Letters in Applied Microbiology 1990, 11, 119-122
CSGJ025
An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aurcus B. M I R H A B I B O L L A H J OI ,Y . L. B R O O K S& R . G . K R O L L *Department of Microbiology, AFRC Institute of Food Research, Shinfield, Reading RG2 9AT, U K Received 12 February I990 and accepted 2 M a y 1990 M I R H A B I B O L L A HB., I , B R O O K S ,J . L . & K R O L L , R.G. 1990. An improved amperometric immunosensor for the detection and enumeration of protein Abearing Staphylococcus aureus. Letters in Applied Microbiology l l , 1 19-122.
An amperometnc immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above lo3 cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.
Immunological methods for detecting bacterial contaminants in foods are usually performed by colorimetric enzyme-linked immunoadsorbent assays (ELISA; Beckers et al. 1988; Mattingly et ul. 1988). However, the development of electrical immunological detection systems, i.e. immunosensors, could have considerable advantages in terms of speed, sensitivity and automation (Green 1987). An enzyme-linked immunosensor able to detect protein A-bearing Staphylococcus uureus in pure cultures and in food samples has been described (Mirhabibollahi et al. 1990). This relied on the amperometric detection of 0, , generated from H,O, by catalase linked to the secondary antibody, by an 0, electrode. Although quite sensitive (ca lo4 cfu/g), this assay was not without problems. It was performed using antibody coated membranes in glass containers and as such was not easily automatable. The electrochemical detection step was time consuming and required the maintenance of a constant temperature and the separation of the reaction mixture from the atmosphere. Furthermore, there were unexplained variations in the background levels of the current increases using different strains of Staph. aureus, which made the system unreliable in the accurate quantification of cell numbers.
* Corresponding author.
Another immunosensor system has been used to detect human orosmucoid glycoprotein by a competitive assay (Doyle et al. 1984). This used alkaline phosphatase labelled antibody, phenylphosphate as the substrate and electrochemical detection of the product, phenol, by a carbon paste electrode incorporating a liquid chromatography separation, Although sensitive (to 1 ng/ml glycoprotein), the assay was quite slow ( > 12 h). A similar procedure using a sandwich assay has been used to detect rabbit IgG at 10 ng/l but this incorporated a separation step on octyldecasilane column (Wehmeyer et ul. 1985). This paper describes an improved immunosensor for detecting protein A-bearing Staph. aureus in pure cultures and foods by a direct electrochemical assay for phenol using an alkaline phosphatase/phenylphosphate/immunosensor system.
Materials and Methods All chemicals were of analytical grade (BDH, Poole) except staphylococcal protein A, disodium phenylphosphate, potassium chloride, Tween 20 and the immunological reagents, which were from Sigma, Poole. Microtitre plates (96-well) were from Western Laboratory Services (Aldershot) and stomacher bags from Seward Medical Ltd (London).
120
B. Mirhabibollahi et al.
B A C T E R I A L C U L T U R E S A N D FOOD S A M P L E S
Broth cultures of Staph. aureus NCDO 949, 1022, 1499 and 2044 were obtained by inoculating and incubating Brain Heart Infusion broth (Oxoid, Basingstoke) for 18 h at 37°C. Plate counts were performed by serial dilution of samples in sterile phosphate buffered saline (PBS, pH 7.3) and surface plating (0.1 ml) on Baird Parker Agar (Oxoid) followed by incubation at 37°C for 2 d. Broth cultures for use as test antigen were diluted in PBST (PBS containing 0.1 v/v Tween 20) containing 1% w/v non-fat dried milk (PBSTM). Samples of semiskimmed pasteurized milk (10 ml) or braising beef (10 g) were inoculated with Staph. aureus NCDO 949. Meat samples were homogenized with 90 ml PBS in stomacher bags and diluted using uncontaminated meat homogenate. Milk samples were diluted with non-inoculated milk.
ELECTROCHEMICAL IMMUNOSENSOR ASSAY
Microtitre plate wells were coated with 200 pl human IgG (10 pg/ml) in 0.05mol/l sodium carbonate buffer (g/l; Na,CO,, 1.5; NaHCO,, 2.93; pH 9-6) by incubation overnight at 4°C and then washed with PBST. Test antigen dilutions (pure cultures, food samples or protein A diluted in PBST) were boiled for 15 min and added (200 pl) to the microtitre plate wells (five replicates). After incubation for 90 min at room temperature, with gentle shaking, the wells were washed with PBST and 200 pl of rabbit antiprotein A antibody (5 pg/ml in PBSTM) was added. The plates were incubated for 1 h, washed with PBSTM and a 1 : lo00 dilution of anti-rabbit IgG-alkaline phosphatase conjugate (200 pl/well) was added. After 30 min the wells were washed with PBST and 200 pl of substrate solution (2.5 mmol/l phenylphosphate in pH 9.6 carbonate buffer) added. After incubation for 1 h at 37°C the product of the reaction (phenol) was detected electrically, using an amperometric platinum electrode system. The platinum anode and Ag/Ag C1 reference electrodes (Rank Bros, Cambridge) were connected to a potentiostat (Ministat, Thomson Electrochem, Newcastle-upon-Tyne). The reaction chamber contained 1 ml of carbonate buffer which was stirred by a magnetic flea and the Ag/AgCl reference electrode was
covered with paper tissue soaked with saturated KCl solution as the electrolyte. The platinum electrode was polarized at +870 mV with respect to the reference electrode (to electrochemically oxidize and detect the phenol). On addition of a sample (195 pl) from the microtitre plate, the current generated was measured as the voltage drop over a 2200 fl resistor by a microvolt digital multimeter (model 177, Keithley Instruments, Reading). The analogue output of the multimeter was plotted on a chart recorder (CR600, J J Loyd Instruments, Southampton). With each sample the current generated reached a maximum value, 1G-15 s after sample addition. Appropriate positive and negative controls were included and the assay took approximately 5 h to complete.
Results and Discussion The detection of phenol produced enzymatically from phenyl phosphate is an attractive feature for incorporation into an immunosensor system. Phenol is rare in biological materials and as such the method should suffer little interference from samples. Phenol can be oxidized electrochemically (> +750 mV), whereas phenylphosphate is electroinactive at positive potentials (Doyle et al. 1984). We have successfully applied this principle directly to samples, without the need for liquid or column chromatography steps (Doyle et al. 1984; Wehmeyer et al. 1985). In this system the amount of current generated is a measure of the amount of phenol oxidized at the platinum anode. This should relate to the number of phosphatase-antibody conjugates remaining which, in turn, should be proportional to the original antigen concentration. This is obviously the case (Fig. 1) with purified protein A, which was reliably detected at 0.01 ng/ml. This represents a 10-fold improvement in sensitivity over the previous immunosensor system (Mirhabibollahi et al. 1990). It is also more sensitive than colorimetric ELISAs for protein A (Fey & Burkhard 1981, 5-10 ng/ml; Bloom et al. 1989, 0.1 ng/ml). Pure cultures of protein Abearing Staph. aureus were detected at ca lo3 cfu/ml and above (Fig. 2), which compared favourably with the catalase-based immunosensor (ca lo4 cfu/ml) and is considerably more sensitive than colorimetric ELISAs for other bacteria which usually require at least lo6 cells/ ml for reliable detection (Beckers el al. 1988).
121
Zmmunosensor for Staphylococcus aureus
7
I
A 0
2.5
I
I
A 00
.
A*
0
L
1.747
$5
++
E:
I
I
-3
-2
' 0' 'I 2' 3'
-I
Protein A concentration (Log,, ng/mll
z Fig. 1. Relationship between protein A concentration and the current generated by the amperometric immunosensor using alkaline phosphatase and phenylphosphate. The product of the reaction, phenol, was detected electrochemically by a Pt electrode poised at +870 mV with respect to the Ag/AgCI reference electrode. Results are the mean values of three experiments.
This number of Staph. aureus in foods is often given as the maximum acceptable level (Anon. 1986), so that this system could be applied directly, without the need for preincubation of samples. The variations in background currents (2G-50 nA/min) with different strains of Staph. aureus reported previously (Mirhabibollahi et al. 1990) are greatly reduced with this system (Fig. 2). Although there are slight variations between strains at the lower limits of detection, these, however, are internally consistent with the corresponding negative controls. Detection and quantification of protein Abearing Staph. aureus NCDO 949 in samples of milk or beef was successful with similar sensitivities (Fig. 3). The other Staph. aureus strains used in the pure culture studies gave similar results (not shown). Thus the improved immunosensor is readily applicable to food samples and does not suffer interference from food ingredients. Again there was a small variation in background currents, but these were consistent with the negative controls. The reliability, repeatability and sensitivity of this system
Fig. 2. Relationship between cell density, as determined by plate counts on Baird Parker Agar, of Staph. aureus NCDO 949 (W), 1022 (a),1499 0) and 2044 (A) and the current generated by the amperometric immunosensor to protein A. Results are the mean values of three experiments. 2.6
-
2.5
-
2 . 4 -~
2.3
-
c
'."I;
L :?
g;
5
I
4
'
5
I
6
'
7
I 8
Cell density (Log,, cfu/g or/rnll
m 0 u
Z
Fig. 3. Relationship between cell density of Staph. aureus NCDO 944 inoculated into samples of milk (A) or beef (w) and the current generated by the amperometric immunosensor to protein A. Results are the mean values of three experiments.
122
B. Mirhabibollahi e t al.
together with its simplicity and few reagents make it amenable to automation. It should be emphasized that we have used the detection of Staph. aureus via protein A as a model system and protein A- strains (of which there are few) will not be detected. This system could be adapted to detect other food-borne pathogens or toxins using appropriate immunological reagents. It may also be applicable for use with other difficult biological samples, e.g. in medical or veterinary analysis. This work was supported by the Ministry of Agriculture, Fisheries and Foods by Open Contract (no. CSA 1200). References ANON.1986 Micro-organisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. Toronto: University of Toronto Press. BECKERS, M.J., TIPS, P.D., SOENTORO,P.S.S., DELFGOU-VAN ASH, E.H.M. & PETERS,R. 1988 The eficiency of enzyme immunoassays for the detection of salmonellas. Food Microbiology 5, 147-156. BLOOM,J.W., WONG,M.F. & MITRA,G. 1989 Detection and reduction of protein A contamination in immobilized protein A purified monoclonal anti-
body preparations. Journal of Immunological Methods 117,83-89. DOYLE, M.J., HALSALL,H.B. & HEINEMAN, W.R. 1984 Enzyme-linked immunoadsorbent assay with electrochemical detection for a,-acid glycoprotein. Analytical Chemistry 56, 2355-2360. FEY, H. & BURKHARD, G.A. 1981 Measurement of staphylococcal protein A and detection of protein A-carrying staphylococcus strains by a competitive ELISA method. Journal of Immunological Methods 47,99-107. GREEN,M.J. 1987 New approaches to electrochemical immunoassays. In Biosensors. Fundamentals and Applications, ed. Turner, A.P.F., Karube, 1. & Wilson, G.S. pp. &70. Oxford: Oxford University Press. MATTINGLY,J.A., BUTMAN, B.T., PLANK, M.C., DURHAM, R.J. & ROBISON, B.J. 1988 Rapid monoclonal antibody-based enzyme-linked immunosorbant assay for detection of Listeria in food products. Journal of the Association of Official Analytical Chemists 71, 679-681. MIRHABIBOLLAHI, B., BROOKS,J.L. & KROLL,R.G. 1990 Development and performance of an enzymelinked amperometric immunosensor for the detection of Staphylococcus aureus in foods. Journal of Applied Bacteriology 68, 577-585. WEHMEYER, K. R., MALSALL, M.B. & HEINEMAN, W.R. 1985 Heterogeneous enzyme immunosassay with electrochemical detection: competitive and ‘sandwich’-type immunoassays. Clinical Chemistry 31, 1546-1549.
CSGJ025
An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aurcus B. M I R H A B I B O L L A H J OI ,Y . L. B R O O K S& R . G . K R O L L *Department of Microbiology, AFRC Institute of Food Research, Shinfield, Reading RG2 9AT, U K Received 12 February I990 and accepted 2 M a y 1990 M I R H A B I B O L L A HB., I , B R O O K S ,J . L . & K R O L L , R.G. 1990. An improved amperometric immunosensor for the detection and enumeration of protein Abearing Staphylococcus aureus. Letters in Applied Microbiology l l , 1 19-122.
An amperometnc immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above lo3 cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.
Immunological methods for detecting bacterial contaminants in foods are usually performed by colorimetric enzyme-linked immunoadsorbent assays (ELISA; Beckers et al. 1988; Mattingly et ul. 1988). However, the development of electrical immunological detection systems, i.e. immunosensors, could have considerable advantages in terms of speed, sensitivity and automation (Green 1987). An enzyme-linked immunosensor able to detect protein A-bearing Staphylococcus uureus in pure cultures and in food samples has been described (Mirhabibollahi et al. 1990). This relied on the amperometric detection of 0, , generated from H,O, by catalase linked to the secondary antibody, by an 0, electrode. Although quite sensitive (ca lo4 cfu/g), this assay was not without problems. It was performed using antibody coated membranes in glass containers and as such was not easily automatable. The electrochemical detection step was time consuming and required the maintenance of a constant temperature and the separation of the reaction mixture from the atmosphere. Furthermore, there were unexplained variations in the background levels of the current increases using different strains of Staph. aureus, which made the system unreliable in the accurate quantification of cell numbers.
* Corresponding author.
Another immunosensor system has been used to detect human orosmucoid glycoprotein by a competitive assay (Doyle et al. 1984). This used alkaline phosphatase labelled antibody, phenylphosphate as the substrate and electrochemical detection of the product, phenol, by a carbon paste electrode incorporating a liquid chromatography separation, Although sensitive (to 1 ng/ml glycoprotein), the assay was quite slow ( > 12 h). A similar procedure using a sandwich assay has been used to detect rabbit IgG at 10 ng/l but this incorporated a separation step on octyldecasilane column (Wehmeyer et ul. 1985). This paper describes an improved immunosensor for detecting protein A-bearing Staph. aureus in pure cultures and foods by a direct electrochemical assay for phenol using an alkaline phosphatase/phenylphosphate/immunosensor system.
Materials and Methods All chemicals were of analytical grade (BDH, Poole) except staphylococcal protein A, disodium phenylphosphate, potassium chloride, Tween 20 and the immunological reagents, which were from Sigma, Poole. Microtitre plates (96-well) were from Western Laboratory Services (Aldershot) and stomacher bags from Seward Medical Ltd (London).
120
B. Mirhabibollahi et al.
B A C T E R I A L C U L T U R E S A N D FOOD S A M P L E S
Broth cultures of Staph. aureus NCDO 949, 1022, 1499 and 2044 were obtained by inoculating and incubating Brain Heart Infusion broth (Oxoid, Basingstoke) for 18 h at 37°C. Plate counts were performed by serial dilution of samples in sterile phosphate buffered saline (PBS, pH 7.3) and surface plating (0.1 ml) on Baird Parker Agar (Oxoid) followed by incubation at 37°C for 2 d. Broth cultures for use as test antigen were diluted in PBST (PBS containing 0.1 v/v Tween 20) containing 1% w/v non-fat dried milk (PBSTM). Samples of semiskimmed pasteurized milk (10 ml) or braising beef (10 g) were inoculated with Staph. aureus NCDO 949. Meat samples were homogenized with 90 ml PBS in stomacher bags and diluted using uncontaminated meat homogenate. Milk samples were diluted with non-inoculated milk.
ELECTROCHEMICAL IMMUNOSENSOR ASSAY
Microtitre plate wells were coated with 200 pl human IgG (10 pg/ml) in 0.05mol/l sodium carbonate buffer (g/l; Na,CO,, 1.5; NaHCO,, 2.93; pH 9-6) by incubation overnight at 4°C and then washed with PBST. Test antigen dilutions (pure cultures, food samples or protein A diluted in PBST) were boiled for 15 min and added (200 pl) to the microtitre plate wells (five replicates). After incubation for 90 min at room temperature, with gentle shaking, the wells were washed with PBST and 200 pl of rabbit antiprotein A antibody (5 pg/ml in PBSTM) was added. The plates were incubated for 1 h, washed with PBSTM and a 1 : lo00 dilution of anti-rabbit IgG-alkaline phosphatase conjugate (200 pl/well) was added. After 30 min the wells were washed with PBST and 200 pl of substrate solution (2.5 mmol/l phenylphosphate in pH 9.6 carbonate buffer) added. After incubation for 1 h at 37°C the product of the reaction (phenol) was detected electrically, using an amperometric platinum electrode system. The platinum anode and Ag/Ag C1 reference electrodes (Rank Bros, Cambridge) were connected to a potentiostat (Ministat, Thomson Electrochem, Newcastle-upon-Tyne). The reaction chamber contained 1 ml of carbonate buffer which was stirred by a magnetic flea and the Ag/AgCl reference electrode was
covered with paper tissue soaked with saturated KCl solution as the electrolyte. The platinum electrode was polarized at +870 mV with respect to the reference electrode (to electrochemically oxidize and detect the phenol). On addition of a sample (195 pl) from the microtitre plate, the current generated was measured as the voltage drop over a 2200 fl resistor by a microvolt digital multimeter (model 177, Keithley Instruments, Reading). The analogue output of the multimeter was plotted on a chart recorder (CR600, J J Loyd Instruments, Southampton). With each sample the current generated reached a maximum value, 1G-15 s after sample addition. Appropriate positive and negative controls were included and the assay took approximately 5 h to complete.
Results and Discussion The detection of phenol produced enzymatically from phenyl phosphate is an attractive feature for incorporation into an immunosensor system. Phenol is rare in biological materials and as such the method should suffer little interference from samples. Phenol can be oxidized electrochemically (> +750 mV), whereas phenylphosphate is electroinactive at positive potentials (Doyle et al. 1984). We have successfully applied this principle directly to samples, without the need for liquid or column chromatography steps (Doyle et al. 1984; Wehmeyer et al. 1985). In this system the amount of current generated is a measure of the amount of phenol oxidized at the platinum anode. This should relate to the number of phosphatase-antibody conjugates remaining which, in turn, should be proportional to the original antigen concentration. This is obviously the case (Fig. 1) with purified protein A, which was reliably detected at 0.01 ng/ml. This represents a 10-fold improvement in sensitivity over the previous immunosensor system (Mirhabibollahi et al. 1990). It is also more sensitive than colorimetric ELISAs for protein A (Fey & Burkhard 1981, 5-10 ng/ml; Bloom et al. 1989, 0.1 ng/ml). Pure cultures of protein Abearing Staph. aureus were detected at ca lo3 cfu/ml and above (Fig. 2), which compared favourably with the catalase-based immunosensor (ca lo4 cfu/ml) and is considerably more sensitive than colorimetric ELISAs for other bacteria which usually require at least lo6 cells/ ml for reliable detection (Beckers el al. 1988).
121
Zmmunosensor for Staphylococcus aureus
7
I
A 0
2.5
I
I
A 00
.
A*
0
L
1.747
$5
++
E:
I
I
-3
-2
' 0' 'I 2' 3'
-I
Protein A concentration (Log,, ng/mll
z Fig. 1. Relationship between protein A concentration and the current generated by the amperometric immunosensor using alkaline phosphatase and phenylphosphate. The product of the reaction, phenol, was detected electrochemically by a Pt electrode poised at +870 mV with respect to the Ag/AgCI reference electrode. Results are the mean values of three experiments.
This number of Staph. aureus in foods is often given as the maximum acceptable level (Anon. 1986), so that this system could be applied directly, without the need for preincubation of samples. The variations in background currents (2G-50 nA/min) with different strains of Staph. aureus reported previously (Mirhabibollahi et al. 1990) are greatly reduced with this system (Fig. 2). Although there are slight variations between strains at the lower limits of detection, these, however, are internally consistent with the corresponding negative controls. Detection and quantification of protein Abearing Staph. aureus NCDO 949 in samples of milk or beef was successful with similar sensitivities (Fig. 3). The other Staph. aureus strains used in the pure culture studies gave similar results (not shown). Thus the improved immunosensor is readily applicable to food samples and does not suffer interference from food ingredients. Again there was a small variation in background currents, but these were consistent with the negative controls. The reliability, repeatability and sensitivity of this system
Fig. 2. Relationship between cell density, as determined by plate counts on Baird Parker Agar, of Staph. aureus NCDO 949 (W), 1022 (a),1499 0) and 2044 (A) and the current generated by the amperometric immunosensor to protein A. Results are the mean values of three experiments. 2.6
-
2.5
-
2 . 4 -~
2.3
-
c
'."I;
L :?
g;
5
I
4
'
5
I
6
'
7
I 8
Cell density (Log,, cfu/g or/rnll
m 0 u
Z
Fig. 3. Relationship between cell density of Staph. aureus NCDO 944 inoculated into samples of milk (A) or beef (w) and the current generated by the amperometric immunosensor to protein A. Results are the mean values of three experiments.
122
B. Mirhabibollahi e t al.
together with its simplicity and few reagents make it amenable to automation. It should be emphasized that we have used the detection of Staph. aureus via protein A as a model system and protein A- strains (of which there are few) will not be detected. This system could be adapted to detect other food-borne pathogens or toxins using appropriate immunological reagents. It may also be applicable for use with other difficult biological samples, e.g. in medical or veterinary analysis. This work was supported by the Ministry of Agriculture, Fisheries and Foods by Open Contract (no. CSA 1200). References ANON.1986 Micro-organisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. Toronto: University of Toronto Press. BECKERS, M.J., TIPS, P.D., SOENTORO,P.S.S., DELFGOU-VAN ASH, E.H.M. & PETERS,R. 1988 The eficiency of enzyme immunoassays for the detection of salmonellas. Food Microbiology 5, 147-156. BLOOM,J.W., WONG,M.F. & MITRA,G. 1989 Detection and reduction of protein A contamination in immobilized protein A purified monoclonal anti-
body preparations. Journal of Immunological Methods 117,83-89. DOYLE, M.J., HALSALL,H.B. & HEINEMAN, W.R. 1984 Enzyme-linked immunoadsorbent assay with electrochemical detection for a,-acid glycoprotein. Analytical Chemistry 56, 2355-2360. FEY, H. & BURKHARD, G.A. 1981 Measurement of staphylococcal protein A and detection of protein A-carrying staphylococcus strains by a competitive ELISA method. Journal of Immunological Methods 47,99-107. GREEN,M.J. 1987 New approaches to electrochemical immunoassays. In Biosensors. Fundamentals and Applications, ed. Turner, A.P.F., Karube, 1. & Wilson, G.S. pp. &70. Oxford: Oxford University Press. MATTINGLY,J.A., BUTMAN, B.T., PLANK, M.C., DURHAM, R.J. & ROBISON, B.J. 1988 Rapid monoclonal antibody-based enzyme-linked immunosorbant assay for detection of Listeria in food products. Journal of the Association of Official Analytical Chemists 71, 679-681. MIRHABIBOLLAHI, B., BROOKS,J.L. & KROLL,R.G. 1990 Development and performance of an enzymelinked amperometric immunosensor for the detection of Staphylococcus aureus in foods. Journal of Applied Bacteriology 68, 577-585. WEHMEYER, K. R., MALSALL, M.B. & HEINEMAN, W.R. 1985 Heterogeneous enzyme immunosassay with electrochemical detection: competitive and ‘sandwich’-type immunoassays. Clinical Chemistry 31, 1546-1549.
