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Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing
Veterinary Microbiology 78 (2001) 319±330
Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing M.R. Gajendragad*, K.N.Y. Kamath, P.Y. Anil, K. Prabhudas, C. Natarajan Indian Veterinary Research Institute Campus, Hebbal, Bangalore 560 024, Karnataka, India Received 29 September 1999; received in revised form 31 May 2000; accepted 14 August 2000
Abstract An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6 MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type speci®c antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than ÿ2.5 Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type speci®cally and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples con®rmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunobiosensor; Piezo electric crystal; Foot and mouth disease virus; Virus typing
1. Introduction Foot and mouth disease (FMD) is one of the most contagious diseases of animals and can cause heavy losses in susceptible cloven-footed animals. There are seven serotypes of *
Corresponding author. Tel.: 91-80-341-0908; fax: 91-80-341-2509. E-mail address: [email protected] (M.R. Gajendragad). 0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 3 0 7 - 2
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FMD virus (FMDV), viz. `O', `A', `C', `Asia 1', `SAT 1', `SAT 2' and `SAT 3'; and within these serotypes many subtypes and strains can be identi®ed. Airborne excretion of the virus, occurs during the acute phase of infection, has been well documented (Sellers and Parker, 1969; Donaldson et al., 1970, 1982; Gloster et al., 1981; Dekker et al., 1996). Ruminants carry the virus following exposure and the virus can persist in the pharynx for up to 2 years (Hargreaves, 1994). The control of FMD in India is mainly through early diagnosis and regular vaccination. Previously the diagnosis of FMD was carried out using the complement ®xation test (CFT), but enzyme-linked immunoabsorbent assay (ELISA), is now the test of choice. ELISA takes about 4±6 h, which is a long time when there is a case of FMD to be diagnosed. This has led to the search for an alternative test/technique which is ef®cient and fast. A literature review shows that biosensors are being used in different diagnostic tests with rapid results, which are speci®c and sensitive. Biosensors basically have two components: biological or sensor molecules and a signal transducer. The biological component consists of an antigen or antibody. The transducer detects the change in one or more physicochemical property of the biological molecule. Hence, increasing attention is being paid to the development of the immunobiosensors especially those that can be used to assay clinical samples (Alder and McCallum, 1983; Guilbault and Jordon, 1988; NgehNgwainbl et al., 1990; Konig and Sratzel, 1993, 1995; Aberl et al., 1994; Morgan et al., 1996). The most commonly used biosensors are the piezo electric (PZ) crystal, immunoelectrode and optic ®ber biosensors. The PZ phenomenon was discovered in 1880 whereby electric dipoles generated in anisotropic natural crystals were subjected to mechanical stress, thus causing them to oscillate at frequencies between 9 and 14 MHz (Morgan et al., 1996). Quartz is the most commonly used PZ material because of its chemical stability in aqueous solutions and resistance to high temperatures without loss of PZ properties. These sensors are constructed by immobilizing a selective binding surface to a transducer. The surface can be an immobilized antigen or an antibody against a speci®c antibody or antigen, respectively. Selective binding of the molecule to the absorptive surface causes the transducer to change one or more of its fundamental signals. The PZ quartz crystal is one such transducer and the crystal oscillates at a very speci®c resonant frequency when placed in an appropriate oscillator circuit. In PZ biosensors, the crystals are coated with an adsorbent that selectively interacts with the analyte of interest; subsequent binding increases the mass of the coated crystal and alters its basic frequency of oscillation. The PZ crystal oscillator can be used as a microbalance to detect a change in mass of the crystal due to the formation of antigen±antibody complex, thus permitting it to be utilized as an immunobiosensor. Electrodes are placed on opposite faces of a PZ crystal and if a potential is applied between these electrodes, forces will be exerted on the bound charges within the crystal and the resultant deformations within the crystal form an electromechanical system which will be vibrated, when properly excited. PZ crystals used in oscillators usually have silver or gold electrodes placed on opposite faces with leads for electrical connections. Examining the cost effectiveness, ease of performance, and feasibility of developing it into a ®eld oriented test, the PZ crystal biosensor was selected to be developed and standardized for rapid diagnosis of FMD and typing the causative virus.
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2. Materials and methods 2.1. FMD virus reference strains Foot and mouth disease virus vaccine strains were grown on BHK21 clone 13 monolayers. The seed viruses were obtained from the FMD vaccine production laboratory of the institute. 2.2. Puri®cation of the viruses and antibody production The 146 s fraction of FMDV was separated by sucrose gradient ultracentrifugation (Suryanarayana et al., 1985). This antigen was used for the production of antisera against each type in rabbits and guinea pigs following the procedure described by Ferris (1988). 2.3. Monoclonal antibodies The monoclonal antibodies against FMDV reference vaccine strains used for the assay were available at the Indian Veterinary Research Institute, Bangalore Campus. 2.4. PZ crystal resonator set-up A simple transistor±transistor logic (TTL) oscillator con®gured around two gates of a SN 7414 IC and a quartz crystal, as the frequency determining element, was assembled. A resistor was used to provide positive feed back from the output of the ®rst gate to input. Two capacitors, one from input and another from output of the ®rst gate were connected to the earth. This circuit worked as a free running oscillator locked to crystal frequency. The second gate merely acted as an inverting buffer to provide a measure of isolation of the ®rst gate from the load connected to the output. Resistor (1 kO) was used for the positive feed back. The capacitors used were 47 and 68 pF. One 6 MHz crystal was used as a frequency-determining element. This crystal was provided with two silver electrodes to which two long leads were connected. The circuit was so assembled that the crystal could be easily connected and disconnected during the experiments. A dc power supply of 6 V was provided to the oscillator by an eliminator assembled in our laboratory. All the components including the crystals were purchased locally for assembling the circuit. The speci®cations of the crystal were as follows: (i) A.T. cut; (ii) fundamental frequency (6 MHz); (iii) silver electrodes; (iv) diameter (8.5 mm); (v) thickness (0.052 mm). The surface of the crystal was made available for immobilization. A frequency counter (Scienti®c, Model HM 50212) was used to measure the frequency of output of the oscillator circuit (Fig. 1). 2.5. Immobilizing antigen/antibody on PZ crystal The antibody against FMDV was coated on a PZ crystal by the following procedure: the PZ crystal was ®rst washed with distilled water and then dipped for few minutes in ethanol. The crystal was then connected to the oscillator circuit and the fundamental
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Fig. 1. Circuit diagram for the PZ immunobiosensor.
frequency (f1) of the crystal was recorded using the frequency meter. The quartz crystal was then rinsed with phosphate-buffered saline-Tween (PBST), pH 7.4. After drying the crystal for 30 min at 378C, 1 ml of Ab was pipetted on both the crystal surfaces using a micropipette. The crystal was washed with PBST, dried at 378C, and the frequency recorded (f2). The crystal was then ready for immunoreaction. Separate crystals were prepared for each of the FMDV serotypes, viz. O, A, C and Asia 1. 2.6. Development of PZ immunobiosensor One microlitre of antigen (tissue culture virus harvested 1 day prior to the experiment with virus titer 105 TCID50) was deposited on the crystal. The crystal was washed with PBST and dried in an incubator at 378C. After drying, the frequency of the oscillation was recorded ( f3).
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Fig. 2. The protocol of the PZ crystal immunobiosensor.
2.7. Standardization of the protocol 2.7.1. Selection of the PZ crystal The experiment was carried out with PZ crystals of various frequencies utilizing antibody of Asia 1 as a model and tissue culture Asia 1 virus with a titre of 105 TCID50 as antigen. PZ crystals of 2, 6 and 8 MHz were coated with the Ab and the assay was carried out. The crystals were assessed for their ease of handling and repeatability of the assay (Fig. 2). 2.7.2. Determination of the positive response The negative or downward shift, Df, for immunoreaction was determined by coating ten 6 MHz PZ crystals with antibody and reacted with homologous tissue culture antigen dilutions ranging from neat to 10ÿ9. A graph of Df was plotted against the dilutions. The experiment was then repeated with monoclonal antibodies. 2.7.3. Repeatability of the assay The standardized assay was repeated using the same reagents, viz. PBST, the tissue culture Ag, and the same crystal. After every assay, the crystal was washed thoroughly and re-coated with the same FMDV type Ab. The test was carried out on each crystal until the results were consistent. 2.7.4. Speci®city of reaction Five crystals were coated with FMDV type Asia 1 Ab. The assay was carried out with different FMDV types as antigens and, for the ®fth crystal, tissue culture medium was
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used as antigen. Similar experiment was carried out by coating the Abs against the FMDV serotypes. 2.8. Shelf-life studies Four crystals were coated with polyclonal antibodies of FMDV type O, A, C and Asia 1, as per the standardized protocol, in order to study the shelf life of the PZ crystal. The crystals were kept in a dry condition at room temperature. The resonant frequencies of the crystals were measured at weekly intervals up to 18 weeks. 3. Results 3.1. Selection of the PZ immunobiosensor Sets of ®ve crystals of known frequency were selected for testing and the mean resonant frequency obtained in each experiment is shown in Tables 1±3. The resonant frequency increases as the size of the crystal decreases. The 6 MHz crystal was convenient to handle and gave satisfactory results and was selected for further studies. The experiments using PZ crystals coated separately with antibodies against serotypes A, C and O of FMDV were carried out with several tissue culture antigens (Ag). The results always showed a similar downward shift in the homologous system. It was found that the 6 MHz crystals were most suitable because they were not too thin to handle and the washing steps could be carried out without breaking them. Furthermore, the assay could be repeated without any signi®cant variations. Thus, the 6 MHz PZ crystals were selected for further standardization. 3.2. Determination of the positive response As can be seen from Fig. 3, up to the dilution of 10ÿ5 we got a mean Df of ÿ2.5 kHz. Therefore, in our experiments a mean Df of ÿ2.5 kHz was taken as positive for diagnosis of FMDV.
Table 1 Results on standardization of 2 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
1998 1999 1998 1999 1998
1992 1996 1996 1992 1992
1989 1994 1992 1990 1989
ÿ3 ÿ2 ÿ4 ÿ2 ÿ3
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Table 2 Results on standardization of 6 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
5997 5994 5996 5996 5996 5994 5994 5994 5995 5992 5998 5998 5995 5998
5990 5984 5993 5991 5992 5990 5990 5990 5992 5987 5982 5992 5992 5991
5984 5982 5982 5986 5989 5987 5988 5987 5988 5983 5980 5983 5982 5983
ÿ6 ÿ2 ÿ11 ÿ5 ÿ3 ÿ3 ÿ2 ÿ3 ÿ4 ÿ5 ÿ2 ÿ9 ÿ10 ÿ8
3.3. Results of the PZ immunobiosensor Table 4 depicts the results of the frequency changes during coating and immunoreaction. It can be seen that, in all the cases, the frequency after antigen exposure was higher than with antibody-coating alone. There was no downward shift of the frequency to indicate a positive response. Table 5 gives the result of frequency changes during coating and immune reaction of a homologous system using tissue culture Ag of titre 105 TCID50. The downward frequency shift was always more than 2.5 kHz in the homologous system indicating a positive immune reaction. The blank frequency (f1) was be very close to the resonant frequency of the crystal. The frequency (f2) shifted downwards after Ab coating and when antigen was added. The frequency (f3) further shifted downwards in the case of a homologous system (reaction of homologous Ag and Ab). The time required to test one sample was approximately 90 min. This was further reduced to 60 min when Ab-coated crystals were prepared prior to the assaying. Table 3 Results on standardization of 8 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
7998 7996 7993 7998 7993
7994 7993 7988 7993 7992
7992 7990 7985 7990 7990
ÿ2 ÿ3 ÿ3 ÿ3 ÿ2
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Fig. 3. Graph showing the cut-off point determination for the PZ crystal immunobiosensor. Table 4 Mean resonant frequency (kHz) with a heterologous system FMDV Ab
FMDV O FMDV A22 FMDV C FMDV Asia 1
Resonant frequency f1
f2
f3
f3 ÿ f2
5996.50 5993.14 5992.40 5995.66
5994.50 5991.20 5989.92 5993.55
5995.33 5991.39 5990.34 5993.84
0.83 0.91 0.42 0.29
Table 6 shows the resonant frequencies observed using the same crystal for the repeated assays. It should be noted that the results were consistent up to the eighth assay and thereafter inconsistent. This could be due to the wear and tear of the crystal surface. This reusability of the crystal minimizes the cost of the assay. Table 7 shows the speci®city of the assay when an FMDV type Asia 1-coated crystal was allowed to react with other FMDV types. It should be noted that there was a downward shift in the resonant frequency by 2.5 kHz only when Asia 1 Ag was used, which proves that the assay was speci®c. The assay was also compared with selected Table 5 Mean resonant frequency (kHz) with homologous TC Ag sample FMDV Ab
FMDV O FMDV A22 FMDV C FMDV Asia 1
Resonant frequency f1
f2
f3
f3 ÿ f2
5995.44 5995.62 5992.10 5993.27
5992.90 5994.92 5989.83 5991.27
5989.40 5991.82 5986.09 5988.02
ÿ3.50 ÿ3.10 ÿ3.74 ÿ3.25
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Table 6 Results of the repeatability of PZ crystal immunobiosensor in homologous system Test number
1 2 3 4 5 6 7 8 9 10 11 12
Resonant frequency (kHz) f1
f2
f3
f3 ÿ f2
5996.20 5997.47 5990.83 5995.94 5995.45 5996.52 5997.8 5996.85 5996.88 5997.98 5996.99 5996.72
5992.80 5992.32 5988.83 5992.45 5993.10 5943.04 5994.98 5985.34 5992.57 5993.01 5991.22 5994.21
5989.80 5990.01 5986.68 5992.77 5990.87 5982.67 5991.76 5986.39 5983.25 5983.00 5983.27 5985.22
ÿ3.00 ÿ2.31 ÿ2.15 ÿ0.32 ÿ2.23 ÿ1.37 ÿ3.22 ÿ1.05 ÿ9.32 ÿ10.01 ÿ7.95 ÿ8.99
clinical samples, the results of which are shown in Table 8. It should be noted that the PZ crystal immunobiosensor could detect the Ag in all samples, including the one that was found negative by ELISA. It could also detect mixed viral infections of type O and Asia 1 in two outbreaks from vaccinated cows (Gajendragad et al., 1999). 3.4. Results on shelf-life studies of the coated PZ crystal There was no change in the resonant frequencies recorded at weekly interval as shown in Table 9. The crystals were used for immunoreaction with their respective antigens in which they showed downward shifts in resonance frequency indicating the formation of Ag±Ab complexes. Satisfactory immunoreactions could be conducted for up to 18 weeks. 4. Discussion The results clearly indicated that the PZ immunobiosensors standardized in the present study could be used for diagnosis of FMDV in ®eld samples and for serotying. Table 7 Mean resonant frequency of the PZ crystals to determine the speci®city of the reaction Experiment number
1 2 3 4 5
Resonant frequency (kHz) f1
f2
f3
5998.27 5998.38 5998.69 5997.98 5998.67
5994.37 5994.99 5994.90 5993.01 5994.76
Ag Ag Ag Ag Ag
f3 ÿ f2
O, 5994.20 A, 5994.54 C, 5994.68 Asia 1, 5990.44 Blank, 5994.36
ÿ0.17 ÿ0.45 ÿ0.22 ÿ2.47 ÿ0.40
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Table 8 Comparison of the results of PZ immunobiosensor and ELISA on certain clinical samples Sample number
Antibody type coated
Resonant frequency (kHz)
f3 ÿ f2
f1
f2
f3
11/95
O A C Asia 1
5981.70 5996.03 5985.37 5998.15
5977.70 5991.89 5981.05 5994.26
5977.70 5995.50 5978.34 5995.37
0.00 3.61 ÿ2.71 1.11
2/95
O A C Asia 1
5982.32 5995.43 5990.22 5995.82
5976.30 5992.60 5988.02 5991.87
5979.04 5994.76 5989.15 5994.85
2.74 2.16 1.13 2.98
JRX
O A C Asia 1
5983.33 5996.82 5995.71 5996.72
5979.27 5990.80 5991.64 5994.49
5974.44 5990.74 5991.64 5992.46
ÿ4.83 ÿ0.06 0.00 ÿ2.03
G
O A C Asia 1
5983.91 5998.32 5986.27 5998.55
5980.47 5995.58 5983.54 5996.05
5980.47 5995.58 5983.54 5991.24
P
O A C Asia 1
5986.88 5996.02 5987.60 5996.72
5985.21 5977.31 5985.22 5994.21
1/92
O A C Asia 1
5990.95 5980.50 5996.24 5991.65
1473
O A C Asia 1
1973
PZ results
ELISA results
C
C
Negative
Negative
O
O
0.00 0.00 0.00 ÿ4.81
Asia 1
Asia 1
5985.20 5983.44 5985.20 5985.22
ÿ0.01 6.13 ÿ0.02 ÿ8.99
Asia 1
Asia 1
5987.33 5974.28 5992.71 5987.22
5984.51 5969.22 5991.91 5991.21
ÿ1.82 ÿ5.06 ÿ0.80 3.99
A22
No results
5976.07 5990.30 5986.80 5996.82
5971.84 5979.73 5980.08 5987.22
5967.30 5982.58 5980.31 5991.21
ÿ4.54 2.85 0.23 3.99
O
O
O A C Asia 1
5986.57 5994.24 5993.61 5996.56
5981.21 5990.70 5985.16 5993.25
5978.84 5989.95 5988.55 5994.13
ÿ2.37 ÿ0.75 3.39 0.88
O
O
IND/C7
O A C Asia 1
5990.83 5988.51 5994.68 5995.31
5988.38 5984.91 5991.83 5992.23
5986.86 5985.00 5991.48 5989.62
ÿ1.52 0.09 ÿ0.35 ÿ2.61
O
O
Asia 1
Asia 1
IDL 27
O A C Asia 1
5996.36 5995.62 5994.69 5996.46
5992.71 5994.29 5987.55 5992.04
5993.53 5990.82 5987.92 5992.39
0.82 ÿ3.47 0.37 0.35
A22
No results
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Table 9 Mean resonant frequency (kHz) of the crystals at different times Week
0 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18
Crys 1
Crys 2
Crys 3
Crys 4
O
A
C
Asia 1
5980.13 5980.11 5980.12 5980.12 5980.11 5980.11 5980.11 5980.10 5980.11 5980.10 5980.11 5980.11 5980.11 5980.11 5980.11 5980.11 5980.10 5976.80
5977.31 5977.31 5977.30 5977.31 5977.31 5977.31 5977.31 5977.30 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5973.27
5979.33 5979.32 5979.32 5979.33 5979.33 5979.33 5979.33 5979.33 5979.32 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5974.80
5992.83 5992.82 5992.82 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5989.80
Immunobiosensors were superior to conventional tests, such as the CFT and ELISA, which is, at present, the test of choice in all the laboratories involved in FMDV diagnosis (Reid et al., 1996). The coated PZ crystals were very stable and could be used for up to 18 weeks. They could also be reused at least eight times for antigen detection. Besides being speci®c, the test was also more sensitive than ELISA was, as it could detect the virus antigen in ELISA negative cases. Another advantage is the economy in the use of reagents, the biosensor required only 2 ml of the reagent whereas in ELISA 50 ml are used. The Ag-capture PCR described by Suryanarayana et al. (1993) is more sensitive than the PZ biosensor but PCR requires costly equipment and reagents, and is cumbersome and time-consuming. Once the coated crystal is ready, the type detection by biosensor takes less than 1 h that is quite rapid compared with ELISA and PCR techniques. The biosensor has the potential to become the test of choice in the immunodiagnosis of viral infections. 5. Conclusions An immunobiosensor for FMDV typing has been developed and standardized using a 6 MHz PZ crystal. The test is simple, speci®c and sensitive. The coated crystals have a shelf-life of 18 weeks and could be reused. They required minimal quantities of reagent, and, hence, are more economical than ELISA. The assay could be performed utilizing a sensitive and cheap frequency meter and proved suitable as an alternative ®eld-based test for FMDV typing.
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Acknowledgements The authors are thankful to the Director IVRI and the Joint Director, IVRI Bangalore Campus for the facilities. They wish to gratefully acknowledge the ®nancial assistance from the Department of Biotechnology. The authors are thankful to Drs. L.D. Misra, Principal Scientist for providing the FMDV vaccine virus strains and S.N. Saha, Senior Scientist for providing the monoclonal antibodies. References Aberl, F., Wolf, H., Kosslinger, C., Drost, S., Wolas, P., Koch, S., 1994. HIV serology using piezo electric immunosensors. Sensors Actuators B Chem. 18, 271±275. Alder, J., McCallum, J., 1983. PZ crystals for mass and chemical measurements. Analyst 108, 1169±1189. Dekker, A., Nielen, M., Molendijk, M., Kroonenbery, F., 1996. Foot and mouth disease airborne transmission prediction model: data and model considerations. Report of European Commission for the Control of Foot and Mouth Disease, pp. 176±182. Donaldson, A.I., Herniman, K.A.J., Parker, J., Sellers, R.F., 1970. Further investigation on the airborne excretion of FMDV. J. Hyg. Camb. 68, 557±564. Donaldson, A.I., Ferris, N.P., Gloster, J., 1982. Air sampling of pigs infected with foot and mouth disease virus: comparison of litton and cyclone samples. Res. Vet. Sci. 33, 384±385. Ferris, N.P., 1988. Selection of foot and mouth disease antisera for diagnosis by ELISA. Rev. Sci. Tech. Off. Int. Epiz. 7, 331. Gajendragad, M.R., Prabhudas, K., Gopalakrishna, S., Suryanarayana, V.V.S., Natarajan, C., 1999. A note on outbreaks due to mixed foot-and-mouth disease virus infections. Acta Virol. 43, 49±52. Gloster, J., Blackall, R.M., Sellers, R.F., Donaldson, A.I., 1981. Forecasting the airborne spread of foot and mouth disease. Vet. Rec. 25, 370±374. Guilbault, G.G., Jordon, J., 1988. Analytical uses of the PZ crystal detector (review). Crit. Rev. Anal. Chem. 19, 1±28. Hargreaves, S.K., 1994. The control of foot and mouth disease in Zimbabwe. OIE Scienti®c Conference on the Control of Foot and Mouth Disease, African Horse Sickness and Contagious Bovine Pleuropneumonia, Gabarone, Botswana, 20±23 April 1994. Konig, B., Sratzel, M., 1993. Detection of viruses and bacteria with piezo electric immunosensors. Anal. Lett. 20, 1567±1585. Konig, B., Sratzel, M., 1995. A piezo electric immunosensor for hepatitis viruses. Anal. Chim. Acta 309, 19±25. Morgan, C.L., Newman, D.J., Price, C.P., 1996. Immunosensors: technology and opportunities in laboratory medicine. Clin. Chem. 42, 193±209. Ngeh-Ngwainbl, J., Suleiman, A., Guilbault, G., 1990. PZ crystals biosensors. Biosens. Bioelectron. 5, 13±26. Reid, S.M., Forsyth, M.A., Ferris, N.P., 1996. Comparison of molecular, antigenic and virus isolation techniques for routine diagnosis of foot and mouth disease. Report of the European Commission for the Control of Foot and Mouth Disease, Kibbutz Ma'ale Hachamisha, Israel, 2±6 September 1996. Sellers, R.F., Parker, J., 1969. Airborne excretion of FMDV. J. Hyg. Camb. 67, 671±677. Suryanarayana, V.V.S., Rao, B.U., Padayatty, J.D., 1985. Cloning and expression of the cDNA for the major antigen of FMDV type `Asia 1' 63/71. Curr. Sci. 54, 1044. Suryanarayana, V.V.S., Srinivas, K., Gajendragad, M.R., Reddy, G.R., Ramakant, Natarajan, C., 1993. Evaluation of ®eld samples by Ag-capture/PCR and solid phase nucleic acid hybridization assay and their comparison with ELISA. In: Proceedings of International Symposium on Virus±Cell Interaction: Cellular and Molecular Responses, p. 139.
Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing M.R. Gajendragad*, K.N.Y. Kamath, P.Y. Anil, K. Prabhudas, C. Natarajan Indian Veterinary Research Institute Campus, Hebbal, Bangalore 560 024, Karnataka, India Received 29 September 1999; received in revised form 31 May 2000; accepted 14 August 2000
Abstract An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6 MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type speci®c antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than ÿ2.5 Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type speci®cally and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples con®rmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunobiosensor; Piezo electric crystal; Foot and mouth disease virus; Virus typing
1. Introduction Foot and mouth disease (FMD) is one of the most contagious diseases of animals and can cause heavy losses in susceptible cloven-footed animals. There are seven serotypes of *
Corresponding author. Tel.: 91-80-341-0908; fax: 91-80-341-2509. E-mail address: [email protected] (M.R. Gajendragad). 0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 3 0 7 - 2
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FMD virus (FMDV), viz. `O', `A', `C', `Asia 1', `SAT 1', `SAT 2' and `SAT 3'; and within these serotypes many subtypes and strains can be identi®ed. Airborne excretion of the virus, occurs during the acute phase of infection, has been well documented (Sellers and Parker, 1969; Donaldson et al., 1970, 1982; Gloster et al., 1981; Dekker et al., 1996). Ruminants carry the virus following exposure and the virus can persist in the pharynx for up to 2 years (Hargreaves, 1994). The control of FMD in India is mainly through early diagnosis and regular vaccination. Previously the diagnosis of FMD was carried out using the complement ®xation test (CFT), but enzyme-linked immunoabsorbent assay (ELISA), is now the test of choice. ELISA takes about 4±6 h, which is a long time when there is a case of FMD to be diagnosed. This has led to the search for an alternative test/technique which is ef®cient and fast. A literature review shows that biosensors are being used in different diagnostic tests with rapid results, which are speci®c and sensitive. Biosensors basically have two components: biological or sensor molecules and a signal transducer. The biological component consists of an antigen or antibody. The transducer detects the change in one or more physicochemical property of the biological molecule. Hence, increasing attention is being paid to the development of the immunobiosensors especially those that can be used to assay clinical samples (Alder and McCallum, 1983; Guilbault and Jordon, 1988; NgehNgwainbl et al., 1990; Konig and Sratzel, 1993, 1995; Aberl et al., 1994; Morgan et al., 1996). The most commonly used biosensors are the piezo electric (PZ) crystal, immunoelectrode and optic ®ber biosensors. The PZ phenomenon was discovered in 1880 whereby electric dipoles generated in anisotropic natural crystals were subjected to mechanical stress, thus causing them to oscillate at frequencies between 9 and 14 MHz (Morgan et al., 1996). Quartz is the most commonly used PZ material because of its chemical stability in aqueous solutions and resistance to high temperatures without loss of PZ properties. These sensors are constructed by immobilizing a selective binding surface to a transducer. The surface can be an immobilized antigen or an antibody against a speci®c antibody or antigen, respectively. Selective binding of the molecule to the absorptive surface causes the transducer to change one or more of its fundamental signals. The PZ quartz crystal is one such transducer and the crystal oscillates at a very speci®c resonant frequency when placed in an appropriate oscillator circuit. In PZ biosensors, the crystals are coated with an adsorbent that selectively interacts with the analyte of interest; subsequent binding increases the mass of the coated crystal and alters its basic frequency of oscillation. The PZ crystal oscillator can be used as a microbalance to detect a change in mass of the crystal due to the formation of antigen±antibody complex, thus permitting it to be utilized as an immunobiosensor. Electrodes are placed on opposite faces of a PZ crystal and if a potential is applied between these electrodes, forces will be exerted on the bound charges within the crystal and the resultant deformations within the crystal form an electromechanical system which will be vibrated, when properly excited. PZ crystals used in oscillators usually have silver or gold electrodes placed on opposite faces with leads for electrical connections. Examining the cost effectiveness, ease of performance, and feasibility of developing it into a ®eld oriented test, the PZ crystal biosensor was selected to be developed and standardized for rapid diagnosis of FMD and typing the causative virus.
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2. Materials and methods 2.1. FMD virus reference strains Foot and mouth disease virus vaccine strains were grown on BHK21 clone 13 monolayers. The seed viruses were obtained from the FMD vaccine production laboratory of the institute. 2.2. Puri®cation of the viruses and antibody production The 146 s fraction of FMDV was separated by sucrose gradient ultracentrifugation (Suryanarayana et al., 1985). This antigen was used for the production of antisera against each type in rabbits and guinea pigs following the procedure described by Ferris (1988). 2.3. Monoclonal antibodies The monoclonal antibodies against FMDV reference vaccine strains used for the assay were available at the Indian Veterinary Research Institute, Bangalore Campus. 2.4. PZ crystal resonator set-up A simple transistor±transistor logic (TTL) oscillator con®gured around two gates of a SN 7414 IC and a quartz crystal, as the frequency determining element, was assembled. A resistor was used to provide positive feed back from the output of the ®rst gate to input. Two capacitors, one from input and another from output of the ®rst gate were connected to the earth. This circuit worked as a free running oscillator locked to crystal frequency. The second gate merely acted as an inverting buffer to provide a measure of isolation of the ®rst gate from the load connected to the output. Resistor (1 kO) was used for the positive feed back. The capacitors used were 47 and 68 pF. One 6 MHz crystal was used as a frequency-determining element. This crystal was provided with two silver electrodes to which two long leads were connected. The circuit was so assembled that the crystal could be easily connected and disconnected during the experiments. A dc power supply of 6 V was provided to the oscillator by an eliminator assembled in our laboratory. All the components including the crystals were purchased locally for assembling the circuit. The speci®cations of the crystal were as follows: (i) A.T. cut; (ii) fundamental frequency (6 MHz); (iii) silver electrodes; (iv) diameter (8.5 mm); (v) thickness (0.052 mm). The surface of the crystal was made available for immobilization. A frequency counter (Scienti®c, Model HM 50212) was used to measure the frequency of output of the oscillator circuit (Fig. 1). 2.5. Immobilizing antigen/antibody on PZ crystal The antibody against FMDV was coated on a PZ crystal by the following procedure: the PZ crystal was ®rst washed with distilled water and then dipped for few minutes in ethanol. The crystal was then connected to the oscillator circuit and the fundamental
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Fig. 1. Circuit diagram for the PZ immunobiosensor.
frequency (f1) of the crystal was recorded using the frequency meter. The quartz crystal was then rinsed with phosphate-buffered saline-Tween (PBST), pH 7.4. After drying the crystal for 30 min at 378C, 1 ml of Ab was pipetted on both the crystal surfaces using a micropipette. The crystal was washed with PBST, dried at 378C, and the frequency recorded (f2). The crystal was then ready for immunoreaction. Separate crystals were prepared for each of the FMDV serotypes, viz. O, A, C and Asia 1. 2.6. Development of PZ immunobiosensor One microlitre of antigen (tissue culture virus harvested 1 day prior to the experiment with virus titer 105 TCID50) was deposited on the crystal. The crystal was washed with PBST and dried in an incubator at 378C. After drying, the frequency of the oscillation was recorded ( f3).
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Fig. 2. The protocol of the PZ crystal immunobiosensor.
2.7. Standardization of the protocol 2.7.1. Selection of the PZ crystal The experiment was carried out with PZ crystals of various frequencies utilizing antibody of Asia 1 as a model and tissue culture Asia 1 virus with a titre of 105 TCID50 as antigen. PZ crystals of 2, 6 and 8 MHz were coated with the Ab and the assay was carried out. The crystals were assessed for their ease of handling and repeatability of the assay (Fig. 2). 2.7.2. Determination of the positive response The negative or downward shift, Df, for immunoreaction was determined by coating ten 6 MHz PZ crystals with antibody and reacted with homologous tissue culture antigen dilutions ranging from neat to 10ÿ9. A graph of Df was plotted against the dilutions. The experiment was then repeated with monoclonal antibodies. 2.7.3. Repeatability of the assay The standardized assay was repeated using the same reagents, viz. PBST, the tissue culture Ag, and the same crystal. After every assay, the crystal was washed thoroughly and re-coated with the same FMDV type Ab. The test was carried out on each crystal until the results were consistent. 2.7.4. Speci®city of reaction Five crystals were coated with FMDV type Asia 1 Ab. The assay was carried out with different FMDV types as antigens and, for the ®fth crystal, tissue culture medium was
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used as antigen. Similar experiment was carried out by coating the Abs against the FMDV serotypes. 2.8. Shelf-life studies Four crystals were coated with polyclonal antibodies of FMDV type O, A, C and Asia 1, as per the standardized protocol, in order to study the shelf life of the PZ crystal. The crystals were kept in a dry condition at room temperature. The resonant frequencies of the crystals were measured at weekly intervals up to 18 weeks. 3. Results 3.1. Selection of the PZ immunobiosensor Sets of ®ve crystals of known frequency were selected for testing and the mean resonant frequency obtained in each experiment is shown in Tables 1±3. The resonant frequency increases as the size of the crystal decreases. The 6 MHz crystal was convenient to handle and gave satisfactory results and was selected for further studies. The experiments using PZ crystals coated separately with antibodies against serotypes A, C and O of FMDV were carried out with several tissue culture antigens (Ag). The results always showed a similar downward shift in the homologous system. It was found that the 6 MHz crystals were most suitable because they were not too thin to handle and the washing steps could be carried out without breaking them. Furthermore, the assay could be repeated without any signi®cant variations. Thus, the 6 MHz PZ crystals were selected for further standardization. 3.2. Determination of the positive response As can be seen from Fig. 3, up to the dilution of 10ÿ5 we got a mean Df of ÿ2.5 kHz. Therefore, in our experiments a mean Df of ÿ2.5 kHz was taken as positive for diagnosis of FMDV.
Table 1 Results on standardization of 2 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
1998 1999 1998 1999 1998
1992 1996 1996 1992 1992
1989 1994 1992 1990 1989
ÿ3 ÿ2 ÿ4 ÿ2 ÿ3
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Table 2 Results on standardization of 6 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
5997 5994 5996 5996 5996 5994 5994 5994 5995 5992 5998 5998 5995 5998
5990 5984 5993 5991 5992 5990 5990 5990 5992 5987 5982 5992 5992 5991
5984 5982 5982 5986 5989 5987 5988 5987 5988 5983 5980 5983 5982 5983
ÿ6 ÿ2 ÿ11 ÿ5 ÿ3 ÿ3 ÿ2 ÿ3 ÿ4 ÿ5 ÿ2 ÿ9 ÿ10 ÿ8
3.3. Results of the PZ immunobiosensor Table 4 depicts the results of the frequency changes during coating and immunoreaction. It can be seen that, in all the cases, the frequency after antigen exposure was higher than with antibody-coating alone. There was no downward shift of the frequency to indicate a positive response. Table 5 gives the result of frequency changes during coating and immune reaction of a homologous system using tissue culture Ag of titre 105 TCID50. The downward frequency shift was always more than 2.5 kHz in the homologous system indicating a positive immune reaction. The blank frequency (f1) was be very close to the resonant frequency of the crystal. The frequency (f2) shifted downwards after Ab coating and when antigen was added. The frequency (f3) further shifted downwards in the case of a homologous system (reaction of homologous Ag and Ab). The time required to test one sample was approximately 90 min. This was further reduced to 60 min when Ab-coated crystals were prepared prior to the assaying. Table 3 Results on standardization of 8 MHz PZ crystal for immunobiosensor Experiment number
1 2 3 4 5
Average frequency of the crystal (kHz) f1
f2
f3
f3 ÿ f2
7998 7996 7993 7998 7993
7994 7993 7988 7993 7992
7992 7990 7985 7990 7990
ÿ2 ÿ3 ÿ3 ÿ3 ÿ2
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Fig. 3. Graph showing the cut-off point determination for the PZ crystal immunobiosensor. Table 4 Mean resonant frequency (kHz) with a heterologous system FMDV Ab
FMDV O FMDV A22 FMDV C FMDV Asia 1
Resonant frequency f1
f2
f3
f3 ÿ f2
5996.50 5993.14 5992.40 5995.66
5994.50 5991.20 5989.92 5993.55
5995.33 5991.39 5990.34 5993.84
0.83 0.91 0.42 0.29
Table 6 shows the resonant frequencies observed using the same crystal for the repeated assays. It should be noted that the results were consistent up to the eighth assay and thereafter inconsistent. This could be due to the wear and tear of the crystal surface. This reusability of the crystal minimizes the cost of the assay. Table 7 shows the speci®city of the assay when an FMDV type Asia 1-coated crystal was allowed to react with other FMDV types. It should be noted that there was a downward shift in the resonant frequency by 2.5 kHz only when Asia 1 Ag was used, which proves that the assay was speci®c. The assay was also compared with selected Table 5 Mean resonant frequency (kHz) with homologous TC Ag sample FMDV Ab
FMDV O FMDV A22 FMDV C FMDV Asia 1
Resonant frequency f1
f2
f3
f3 ÿ f2
5995.44 5995.62 5992.10 5993.27
5992.90 5994.92 5989.83 5991.27
5989.40 5991.82 5986.09 5988.02
ÿ3.50 ÿ3.10 ÿ3.74 ÿ3.25
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Table 6 Results of the repeatability of PZ crystal immunobiosensor in homologous system Test number
1 2 3 4 5 6 7 8 9 10 11 12
Resonant frequency (kHz) f1
f2
f3
f3 ÿ f2
5996.20 5997.47 5990.83 5995.94 5995.45 5996.52 5997.8 5996.85 5996.88 5997.98 5996.99 5996.72
5992.80 5992.32 5988.83 5992.45 5993.10 5943.04 5994.98 5985.34 5992.57 5993.01 5991.22 5994.21
5989.80 5990.01 5986.68 5992.77 5990.87 5982.67 5991.76 5986.39 5983.25 5983.00 5983.27 5985.22
ÿ3.00 ÿ2.31 ÿ2.15 ÿ0.32 ÿ2.23 ÿ1.37 ÿ3.22 ÿ1.05 ÿ9.32 ÿ10.01 ÿ7.95 ÿ8.99
clinical samples, the results of which are shown in Table 8. It should be noted that the PZ crystal immunobiosensor could detect the Ag in all samples, including the one that was found negative by ELISA. It could also detect mixed viral infections of type O and Asia 1 in two outbreaks from vaccinated cows (Gajendragad et al., 1999). 3.4. Results on shelf-life studies of the coated PZ crystal There was no change in the resonant frequencies recorded at weekly interval as shown in Table 9. The crystals were used for immunoreaction with their respective antigens in which they showed downward shifts in resonance frequency indicating the formation of Ag±Ab complexes. Satisfactory immunoreactions could be conducted for up to 18 weeks. 4. Discussion The results clearly indicated that the PZ immunobiosensors standardized in the present study could be used for diagnosis of FMDV in ®eld samples and for serotying. Table 7 Mean resonant frequency of the PZ crystals to determine the speci®city of the reaction Experiment number
1 2 3 4 5
Resonant frequency (kHz) f1
f2
f3
5998.27 5998.38 5998.69 5997.98 5998.67
5994.37 5994.99 5994.90 5993.01 5994.76
Ag Ag Ag Ag Ag
f3 ÿ f2
O, 5994.20 A, 5994.54 C, 5994.68 Asia 1, 5990.44 Blank, 5994.36
ÿ0.17 ÿ0.45 ÿ0.22 ÿ2.47 ÿ0.40
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Table 8 Comparison of the results of PZ immunobiosensor and ELISA on certain clinical samples Sample number
Antibody type coated
Resonant frequency (kHz)
f3 ÿ f2
f1
f2
f3
11/95
O A C Asia 1
5981.70 5996.03 5985.37 5998.15
5977.70 5991.89 5981.05 5994.26
5977.70 5995.50 5978.34 5995.37
0.00 3.61 ÿ2.71 1.11
2/95
O A C Asia 1
5982.32 5995.43 5990.22 5995.82
5976.30 5992.60 5988.02 5991.87
5979.04 5994.76 5989.15 5994.85
2.74 2.16 1.13 2.98
JRX
O A C Asia 1
5983.33 5996.82 5995.71 5996.72
5979.27 5990.80 5991.64 5994.49
5974.44 5990.74 5991.64 5992.46
ÿ4.83 ÿ0.06 0.00 ÿ2.03
G
O A C Asia 1
5983.91 5998.32 5986.27 5998.55
5980.47 5995.58 5983.54 5996.05
5980.47 5995.58 5983.54 5991.24
P
O A C Asia 1
5986.88 5996.02 5987.60 5996.72
5985.21 5977.31 5985.22 5994.21
1/92
O A C Asia 1
5990.95 5980.50 5996.24 5991.65
1473
O A C Asia 1
1973
PZ results
ELISA results
C
C
Negative
Negative
O
O
0.00 0.00 0.00 ÿ4.81
Asia 1
Asia 1
5985.20 5983.44 5985.20 5985.22
ÿ0.01 6.13 ÿ0.02 ÿ8.99
Asia 1
Asia 1
5987.33 5974.28 5992.71 5987.22
5984.51 5969.22 5991.91 5991.21
ÿ1.82 ÿ5.06 ÿ0.80 3.99
A22
No results
5976.07 5990.30 5986.80 5996.82
5971.84 5979.73 5980.08 5987.22
5967.30 5982.58 5980.31 5991.21
ÿ4.54 2.85 0.23 3.99
O
O
O A C Asia 1
5986.57 5994.24 5993.61 5996.56
5981.21 5990.70 5985.16 5993.25
5978.84 5989.95 5988.55 5994.13
ÿ2.37 ÿ0.75 3.39 0.88
O
O
IND/C7
O A C Asia 1
5990.83 5988.51 5994.68 5995.31
5988.38 5984.91 5991.83 5992.23
5986.86 5985.00 5991.48 5989.62
ÿ1.52 0.09 ÿ0.35 ÿ2.61
O
O
Asia 1
Asia 1
IDL 27
O A C Asia 1
5996.36 5995.62 5994.69 5996.46
5992.71 5994.29 5987.55 5992.04
5993.53 5990.82 5987.92 5992.39
0.82 ÿ3.47 0.37 0.35
A22
No results
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Table 9 Mean resonant frequency (kHz) of the crystals at different times Week
0 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18
Crys 1
Crys 2
Crys 3
Crys 4
O
A
C
Asia 1
5980.13 5980.11 5980.12 5980.12 5980.11 5980.11 5980.11 5980.10 5980.11 5980.10 5980.11 5980.11 5980.11 5980.11 5980.11 5980.11 5980.10 5976.80
5977.31 5977.31 5977.30 5977.31 5977.31 5977.31 5977.31 5977.30 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5977.31 5973.27
5979.33 5979.32 5979.32 5979.33 5979.33 5979.33 5979.33 5979.33 5979.32 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5979.33 5974.80
5992.83 5992.82 5992.82 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5992.81 5989.80
Immunobiosensors were superior to conventional tests, such as the CFT and ELISA, which is, at present, the test of choice in all the laboratories involved in FMDV diagnosis (Reid et al., 1996). The coated PZ crystals were very stable and could be used for up to 18 weeks. They could also be reused at least eight times for antigen detection. Besides being speci®c, the test was also more sensitive than ELISA was, as it could detect the virus antigen in ELISA negative cases. Another advantage is the economy in the use of reagents, the biosensor required only 2 ml of the reagent whereas in ELISA 50 ml are used. The Ag-capture PCR described by Suryanarayana et al. (1993) is more sensitive than the PZ biosensor but PCR requires costly equipment and reagents, and is cumbersome and time-consuming. Once the coated crystal is ready, the type detection by biosensor takes less than 1 h that is quite rapid compared with ELISA and PCR techniques. The biosensor has the potential to become the test of choice in the immunodiagnosis of viral infections. 5. Conclusions An immunobiosensor for FMDV typing has been developed and standardized using a 6 MHz PZ crystal. The test is simple, speci®c and sensitive. The coated crystals have a shelf-life of 18 weeks and could be reused. They required minimal quantities of reagent, and, hence, are more economical than ELISA. The assay could be performed utilizing a sensitive and cheap frequency meter and proved suitable as an alternative ®eld-based test for FMDV typing.
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Acknowledgements The authors are thankful to the Director IVRI and the Joint Director, IVRI Bangalore Campus for the facilities. They wish to gratefully acknowledge the ®nancial assistance from the Department of Biotechnology. The authors are thankful to Drs. L.D. Misra, Principal Scientist for providing the FMDV vaccine virus strains and S.N. Saha, Senior Scientist for providing the monoclonal antibodies. References Aberl, F., Wolf, H., Kosslinger, C., Drost, S., Wolas, P., Koch, S., 1994. HIV serology using piezo electric immunosensors. Sensors Actuators B Chem. 18, 271±275. Alder, J., McCallum, J., 1983. PZ crystals for mass and chemical measurements. Analyst 108, 1169±1189. Dekker, A., Nielen, M., Molendijk, M., Kroonenbery, F., 1996. Foot and mouth disease airborne transmission prediction model: data and model considerations. Report of European Commission for the Control of Foot and Mouth Disease, pp. 176±182. Donaldson, A.I., Herniman, K.A.J., Parker, J., Sellers, R.F., 1970. Further investigation on the airborne excretion of FMDV. J. Hyg. Camb. 68, 557±564. Donaldson, A.I., Ferris, N.P., Gloster, J., 1982. Air sampling of pigs infected with foot and mouth disease virus: comparison of litton and cyclone samples. Res. Vet. Sci. 33, 384±385. Ferris, N.P., 1988. Selection of foot and mouth disease antisera for diagnosis by ELISA. Rev. Sci. Tech. Off. Int. Epiz. 7, 331. Gajendragad, M.R., Prabhudas, K., Gopalakrishna, S., Suryanarayana, V.V.S., Natarajan, C., 1999. A note on outbreaks due to mixed foot-and-mouth disease virus infections. Acta Virol. 43, 49±52. Gloster, J., Blackall, R.M., Sellers, R.F., Donaldson, A.I., 1981. Forecasting the airborne spread of foot and mouth disease. Vet. Rec. 25, 370±374. Guilbault, G.G., Jordon, J., 1988. Analytical uses of the PZ crystal detector (review). Crit. Rev. Anal. Chem. 19, 1±28. Hargreaves, S.K., 1994. The control of foot and mouth disease in Zimbabwe. OIE Scienti®c Conference on the Control of Foot and Mouth Disease, African Horse Sickness and Contagious Bovine Pleuropneumonia, Gabarone, Botswana, 20±23 April 1994. Konig, B., Sratzel, M., 1993. Detection of viruses and bacteria with piezo electric immunosensors. Anal. Lett. 20, 1567±1585. Konig, B., Sratzel, M., 1995. A piezo electric immunosensor for hepatitis viruses. Anal. Chim. Acta 309, 19±25. Morgan, C.L., Newman, D.J., Price, C.P., 1996. Immunosensors: technology and opportunities in laboratory medicine. Clin. Chem. 42, 193±209. Ngeh-Ngwainbl, J., Suleiman, A., Guilbault, G., 1990. PZ crystals biosensors. Biosens. Bioelectron. 5, 13±26. Reid, S.M., Forsyth, M.A., Ferris, N.P., 1996. Comparison of molecular, antigenic and virus isolation techniques for routine diagnosis of foot and mouth disease. Report of the European Commission for the Control of Foot and Mouth Disease, Kibbutz Ma'ale Hachamisha, Israel, 2±6 September 1996. Sellers, R.F., Parker, J., 1969. Airborne excretion of FMDV. J. Hyg. Camb. 67, 671±677. Suryanarayana, V.V.S., Rao, B.U., Padayatty, J.D., 1985. Cloning and expression of the cDNA for the major antigen of FMDV type `Asia 1' 63/71. Curr. Sci. 54, 1044. Suryanarayana, V.V.S., Srinivas, K., Gajendragad, M.R., Reddy, G.R., Ramakant, Natarajan, C., 1993. Evaluation of ®eld samples by Ag-capture/PCR and solid phase nucleic acid hybridization assay and their comparison with ELISA. In: Proceedings of International Symposium on Virus±Cell Interaction: Cellular and Molecular Responses, p. 139.