Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-08-31
2002-12-24
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S001100, C435S001300, C435S006120, C435S040500, C435S040520, C435S176000, C435S325000, C436S172000, C422S020000, C422S052000, C422S078000, C422S105000, C427S002130, C427S543000, C250S250000, C250S36100C, C250S459100, C219S687000
Reexamination Certificate
active
06498016
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a rapid method for carrying out microwave mediated ELISA (MELISA). More particularly, this invention relates to a rapid and efficient method for microwave mediated ELISA (MELISA) wherein all the major steps of ELISA can be performed under microwave irradiation in short time. This method is useful in clinical diagnostics, molecular biology, agriculture, food technology, environmental science etc.
The invented ELISA method is simple, time saving and obviates the time consuming cumbersome procedure. This method has the potential for automation.
This method has the advantage over the existing methods for ELISA which take long time usually ranging from several hours to 2 days whereas the invented method takes less than 10 minutes. It is particularly useful for disease diagnosis where quick results are required.
BACKGROUND OF THE INVENTION
Enzyme linked immunosorbent assay (ELISA) is a very sensitive technique used for semiquantitative or quantitative determination of the concentration of certain antigens and antibodies. ELISA has become a useful tool in disease diagnosis in both animals and plants. Apart from this, its other applications include screening of monoclonal antibodies during the course of their production (Douillard, J. Y. and Hoffman, T., 1983), pesticide residue detection in crop produce (Van Emon, J. M. and Lopez-Avila, V., 1992) and environmental samples like soil and water (Linde, D. G. and Goh, K. S., 1995), detection of apoptosis in tissue culture etc. (Salgame, P. et al, 1996). Polystyrene microtitre plate is used universally for carrying out ELISA, as it is transparent, cheap, easily available, can be moulded to any desired shape and has a property of binding proteins through adsorption. Conventional methods of ELISA are based on the immobilization of antigen or antibody onto the surface of the wells of a polystyrene microtitre plate through adsorption. This is attributed to the non-covalent interaction between the biomolecule and the polystyrene surface.
However, adsorption is usually too inefficient a process to give good yields and doesn't always proceed in a dose dependent manner. To overcome the inefficiency of conventional methods covalent immobilization of biomolecules onto the microtitre plate has been carried out by many (Satoh, A. et al, 1999). Covalent binding of immunogens to grafted plastic surfaces has also been reported (Larsson, P. H. et al, 1987). Despite this, the conventional ELISA method requires very long time varying from several hours to 2 days for completion. This is the main drawback of different ELISA methods, either based on adsorption or covalent binding. In case of medical urgency precious time is lost in diagnosis before the patient could be given medication. In agriculture, ELISA is usefull for detecting pesticide residues in crop produce and environmental samples. Export and marketing of crop produce can be delayed as a consequence of this handicap in the ELISA method, which contributes to loss of valuable foreign exchange.
The applicants have developed a novel and unique method whereby ELISA can be carried out rapidly by the use of microwaves. Microwaves are known for accelerating immunohistochemistry for about a decade (Boon, M. E. and Kok, L. P., 1992; Boon, M. E. et al, 1989; Boon, M. E. et al, PCT patent application WO 89/ 03038; Chiu, K. Y. and Chan, K. W., 1987; Hjerpe, A. et al, 1988).
Covalent immobilization of antigen or antibody on to a polystyrene surface by microwave irradiation has not been reported so far. In fact, all the major steps of ELISA by microwave irradiation in such a short time to detect minute quantities of antigen or antibody by measuring optical density was not known in the prior art.
However, there are attempts for doing one of the steps of ELISA by microwave irradiation (Hierpe, A. et al, 1988) where polystyrene ELISA plates were coated first with rabbit anti-carcinoembryonic antigen by incubating over night at 4° C., followed by incubation of the antigen (CEA). In the subsequent step i.e. after the addition of enzyme-linked antibodies the authors studied the effect of microwave irradiation on antigen-antibody reactions.
In another experiment by the same authors, ELISA plates were first coated with normal mouse serum by incubating over night at 4° C., followed by overnight incubation with non-labeled rabbit anti-mouse Ig. In the subsequent step they added mouse PAP (peroxidase-antiperoxidase) complexes and determined the effect of microwave irradiation on the reactivity constants of reactions of this last step.
In third experiment Hjerpe et.al first coated the plates with non-specific mouse serum followed by incubation with biotinylated horse-anti-mouse IgG. The plates were then used to study the effect of microwaves upon the subsequent binding of biotin-avidin complexes.
The reaction yields in all the above experiments were smaller in samples subjected to microwave irradiation as compared to those processed without microwave stimulation. The experiments showed that the microwaves caused a major loss of reactivity and the total yields were approximately 10% to 15% compared to conventional one that was carried out outside the microwave oven. According to authors, the diminished value in the microwave technique may be due to too high temperatures in the wells, despite the fact that a water load (a beaker of water to absorb excess microwave energy) and chilled bottom plate were taken as a precautionary measure.
In further ELISA experiment authors (Koh and Boon, 1992) used a fiberoptic thermometer to restrict the temperature below 40° C. Here also a water load of 200 ml tap water was taken. Besides, the fluid in the wells was stirred by slowly blowing air through the solution via thin plastic tips, which were inserted into the wells. In an experiment the authors carried out only two steps namely, antibody and conjugate binding steps by microwave irradiation for 6 minutes at 150 watts each.
In another experiment, antibody, antigen and conjugate binding steps were carried out in 15, 30 and 30 minutes respectively using 45-50% microwave power in each step.
Remaining steps in both the experiments were carried out by conventional procedure. But the ELISA values were found to be much less in the above experiments than the conventional method.
According to authors, the longer exposure time gave higher extinction value (ELISA value) but the time gain was not attractive. In fact there was no benefit when exposure time of 30 minutes or more were used. Too short exposure times led to extinction values which, were rather low and could not be used.
The reported methods of ELISA by microwave exposure has several drawbacks such as (1) the results (ELISA value) were much less than the conventional procedure, (2) the time gain was not attractive. It may be possible to get comparable results (ELISA value) by doing the ELISA out side microwave oven in the same time, (3) procedure required water load, (4) it required cooling system or chilled bottom plate, (5) it needed a stirring system in the well of the microtitre plate, (6) not all the steps were carried out by microwave energy and the reported ELISA procedure has little or no potential for automation.
The applicants in the present invention have overcome all the above drawbacks. In fact, like thermal energy microwave can also activate or inactivate a biomolecule. Without proper conditions the microwave may cause partial or total destruction of the biomolecule leading to low or undesirable ELISA value. In the invented procedure proper conditions were found, most of, which are contrary to the reported method, or not known in the prior art. In the invented method all the steps except color development are carried out by microwave stimulation. Blocking step was not carried out by microwave irradiation in the published procedure as it gave nonspecific binding, whereas the applicants have invented a method where blocking step is carried out in short time by microwave irradiation. Longer microwave exposure time gave highe
Bora Utpal
Nahar Pradip
Sharma Gainda Lal
Chin Christopher L.
Cook Lisa V.
Council of Scientific and Industrial Research
Ladas & Parry
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