Title of Invention

"A NOVEL IMMUNOBIOSENSOR APPARATUS FOR RAPID DIAGNOSIS OF FOOD AND MOTH DISEASE (FMD) IN LIVESTOCK"

Abstract

This invention relates to an immunobiosensor apparatus for use in identifying Foot and Mouth Disease (FMD) with an indication to the type of virus involved in livestocks, comprising a signal transducer capable of oscillating at a specific resonant frequency when connected to an osicillator circuit; a Transistor-Transistor Logic Oscillator Circuit (TTL) configured around said signal transducer, said circuit being supplied with 6V D.C. power supply from an external source, a plurality of electrodes placed on opposite faces of said signal transducer provided with a plurality of long leads; and a frequency counter to measure the output frequency of said oscillator circuit, wherein a fundamental frequency (f1) of said signal transducer being measured by connecting said oscillator circuit to said transducer, characterised in that said signal transducer is a 6 Mhz PZ crystal emerging as a frequency-determining element.

Full Text

FIELD OF INVENTION The invention relates to a novel immunobiosensor apparatus agnosiS Of Foot And Mouth Disease (FMD)in livestockin particular to an apparatus for diagnosis of FMD in livestocks with indication to the exact type of virus involved. PRIOR ART The control of FMD in livestocks in India is mainly through early diagnosis and regular vaccination. Previously the diagnosis of FMD was carried out using the complement fixation 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 effiecient and fast. Biosensors are being used in different diagnostic tests with rapid results, which are specific and sensitive. Biosensors basically have two componentsi biological or sensor molecules and a signal transducer. The biological component consists of an antigen or antibody. The transducer det'ects 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 Jordan, 1988; Ngeh-Ngwainbl et al. 1990; Konig and Bratzel, 1993 and 1999; Aberl et al. 1994; Morgan et al. 1996). The most commonly used biosensors are the Piezo electric (PZ) crystal, immunoelectrode and optic fiber biosensors. The piezoelectric phenomenon was discovered in 1880 whereby electric dipoles generated in an isotropic 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 piezoelectric material because of its chemical stability in aqueous solutions and resistance to high temperatures without loss of piezoelectric properties. These sensors are constructed by immobilizing a selective binding surface to a transducer. The surface can be an immobilized antigen or antibody against a specific 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 •i, ^ transducer and the crystal oscillates at a very specific 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 being increases the mass of the coated crystal and alters its frequency of oscillation. The PZ crystal oscillator can be used as a micro-balance 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). The working of an immunobiosensor can generally be described whereby the 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 deformation within the srystal form an electromechanical system which will be vibrated, when properly excited. PZ crystals used in oscillators normally-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 field oriented test, the PZ crystal biosensor was selected to be developed and standardized for rapid diagnosis of RID and typing the causative virus. Internationally and nationally the use of immunobiosensors are restricted to the diagnosis of doping in the field of sports. The application of the assay for the diagnosis have been tried for certain viral diseases of humans but by and large, it is EL ISA which is the test of choice in many diagnostic laboratories. OBJECTS QF THE INVENTION An object of thi» invention im to propose a novel immunobiosensor apparatus for the diagnosis of Foot and Mouth Disease (FMD) in livestocks, with an indication to the exact type of virus involved. Another object of this invention is to propose an immunobiosensor diagnosis kit for the diagnosis of FMD. SUMMARY QF_ THE INVENTION A novel immunobiosensor apparatus developed and standardized according to the invention for diagnosis of Foot and Mouth Disease (FMD) in livestocks with indication to the exact type of virus involved, comprisies a signal transducer capable of oscillating at a specific resonant frequency when connected to an osicillator circuit! a Transistor-Transistor Logie oscillator circuit (TTL) configured around said signal transducer, said circuit being supplied with 6V D.C. power supply from an external sourcei a plurality of electrodes placed on opposite faces of said signal transducer provided with a plurality of long leads; and a frequency counter to measure the output frequency of siad oscillator circuit, wherein a fundamental frequency (f1) of said signal transducer being measured by connecting said oscillator circuit to said transducer, further frequencies (f2 , f3 ) being determined on application of an antibody and an antigen on the surfaces of said transducer, said disease being diagnosed by evaluating the downward shift in mean resonance frequency (A f> for immunoreaction and comparing the came with a standardised A*» charaterized in that said signal transducer is a 6 Mhz PZ crystal emerging as a frequency-determining element rtuseable for example, 6 to 8 times, said diagnosis requiring a time of 90-100 minutes, consumption per test of said antibody or said antigen being 2-5 µl, and said frequency counter providing mathematical value for exact typing of said virus. In accordance with the invention, the FMDV vaccine strains were grown on BHK 21 clone 13 monolayers.A fraction of FMDV was separated by sucrose gradient ultracentrifuation. This antigen was used for production of antisera following the procedure described by Ferris (1988). PZ crystal resonator set-up A transistor-transistor logic (TTL) oscillator a configured around two gates of a SN 7414 IC and a quartz crystal, used as the frequency determining element, were assembled. A resistor was used to provide positive feed back from the output of the first gate to input. Two capacitors, one from input and another from output of the first 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 « measure of isolation of the first gate from the load connected to the output. Ik Ω resistor was used for the positive feed back. The capacitors used were 47 Pf and 68 Pf. One 6 MHz crystal was used as a frequency-determining element. This crystal was provided with two silver electrodes to which twolong 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 external source. The surface of the crystal was made available for immobilization. A scientific frequency counter was used to measure the frequency of output of the oscillator circuit. Immobilising antigen/antibody on PZ crysta1 The antibody against FMDV was coated on a PZ crystal by the following procedure. The PZ crystal was first washed with distilled water and then dipped in ethanol. The crystal was then connected to the oscillator circuit and the fundamental frequency (f 1) 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 37 º C, 1 µl of antibody was pipetted on both the crystal surfaces using a micropipette. The crystal was washed with PBST, dried at 37 ºC, and the frequency (f 2) recorded. The crystal was then ready for immune reaction. Separate crystals were used for each of the FMDV serotypes, viz., '0', 'A', *C and Asia 1'. Development of PZ immunobiosensor One ul of antigen (tissue culture virus harvested a day prior to the experiment with virus titer 10 5 TCID) was deposited on the crystal. The crystal was washed with PBST and dired in an incubator at 37 º C. After drying, the frequency of the oscillation was recorded (f3). Standardization of the Protocol Selection of the PZ crystal was carried out with PZ crystals of various frequencies utilising antibody of Asia 1 as a model and 3 tissue culture Asia 1 virus with a titre of 10 TCID as 50 antigen. PZ crystal of 2,6 and 8 MHz were coated with the antibody and the assay was carried out. The crystal were assessed for their ease of handling and repeatability of the assay. Determination of the positive response: The negative or downward shift,Δf, for immunore*ction was determined by coating ten 6 MHz PZ crystals with antibody and reactd with hmomologous tissue culture antigen dilutions ranging from neat to 10-9 . The process was then repeated with monoclonal antibodies. Repeatability of the Assayt The standardised assay was repeated using the same reagents viz., PBST, the tissue culture antigen and the same crystal. After every assay, the crystal was washed thoroughly and re-coated with the same FMDV type antobody. The test was carried out an each crystal until the results were consistent. Specificity of reaction: Five crystals were coated with FMDV type Asia 1 Antibody. The assay was carried out with different FMDV types as antigens and, for the fifth crystal, tissue culture medium was used as antigen. Similar experiment was carried out by coating the Antibody against the FMDV serotypes. Self-life studies Four crystals were coated with polyclonal antibodies of FMDV type '0', 'A', 'C' and 'Asia 1', as per the standardised 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. Results Selection of the PZ immunobiosensor Sets of the crystals of known frequency were selected for testing and the mean resonant frequency obtained in each experiment. Table 1: Results on standardisation of 2 MHz PZ crystal for immunobiosensor (Table Removed) Table 2:Results on standardisation of 6 MHz PZ crystal for immunobiosensor (Table Removed) Table 3: Results on standardisation of 8 Mhz PZ crystal for immunobiosensor (Table Removed) It was observed that the resonant frequency increases as the size of the crystal decreases. The 6 MHz crystal was convneient 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 0 of FMDV were carried out with with several tissue culture antigens. 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. Further more, the assay could be repeated without any significant variations. Thus the 6 MHz PZ crystals were selected for further standardisation. Determination to the positive response It is found that up to the silution of 10-5 a mean of -2.5 kHz emerged. Therefore, a mean Δ f of -2.5 kHz was taken as positive for diagnosis of FMDV. Results of the PZ immunobiosensor 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 4 depicts the result of the frequency changes during coating and immunoreaction. Table 4: Mean resonant frequency in kHz a heterologous system (Table Removed) The result of frequency changes during coating and immune reaction of a homologous system using tissue culture antigen of titre 105 TCID50 , is shown in Table 3. Table 5: Mean resonant frequency in kHz with homologous TC Ag sample (Table Removed) It was observed that 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 very close to the resonant frequency of the crystal. The frequency (f 2 shifted downward* after antibody coating and when antigen was added. The frequency (f3 ) further shifted downwards in the case of a homologous system (reaction of homologous antigen and antibody). The time required to test one sample was approximately 90 min. This was further reduced to 60 min when antibody-coated crystals were prepared prior to the assaying. The resonant frequencies observed using the same crystal for repeated assays are shown in Table 6. Table 6: Results of the repeatability of PZ crystal immunobiosensor in homologous system. (Table Removed) It should be noted that the results were consistent up to the 8th assay and therefore inconsistent. This could be due to the wear and tear of the crystal surface. This reusability of the crystal minimises the cost of the assay. The specificity of the assay was established from the expreiment results as depicted in Table 7. Table 7: Mean resonant frequency of the PZ crystals to determine the specificity of the reaction. (Table Removed) 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 ' Antigen was used, which proves that the assay was specific. The assay was also compared with selected samples. It should be noted that the PZ crystal immunobiosensor could detect the antigen in all samples, including the one that was found negative by ELISA. It could also detect mixed viral infections of type '0' and 'Asia 1' in two outbreaks from vaccinated cows. (Gajendragad et al., 1999). Results on shelf-1ife studies of the coated PZ crystal There was no change in the resonant frequencies recorded at weekly intervalas shown in Table 8. Table 8: Meann resonant frequency in kHz of the crystals at different times (Table Removed) 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.) The invention is explained in more details hereinbelow with reference to the accompanying figures. In detaili Fig. 1 Shows a circuit diagram for the pietoelectric immunobiosensor according to the invention, 16 Fig. 2 Shows a protocol of the PZ crystal immunobiosensor, Fig. 3 Shows graphical determination of the cut-off point for the PZ crystal of immunobiosensor, A transistor-transistor logic oscillator (Fig. 1) was constructed to cause the selected signal transducer in the form of Pietoelectric crystal to oscillator at a specific resonant frequency under mechanical stress being generated by electric dipoltmt when the crystal is placed on the oscillator circuit provided with power supply from an external source. A secientific frequency counter connected to the oscillator circuit measuring the oscillating frequency of the crystal. The PZ crystal used for the apparatus was selected and standardized after repeated determination of the mean resonant frequency of a plurality of crystals of different denomination for example, 2 MH z, 6 MHz and 8 Mhz . The experiment each time was carried out using PZ crystals coated separately with antibodies against serotype of FMDV with plurality tissue culture antigens. The 6 MH PZ crystal was then selected for the novel z apparatus being free of significant variations and being convient from useability point of view. The shelf-life of different crystals of different sizes were also studied through measuring the resonant frequencies of the crystals at weekly intervals. Results of standardisation of PZ crystals are depicted in figures 3 to 5. It is observed that a similar downward shift in resonant frequency occurs in the case of selected crystal (6 MHz ) in the homologous system. Determination of the positive response was carried-out through repeated evaluation of the negative or downward shift ( f) for immunoreaction using plurality of 6 MHz PZ crystals with antibody reacted with antigen of various dilutions. It is ascertained from the graphical representation (Fig. 6) of the results that upto the dilution of 10 -5 , a mean Δf of -2.5 kH indicates the positive diagnosis of FMDV (Fig.-6). The immunobiosensor are constructed by immobilising antibody against FMDV coated on the selected and standardised signal transducer. The blank PZ crystal was washed with distilled water and then dipped in ethanol. The fundamental frequency (f1 ) of the blank crystal was then recorded in the frequency counter by placing the crystal on the oscillator circuit. The crystal was then dis-engaged from the oscillator circuit and rinsed with PBBT and dried in an incubator. Antibody was coated on the crystal, washed and dried before the frequency (f2 ) was further recorded on re-engaing the crystal on the oscillator circuit. The crystal was then ready for immunoreaction for which antigen was deposited on the crystal. The crystal was further washed with PBST and dried in the incubator before being placed for measuring the frequency (f3 ). The downward shift (Δf) in the resonant frequency when ascertained above a standardised negative shift, a positive result is established. We Claim; 1. An immunobiosensor apparatus for use in identifying Foot and Mouth Disease (FMD) with an indication to the type of virus involved in livestocks, comprising a signal transducer capable of oscillating at a specific resonant frequency when connected to an osicillator circuit; a Transistor-Transistor Logie Oscillator Circuit (TTL) configured around said signal transducer, said circuit being supplied with 6V D.C. power supply from an external source, a plurality of electrodes placed on opposite faces of said signal transducer provided with a plurality of long leads; and a frequency counter to measure the output frequency of said oscillator circuit, wherein a fundamental frequency (f1) of said signal transducer being measured by connecting said oscillator circuit to said transducer, characterised in that said signal transducer is a 6 Mhz PZ crystal emerging as a frequency-determining element. 2. The immouobiosensor apparatus as claimed in claim 1, wherein said TTL-oscillator circuit comprises a plurality of capacitors, a resistor to provide a positive feed back, plurality of electrodes; plurality of internal circuits (Ics) to amplify the input voltage; and an external D.C. supply of 6-volt being provided to the circuit. 3. The immouobiosensor apparatus as claimed in claim 1 wherein said electrodes are made of silver.

Documents:

1245-del-2002-abstract.pdf

1245-del-2002-claims.pdf

1245-del-2002-correspondence-others.pdf

1245-del-2002-correspondence-po.pdf

1245-del-2002-description (complete).pdf

1245-del-2002-form-1.pdf

1245-del-2002-form-19.pdf

1245-del-2002-form-2.pdf

1245-del-2002-form-3.pdf

1245-del-2002-form-4.pdf

1245-del-2002-form-5.pdf

1245-del-2002-gpa.pdf

1245-del-2002-petition-137.pdf