18 and SEA-86 were 12.5 and 6.25 ng,respec?tively.When McAbs SEA-7 was used,a higher detection sensitivity was obtained.Only 1.56 ng SEA could be analyzed.We also used McAbs SEA-7HRPto recognize SEA directly,and positive signals were obtained when more than 6.25 ng of SEA was loaded(Fig.5).These results indicated that SEA-7 could be well used as a primary anti?body to identify SEA by Western blotting with a high sensitivity.

Fig.5 SEA in aqueous solution detected by Western blotting.SEA was separated by 12%SDS-PAGE.The blots were developed with McAb SEA-7HRP(A)and with SEA-7 as primary antibody and HRP-conjugated goat against mouse IgG as secondary antibody(B).

2.6 Detection of SEA in food samples

In order to demonstrate that the Western blotting method established above could be used to detect SEA-contaminated food,ham and porridge were homogenized and their supernatants were spiked with SEA 0.5 mg·L-1.The result of Western blotting showed that SEA was clearly observed in each food sample detected,with positive band given by the standard sample of SEA in water(Fig.6).When the samples without SEA were analyzed at the same time,no positive signals were observed,indicating that McAb SEA-7 could well recognize SEA in these food samples with a higher detection sensitivity and specificity.

Fig.6 Western blotting analysis of food samples spiked with SEA.The supernatant of ham homogenate and porridge was spiked with SEA.10 μL sample was loaded onto the 12%SDS gel and assayed by Western blotting.McAb SEA-7 and HRP-conjugated secondary antibody were respectively used as first and second antibody.Lane 1，3 and 5 was water，ham and porridge without SEA，respectively.Lane 2，4 and 6 was water，ham and porridge with SEA 0.5 mg·L-1,respectively.

3 DISCUSSION

Food-borne diseases have a major public health impact.Staphylococcal food-borne diseases resulting from SEs contamination are the second most common cause of reported food-borne illness.A meager amount of enterotoxin is sufficient to cause intoxication.Among the SEs,SEA is the most common toxin responsible for staphylococcal food poisoning[15].

Several methods have been developed for the detection of SEA contamination in food,such as ELISA and PCR,piezoelectric immunosensor andin vitrocell proliferation assay[16-19].Among these methods,ELISA is commonly used thanks to its simplicity,sensitivity,and easy availability.But it has several drawbacks.For example,SEA in heat-processed food does not react well with the antibodies used in ELISA and some unrelated antigens in food often cause false positive results.Surface plasmon resonance(SPR)-based immu?nosensor has been used for determining SEA over the past few years,but the self-assembled monolayer(SAM)of the sensor surface may bring stereospecific blockade,thus decreasing the detection sensitivity.And low contaminated samples need pretreatment before the proposed method would be applicable.

In recent years,assays based on PCR detection and genotyping ofsegenes ofS.aureusisolated from food materials have gained much atten?tion[4,11-12].Despite being sensitive and specific,they fail to provide information on viability of theS.aureus.Researchers have improved the conven?tional PCR method and developed many new approaches to detectenterotoxin genes ofS.aureus,such as electrochemical PCR-EIA,5′nuclease multiplex PCR assay,RFLP-PCR and real-time PCR.Although they entail some advan?tages over conventional PCR,their implementa?tion requires specific primers and equipment.Also,it is not clear whether allsegene-positive strains actually cause food poisoning.

Western blotting is a widely used method for protein analysis and has been used to detect SEA.Proteins treated by SDS are solubilized and refolded and could be well separated by SDS-PAGE according their relative molecular mass.As a result,SEA spiked in food could be analyzed by Western blotting without the interfer?ence from other proteins,while SEA in water is also detected at the same time.An antibody with high affinity and specificity to recognize SEA is the most important reagent to establisha a Western blotting method for SEA detection.

In this study,we prepared 3 strains of anti-SEA McAbs,which all belong to IgG1subclass and could specifically recognize and combine with SEA.We labeled anti-SEA McAbs with HRP,which could recognize SEA at high titers(1∶32 000)and could directly detect SEA in one step.Thus,the detection time could be greatly shortened.In order to increase the detection sensitivity,we used McAb SEA-7 as the first antibody to recognize SEA and HRP-conjugated anti-mouse IgG as the second antibody to identify the McAb SEA-7.Using this protocol,SEA 1.56 ng in water and SEA 5 ng in ham and porridge homogenate could be analyzed.

We established the Western blotting protocol as an assay for SEA.The results showed that the anti-SEA McAbs had high specificity and affinity.The McAb SEA-7 could be used to detect SEA in water and food samples by Western blotting.Our study provides a reliable and sensitive method to detect SEA and might offer reference for other researchers.