- Email: [email protected]
Application of a rapid automated immunosensor for the detection of Staphylococcus aureus enterotoxin B in cream
International
Journal
of Food
Microbiology
35 (1997) 293-297
Short communication
Application of a rapid automated immunosensor for the detection of Staphylococcus aureus enterotoxin B in cream N.J.C.
Strachan
‘,*, P.G. John b, I.G. Millar a
aFood Science and Technology Research Centre, School of Applied Sciences, Robert Gordon University, St. Andrex Street, Aberdeen, AB25 IHG, UK b CSL, Food Science Laboratory Tarry, P.O. Box 31, 135 Abbey Road, Aberdeen, AB9 8DG, UK Received
31 August
1996; received
in revised form
30 December
1996; accepted
30 December
1996
Abstract An automated immunosensor has been applied to the detection of Staphylococcus aurcus enterotoxin B in cream. It was capable of detecting the toxin down to a level of 5 rig/g in approximately 10 min. This paper describes the immunosensor method used and how it compares with conventional ELISA (enzyme linked immunosorbent assay) methods. 0 1997 Elsevier Science B.V.
Keywords: Immunosensor;
Staphylococcus aureus enterotoxin
1. Introduction Staphylococcus aureus is a common comensal organism in humans, with an estimated 20-50% having the organism in the upper respiratory tract (Adams and Moss, 1995; Bergdoll, 1989). Strains of S. aureus can produce one of seven different
* Corresponding author. Tel.: + 44 1224 2662839; fax: + 44 1224 262828; e-mail: [email protected] 016%1605/97/$17.00
0 1997 El sevier Science
PIrSO168-1605(97)01252-X
B.V. All rights
reserved.
B; Toxin;
Cream;
ELISA
types of enterotoxins (A, B, C,, C,, C,, D and E), all of which can cause food poisoning characterised mainly by vomiting and diarrhoea, but which in rare instances can cause enteritis, fever, leukocytosis and death (Bergdoll, 1989). Only very small doses of toxin are required to cause illness (as little as 1 pg of pure toxin in susceptible individuals). The toxins are small single-chain polypeptides (M,, 27.5-30 kDa) which are resistant to gut proteases. They are relatively heat stable and thus the toxins can be found in
294
N.J.C.
Strachm
et 01. International
Journal of Food Microhiolog~
cooked foods. The most common food vehicles for S. aureu~ intoxication include milk products such as cream, cheese, custard and cooked meats (Wieneke et al., 1993). This is usually caused by one or a combination of factors including cross contamination, poor hygiene and temperature abuse during the preparation of the foods. The standard AOAC method for staphylococcal enterotoxins in foods, the microslide gel double diffusion test, is a qualitative assay with a claimed sensitivity of lo&100 ng/ml of enterotoxin in culture fluids and concentrated food extracts (Andrews and Messer, 1990). There are currently a number of Enzyme-Linked ImmunoSorbent Assay (ELISA) methods available for detecting S. UL~Y~USenterotoxins but these can take between 1.5 and 7 h to give a result and usually require coating microplates with antibodies overnight. These have varying sensitivities ( - 1 rig/g)) and specificities to the different toxins (Bhatti et al., 1994; Morissette et al., 1991; Park et al., 1993, 1994). Fibre-optic fluorimetric sensors which utilise the evanescent wave principle have been developed to detect antigen/antibody binding sandwich immunoassays for the detection of botulinurn toxin (Ogert et al., 1992; Kumar et al., 1994) and these take only a few minutes to perform. However a major problem with these sensors is that the solid phase, the optical fibre, has to be replaced manually after each assay. Attempts have been made at regenerating the fibre by breaking the antigen/antibody binding alkaline solutions using either acid or (Wijesuriya et al., 1995). However this in general has not been very successful. Sapidyne (ID, USA) have developed an immunosensor which employs disposable particles as the solid phase (Glass and Lackie, 1995). During each assay the immunosensor loads new particles and then pumps the reagents and test sample entirely automatically. of the This paper describes the application to detect staphyloparticle based immunosensor coccal enterotoxin B (SEB). The results of this technique are compared with existing methods.
2. Materials
3.5 (1997) 293-297
and methods
2.1. Reagents
and chemicals
S. uureus enterotoxin B (SEB), anti-enterotoxin B (rabbit) antibodies, phosphate buffered saline (PBS), Fluorotag FITC conjugation kit. goat serum and hexane were purchased from Sigma (Poole, Dorset, UK). Polymethylmethacrylate (PMC) beads (98 pm) were obtained from Bangs Laboratories (Carmel, IN). Samples of single cream were purchased from local supermarkets. 2.2. Automated
particle
based immunosensor
The particle based immunoassay system (Sapidyne) consists of a fluorimeter containing a 1.5 mm diameter glass capillary within the final lens (Fig. 1). The 98 /lrn PMC beads were coated with antibodies (see below) and used as the solid phase in the immunoassay. A syringe pump flowed the PMC beads through the system where they were trapped against a 53 pm filter (Cole Parmer, Niles, IL) situated within the flow cell. By use of a rotary valve, the reagents for each assay could be selected and flowed past the antibody-coated PMC beads using a syringe pump. Once the assay
Flow
Excitation Light from
Particles
Screen Flourescent Emission Light to Detector
Fig. I. A schematic munosensor.
representation
of the particle
based
im-
N.J.C.
Strarhan et al. /International
Journal of Food Microbiology 35 (1997) 293-297
bl
295
The sample is added. Any Antigens present stick to the Antibodies.
Labelled Antibody is added. This sticks to the Antigens.
Fig. 2. Sandwich
immunoassay
was complete, the PMC beads were removed by back-flushing using a peristaltic pump. The entire assay procedure is entirely automated and controlled via software on a personal computer. 2.3. SEB ussuy SEB antibodies were conjugated to FITC using the Fluorotag kit. The 98 pm PMC beads were coated with 1 ml of unconjugated antibodies against SEB (1 mg/ml) by agitation for 1 h on a rocker (Camlab, Cambridge, UK). Non-specific binding sites were blocked by incubating for 1 h in a 10% solution of goat serum and then washed 3 times with PBS, after which the mixture was added to 29 ml PBS and stored at 4°C for up to 4 weeks. This solution of particles enabled 30 assays to be performed on the immunosensor. Different concentrations of SEB (O-40 rig/g)) were individually made up in 10 g aliquots of cream and left overnight at 4°C. Each sample was then mixed with 15 ml of PBS, centrifuged for 10 min at 4750 g and the resulting supernatant was assayed.
format
for SEB.
SEB was detected using a sandwich immunoassay format (Fig. 2). Approximately 100 antibody-coated PMC beads were pumped into the flow cell and trapped on the filter resulting in the generation of a miniature immunoaffinity column approximately 2 mm in height and 1 mm in diameter. This was followed by 4 ml sample for 240 s, 3 ml fluorescently labelled antibody for 240 s, then 4.5 ml PBS for 180 s to wash out excess label.
Fig. 3. The detection
of SEB in cream
by the immunosensor.
296
N.J.C.
Strachan
et al. i International
_.
008-
Fig. 4. Calibration
Journal qf Food Microbiology
;
plot of SEB in cream.
3. Results and discussion Fig. 3 shows a typical output from the immunosensor. It should be noted that the cream sample solutions are themselves fluorescent and can be seen as an increase in voltage during the first 4 min of the assay. When the fluorescent label is added the voltage initially decreases because this solution is less fluorescent than the sample. The signal then increases at a rate proportional to the amount of bound toxin. The signal then decreases when PBS is added to wash away excess label. The amount of binding to the sensor can be calculated by measuring the difference in immunosensor response (V) between the start (average voltage was taken during the first 10 s) and the end (average voltage was taken during the last 10 s) of the assay (endpoint delta). Three replicates of the assay for each sample were performed and a calibration curve was generated (Fig. 4). This shows that the lower limit for detection is 5 rig/g.. Similar sensitivity was obtained for contaminated milk (data not shown). Thus the immunosensor is at least twice as sensitive as the standard AOAC method (Andrews and Messer, 1990) and is slightly less sensitive than the 1 rig/g claimed by some conventional ELISAs (Park et al., 1994; Morissette et al., 1991). This could be due to the particular antibodies used as well as the efficiency of the conjugation procedure. It should be noted that the antibodies on the immunosensor are specific to SEB only. However, antibodies are commercially available which can detect a number of other staphylococcal enterotoxins and these
35 (1997) 293-297
could be used in combination on the immunosensor to screen for these toxins in a single assay. Compared to conventional microtitre plate based ELISA methods for staphylococcal enterotoxins, the particle based immunoassay system is much faster (10 min per sample) compared to 1.5-5 h for conventional ELISAs (Bhatti et al., 1994) and is totally automated. The Sapidyne system used here is a prototype and is limited to analysing four samples sequentially, which is less than the potential throughput on a microtitre plate. However it could easily be adapted to handle larger numbers of samples, e.g. by use of a carousel type autosampler. The Sapidyne system could be readily adapted for use as a true on-line sensor, allowing real time analysis of foods during processing and thus giving the potential for positive release of products from the factory prior to retail sale. The immunosensor also has the potential of being made portable and as such could be operated outside the laboratory.
Acknowledgements Technical assistance pro\iided by Dr Tom Glass (Sapidyne) is gratefully acknowledged. This work was performed at the Food Science Laboratory, Torry with the financial support of the UK Ministry of Agriculture, Fisheries and Foods (MAFF). The views expressed in this paper are those of the authors and do not necessarily reflect those, or the poiicy, of MAFF.
References Adams, M.R. and Moss. M.O. (1995) Food Microbiology. The Royal Society of Chemistry, Cambridge, pp. 398. Andrews, W.H. and Messer, J. (1990) Microbiological Methods. In: K. Helrich (editor) Official Methods of Analysis, 15th edition. AOAC, Arlington, VA, USA, pp. 451-454. Bergdoll, M.S. (1989) Staphylococcus oweus In: M.P. Doyle (editor), Foodborne Bacterial Pathogens. Marcel Dekker, New York, pp. 463-523. Bhattl, A.R., Siddjqui, Y.M. and Micussan, V.V. (1994) Highly of staphylococcal enterotoxin B. J. Microbial. Methods 19, 179-187. Glass, T.R. and Lackie, S. (1995) Theory and application of KinExATM. a new immunoassay method. Sapidyne Instruments, Idaho City. Idaho, USA, pp. 7.
N.J.C.
Strachan
et al. /International
Journal of Food Microbiology
Kumar, P., Colston, J.T. and Chambers, J.P. (1994) Detection of botulinurn toxin using an evanescent wave immunosenSOT. Bios. Bioelect. 9, 57763. Morissette, C., Comet, J. and Lamoureux, G. (1991) Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese. Appl. Environ. Microbial. 57, 8366842. Ogert, R.A., Brown, J.E., Singh, B.R., Shriver-Lake, L.C. and Ligler, F.S. (1992) Detection of Clostridium botulinurn toxin A using a fibre optic based biosensor. Anal. Biochem. 205, 3066312. Park, C.E., Akhtar, M. and Rayman, M.K. (1993) Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immuno-
3.5 (1997) 293-297
297
sorbent assay kit (TECRA). Appl. Environ. Microbial. 59, 2210-2213. Park, C.E., Akhtar, M. and Rayman, M.K. (1994) Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D and E in foods. Appl. Environ. Microbial. 60, 6777681. Wieneke, A.A., Roberts, D. and Gilbert, R.J. (1993) Staphylococcal food poisoning in the United Kingdom, 1969-90. Epidemiol. Infect. 110, 519-531. Wijesuriya, D., Breslin, K., Anderson, G., Shriver-Lake, L. and Ligler, F.S. (1995) Regeneration of immobilised antibodies on fiber optic probes. Biosens. Bioelect. 9, 5855 592.
Journal
of Food
Microbiology
35 (1997) 293-297
Short communication
Application of a rapid automated immunosensor for the detection of Staphylococcus aureus enterotoxin B in cream N.J.C.
Strachan
‘,*, P.G. John b, I.G. Millar a
aFood Science and Technology Research Centre, School of Applied Sciences, Robert Gordon University, St. Andrex Street, Aberdeen, AB25 IHG, UK b CSL, Food Science Laboratory Tarry, P.O. Box 31, 135 Abbey Road, Aberdeen, AB9 8DG, UK Received
31 August
1996; received
in revised form
30 December
1996; accepted
30 December
1996
Abstract An automated immunosensor has been applied to the detection of Staphylococcus aurcus enterotoxin B in cream. It was capable of detecting the toxin down to a level of 5 rig/g in approximately 10 min. This paper describes the immunosensor method used and how it compares with conventional ELISA (enzyme linked immunosorbent assay) methods. 0 1997 Elsevier Science B.V.
Keywords: Immunosensor;
Staphylococcus aureus enterotoxin
1. Introduction Staphylococcus aureus is a common comensal organism in humans, with an estimated 20-50% having the organism in the upper respiratory tract (Adams and Moss, 1995; Bergdoll, 1989). Strains of S. aureus can produce one of seven different
* Corresponding author. Tel.: + 44 1224 2662839; fax: + 44 1224 262828; e-mail: [email protected] 016%1605/97/$17.00
0 1997 El sevier Science
PIrSO168-1605(97)01252-X
B.V. All rights
reserved.
B; Toxin;
Cream;
ELISA
types of enterotoxins (A, B, C,, C,, C,, D and E), all of which can cause food poisoning characterised mainly by vomiting and diarrhoea, but which in rare instances can cause enteritis, fever, leukocytosis and death (Bergdoll, 1989). Only very small doses of toxin are required to cause illness (as little as 1 pg of pure toxin in susceptible individuals). The toxins are small single-chain polypeptides (M,, 27.5-30 kDa) which are resistant to gut proteases. They are relatively heat stable and thus the toxins can be found in
294
N.J.C.
Strachm
et 01. International
Journal of Food Microhiolog~
cooked foods. The most common food vehicles for S. aureu~ intoxication include milk products such as cream, cheese, custard and cooked meats (Wieneke et al., 1993). This is usually caused by one or a combination of factors including cross contamination, poor hygiene and temperature abuse during the preparation of the foods. The standard AOAC method for staphylococcal enterotoxins in foods, the microslide gel double diffusion test, is a qualitative assay with a claimed sensitivity of lo&100 ng/ml of enterotoxin in culture fluids and concentrated food extracts (Andrews and Messer, 1990). There are currently a number of Enzyme-Linked ImmunoSorbent Assay (ELISA) methods available for detecting S. UL~Y~USenterotoxins but these can take between 1.5 and 7 h to give a result and usually require coating microplates with antibodies overnight. These have varying sensitivities ( - 1 rig/g)) and specificities to the different toxins (Bhatti et al., 1994; Morissette et al., 1991; Park et al., 1993, 1994). Fibre-optic fluorimetric sensors which utilise the evanescent wave principle have been developed to detect antigen/antibody binding sandwich immunoassays for the detection of botulinurn toxin (Ogert et al., 1992; Kumar et al., 1994) and these take only a few minutes to perform. However a major problem with these sensors is that the solid phase, the optical fibre, has to be replaced manually after each assay. Attempts have been made at regenerating the fibre by breaking the antigen/antibody binding alkaline solutions using either acid or (Wijesuriya et al., 1995). However this in general has not been very successful. Sapidyne (ID, USA) have developed an immunosensor which employs disposable particles as the solid phase (Glass and Lackie, 1995). During each assay the immunosensor loads new particles and then pumps the reagents and test sample entirely automatically. of the This paper describes the application to detect staphyloparticle based immunosensor coccal enterotoxin B (SEB). The results of this technique are compared with existing methods.
2. Materials
3.5 (1997) 293-297
and methods
2.1. Reagents
and chemicals
S. uureus enterotoxin B (SEB), anti-enterotoxin B (rabbit) antibodies, phosphate buffered saline (PBS), Fluorotag FITC conjugation kit. goat serum and hexane were purchased from Sigma (Poole, Dorset, UK). Polymethylmethacrylate (PMC) beads (98 pm) were obtained from Bangs Laboratories (Carmel, IN). Samples of single cream were purchased from local supermarkets. 2.2. Automated
particle
based immunosensor
The particle based immunoassay system (Sapidyne) consists of a fluorimeter containing a 1.5 mm diameter glass capillary within the final lens (Fig. 1). The 98 /lrn PMC beads were coated with antibodies (see below) and used as the solid phase in the immunoassay. A syringe pump flowed the PMC beads through the system where they were trapped against a 53 pm filter (Cole Parmer, Niles, IL) situated within the flow cell. By use of a rotary valve, the reagents for each assay could be selected and flowed past the antibody-coated PMC beads using a syringe pump. Once the assay
Flow
Excitation Light from
Particles
Screen Flourescent Emission Light to Detector
Fig. I. A schematic munosensor.
representation
of the particle
based
im-
N.J.C.
Strarhan et al. /International
Journal of Food Microbiology 35 (1997) 293-297
bl
295
The sample is added. Any Antigens present stick to the Antibodies.
Labelled Antibody is added. This sticks to the Antigens.
Fig. 2. Sandwich
immunoassay
was complete, the PMC beads were removed by back-flushing using a peristaltic pump. The entire assay procedure is entirely automated and controlled via software on a personal computer. 2.3. SEB ussuy SEB antibodies were conjugated to FITC using the Fluorotag kit. The 98 pm PMC beads were coated with 1 ml of unconjugated antibodies against SEB (1 mg/ml) by agitation for 1 h on a rocker (Camlab, Cambridge, UK). Non-specific binding sites were blocked by incubating for 1 h in a 10% solution of goat serum and then washed 3 times with PBS, after which the mixture was added to 29 ml PBS and stored at 4°C for up to 4 weeks. This solution of particles enabled 30 assays to be performed on the immunosensor. Different concentrations of SEB (O-40 rig/g)) were individually made up in 10 g aliquots of cream and left overnight at 4°C. Each sample was then mixed with 15 ml of PBS, centrifuged for 10 min at 4750 g and the resulting supernatant was assayed.
format
for SEB.
SEB was detected using a sandwich immunoassay format (Fig. 2). Approximately 100 antibody-coated PMC beads were pumped into the flow cell and trapped on the filter resulting in the generation of a miniature immunoaffinity column approximately 2 mm in height and 1 mm in diameter. This was followed by 4 ml sample for 240 s, 3 ml fluorescently labelled antibody for 240 s, then 4.5 ml PBS for 180 s to wash out excess label.
Fig. 3. The detection
of SEB in cream
by the immunosensor.
296
N.J.C.
Strachan
et al. i International
_.
008-
Fig. 4. Calibration
Journal qf Food Microbiology
;
plot of SEB in cream.
3. Results and discussion Fig. 3 shows a typical output from the immunosensor. It should be noted that the cream sample solutions are themselves fluorescent and can be seen as an increase in voltage during the first 4 min of the assay. When the fluorescent label is added the voltage initially decreases because this solution is less fluorescent than the sample. The signal then increases at a rate proportional to the amount of bound toxin. The signal then decreases when PBS is added to wash away excess label. The amount of binding to the sensor can be calculated by measuring the difference in immunosensor response (V) between the start (average voltage was taken during the first 10 s) and the end (average voltage was taken during the last 10 s) of the assay (endpoint delta). Three replicates of the assay for each sample were performed and a calibration curve was generated (Fig. 4). This shows that the lower limit for detection is 5 rig/g.. Similar sensitivity was obtained for contaminated milk (data not shown). Thus the immunosensor is at least twice as sensitive as the standard AOAC method (Andrews and Messer, 1990) and is slightly less sensitive than the 1 rig/g claimed by some conventional ELISAs (Park et al., 1994; Morissette et al., 1991). This could be due to the particular antibodies used as well as the efficiency of the conjugation procedure. It should be noted that the antibodies on the immunosensor are specific to SEB only. However, antibodies are commercially available which can detect a number of other staphylococcal enterotoxins and these
35 (1997) 293-297
could be used in combination on the immunosensor to screen for these toxins in a single assay. Compared to conventional microtitre plate based ELISA methods for staphylococcal enterotoxins, the particle based immunoassay system is much faster (10 min per sample) compared to 1.5-5 h for conventional ELISAs (Bhatti et al., 1994) and is totally automated. The Sapidyne system used here is a prototype and is limited to analysing four samples sequentially, which is less than the potential throughput on a microtitre plate. However it could easily be adapted to handle larger numbers of samples, e.g. by use of a carousel type autosampler. The Sapidyne system could be readily adapted for use as a true on-line sensor, allowing real time analysis of foods during processing and thus giving the potential for positive release of products from the factory prior to retail sale. The immunosensor also has the potential of being made portable and as such could be operated outside the laboratory.
Acknowledgements Technical assistance pro\iided by Dr Tom Glass (Sapidyne) is gratefully acknowledged. This work was performed at the Food Science Laboratory, Torry with the financial support of the UK Ministry of Agriculture, Fisheries and Foods (MAFF). The views expressed in this paper are those of the authors and do not necessarily reflect those, or the poiicy, of MAFF.
References Adams, M.R. and Moss. M.O. (1995) Food Microbiology. The Royal Society of Chemistry, Cambridge, pp. 398. Andrews, W.H. and Messer, J. (1990) Microbiological Methods. In: K. Helrich (editor) Official Methods of Analysis, 15th edition. AOAC, Arlington, VA, USA, pp. 451-454. Bergdoll, M.S. (1989) Staphylococcus oweus In: M.P. Doyle (editor), Foodborne Bacterial Pathogens. Marcel Dekker, New York, pp. 463-523. Bhattl, A.R., Siddjqui, Y.M. and Micussan, V.V. (1994) Highly of staphylococcal enterotoxin B. J. Microbial. Methods 19, 179-187. Glass, T.R. and Lackie, S. (1995) Theory and application of KinExATM. a new immunoassay method. Sapidyne Instruments, Idaho City. Idaho, USA, pp. 7.
N.J.C.
Strachan
et al. /International
Journal of Food Microbiology
Kumar, P., Colston, J.T. and Chambers, J.P. (1994) Detection of botulinurn toxin using an evanescent wave immunosenSOT. Bios. Bioelect. 9, 57763. Morissette, C., Comet, J. and Lamoureux, G. (1991) Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese. Appl. Environ. Microbial. 57, 8366842. Ogert, R.A., Brown, J.E., Singh, B.R., Shriver-Lake, L.C. and Ligler, F.S. (1992) Detection of Clostridium botulinurn toxin A using a fibre optic based biosensor. Anal. Biochem. 205, 3066312. Park, C.E., Akhtar, M. and Rayman, M.K. (1993) Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immuno-
3.5 (1997) 293-297
297
sorbent assay kit (TECRA). Appl. Environ. Microbial. 59, 2210-2213. Park, C.E., Akhtar, M. and Rayman, M.K. (1994) Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D and E in foods. Appl. Environ. Microbial. 60, 6777681. Wieneke, A.A., Roberts, D. and Gilbert, R.J. (1993) Staphylococcal food poisoning in the United Kingdom, 1969-90. Epidemiol. Infect. 110, 519-531. Wijesuriya, D., Breslin, K., Anderson, G., Shriver-Lake, L. and Ligler, F.S. (1995) Regeneration of immobilised antibodies on fiber optic probes. Biosens. Bioelect. 9, 5855 592.