Title of Invention

AN ENZYME-LINKED IMMUNOSORBENT ASSAY PROCEDURE BY SONICATION

Abstract

The present invention relates to a rapid enzyme-linked immunosorbent assay procedure for the detection of small quantities of biomolecule such as antigen or antibody by sonication. This invention particularly relates to the sonication -assisted enzyme-linked immunosorbent assay (Sono-ELISA or SELISA) which comprises of immobilization of antigen or antibody onto the solid surface and performing subsequent ELISA steps by sonication at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes and at a temperature ranging from 30°C to 60°C. The ELISA method described in the present invention can be achieved very fast preferably in the time range of 4 - 40 minutes. The invented procedure is useful for performing ELISA required in diagnostics, agriculture, food technology, environmental science, research work etc. The invented ELISA procedure is rapid, economical, reproducible and simple and has the potential for automation.

Full Text

FIELD OF INVENTION The present invention relates to an enzyme-linked immunosorbent assay procedure by sonication. More particularly, the present invention relates to a simple method for enzyme-linked immunosorbent assay technique on a solid surface by ultrasound waves. BACKGROUND AND PRIOR ART The Enzyme-linked immunosorbent assay (ELISA) is an important tool for clinical diagnostics for detection of antigens or antibodies in the diseased sample of both animals (Alois! et.al, Am.J.Med Technol (1981); An ELISA method for detecting unexpected antibodies; 47; 913-918) and plants. Other applications of ELISA include detection of bio-threat agents (Turner et.al, Biosens Bioelectron (2001); Substitution of antibodies and receptors with molecularly imprinted polymers in enzyme-linked and flurorescent assays; 16; 701-707, Reynolds et.al. J. immunol. Methods (2002); Detection strategies for catalytic antibodies; 269; 125-31, Huels et.al. Drug Discovery Today (2002); The impact of protein biochips and microarrays on the drug development process; S119-S124), biomedica! research and high throughput screening for discovery of drug or drug targets (Kricka et.al. Clin.Chem (1980); Variability in adsorption properties of microltitre plates used as solid supports in enzyme immunoassay; 26; 741-744). Conventional ELISA method takes very long time varying from several hours to 2 days for completion. This is one of the major drawbacks of the ELISA procedure required for many important applications. Rapid ELISA can facilitates proper medication by quick diagnosis, accelerates the pace of research including drug screening leading to drug discovery. Rapid ELISA is also necessary for detecting bio-threat agent. Many attempts have been made to shorten ELISA timing. Bora et al. J. Immunol.Methods (2002); Covalent immbolization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures; 268;171-177 have reported a rapid and simple method for a double-antibody sandwich (DAS) ELISA and a direct antigen coating (DAC) ELISA by covalently immobilizing antigen or antibody onto the activated polystyrene surface, developed by Nahar et.al. Anal. Bioechem (2001); Light-Induced Activation of an Inert Surface for Covalent Immobilization of a Protein Ligand; 294; 148-153. Detection of antibodies at lower concentrations was also done successfully using such activated surface. The ELISA value obtained in about eight hours by this method was found to be similar with the values obtained in the conventional ELISA method carried out in twenty hours. ELISA timing was further shortened by Bora et.al by carrying out ELISA at an elevated temperature. Thus, Bora et al J. Immunol. Methods. (2004); Heat-mediated enzyme-linked immunosorbent assay procedure on a photoactivated surface ;293 ; 43-50., carried out heat-mediated ELISA (HELISA) at temperature ranging from 40-50°C in about 3 hours on an activated polymer surface (US 2004/0191831 A1). However, dramatic decrease in ELISA timing was reported by Nahar et.al (US 6498016 B1) where microwave-mediated ELISA (MELISA) decreased the ELISA timing to less than 10 minutes. MELISA is based on dielectric polarization of the molecule (s); hence composition of the incubation fluids is of significant importance. Absorption of microwave energy and thus increase in temperature depends on molecules present in the incubation fluids. MELISA depends on microwaves which have two different effects acting simultaneously on the bio molecules. These effects are microwave thermal and microwave specific or microwave non-thermal effect. Microwave specific effect or non-thermal effect is an effect which can activate or deactivate a biomolecule even if temperature is not enhanced (Andre Loupy et al. Pure Appl. Chem (2001), Reactivity and selectivity under microwaves in organic chemistry. Relation with medium effects and reaction mechanisms 73; 161-166, S.C. Sudrik et al. J.Org.Chem (2002)., Microwave Specific Wolff Rearrangement of Diazoketones and Its Relevance to the Nonthermal and Thermal Effect; 67; 1574-1579). MELISA is a sensitive technique where a slight change in microwave frequency, output power or time may give non-specific binding leading to non productive assay. Therefore, there is a need for a rapid ELISA method where slight change in frequency, output power or time will not enhance non-specific binding. The applicants in the present invention have invented a novel ELISA technique using ultrasound waves. This sound wave- mediated ELISA or Sono-ELISA (SELISA) is not depended on reagents, neither a slight change in parameters like time affects the ELISA value considerably. Ultrasound waves have found applications in many fields including chemical (W.M. de Azevedo et.al Ultrasonic Sonochemistrv (2006) The effect of ultrasonic waves in conducting polymer solution; 13 (5); 433-437 and V. Calvino et.al Applied Surface Science (2006); Ultrasound accelerated Claisen-Schmidt condensation: A green route to chalcones ; 252 (17); 6071-6074) and biological fields. Ultrasound have been applied for enhancement of the reaction rate of agglutination test (Thomas et.al, Ultrasound in medicine and Biology. (1999): Ultrasonic enhancement of coated particle agglutination immunoassay: Influence of particle density and compressibility; 25 (3) 443-450), for red blood cell agglutination IN VITRO (Doubrovski, Ultrasound in Medicine and Biology (2000); Ultrasonic wave action upon the red blood cell agglutination IN VITRO ; 26 (4) 655-659) and latex immuno agglutination assay (Bhaskar et.al, Journal of Immunoloaical Methods (2002) 262 181-186, for plasma preparation from whole blood (Cousins et.al Ultrasound in Medicine and Biology (2000); Plasma preparation from whole blood using ultrasound; 881-888), for elution of antigen from dried blood (Takkouche et.al. . Immunology letters (1995); Detection of Brucella antibodies in eluted dried blood: a validation study. Immunology letters; 107-108) and mycobacterium cell (Speer et.al. Clinical and vaccine immunology (2006); A Novel enzyme linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. parartuberculosis infections (Johne's disease) in cattle; 535-540) and arrangement of particles in a gel matrix (Gherardini et.al, (2004) Ultrasound in Med. & Biol. 31 (2) 261-272. They are also used in membrane technology to enhance filtration rate (Masselin et.al, Journal Of Membrane Science (2001V 181. 213-220 In the present application ultrasound waves were used for immobilizing antigen or antibody onto the solid surface followed by carrying out subsequent ELISA steps by controlled sonication. Sonication-mediated ELISA has not been reported in the prior art. The present invention relates to an efficient sonication-mediated ELISA method for detection of antigen, antibody etc. This procedure has reduced the total time required for ELISA ranging from 4 min to 40 minutes from 12 to 20 hours in conventional ELISA. Novelty of the present invention is that sonication- mediated ELISA (SELISA) procedure is carried out on an activated surface in 4 - 40 min at a temperature ranging from 30°C to 60°C. All the steps of ELISA such as antigen binding, blocking, antibody and enzyme-conjugated antibody binding are carried out by ultrasound energy generated in a sonicator bath. The invented SELISA procedure is rapid with comparable ELISA value than the conventional method. Both the thermal and non-thermal effect of ultrasound energy is utilized in sonication -mediated ELISA. OBJECTS OF THE INVENTION Thus the main object of the present invention is to provide a novel sonication mediated enzyme-linked immunosorbent assay procedure. Another object of the present invention is to provide a process that shortens the time period of classical ELISA procedure. Still another object of the present invention is to use an alternate source of energy to carry out ELISA technique which has the potential for automation. Yet another object of the present invention is to use sonication energy and an activated surface for rapid ELISA. SUMMARY OF THE INVENTION The present invention provides a rapid enzyme-linked immunosorbent assay procedure for the detection of small quantities of biomolecule such as antigen or antibody by sonication. This invention particularly relates to the sonication -assisted enzyme-linked immunosorbent assay (Sono-ELISA or SELISA) which comprises of immobilization of antigen or antibody onto the solid surface and performing subsequent ELISA steps by sonication at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes and at a temperature ranging from 30°C to 60°C. The invented ELISA can be achieved very fast preferably in the time range of from 4-40 minutes. The invented procedure is useful for performing ELISA required in diagnostics, agriculture, food technology, environmental science, research work etc. The invented ELISA procedure is rapid, economical, reproducible and simple and has the potential for automation. Accordingly, the present invention provides novel sonication mediated enzyme-linked immunosorbent assay process characterized by sonication-mediated binding of biomolecules, wherein the said method comprising: (a) providing an activated solid support; (b) loading a biomolecule onto the activated support of step (a); (c) binding the loaded biomolecule of step (b) onto the activated solid support of step (a) by sonicating with ultrasound waves at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C followed by washing with a buffer; (d) blocking the free surface of the activated solid support having an immobilized biomolecule as obtained from step (c) by loading a blocking solution into the said support and sonicating at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C and washing with buffer; (e) loading another biomolecule complementary to that loaded in step (b) onto the activated support as obtained from step (d) followed by sonicating at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C followed by washing with buffer; (f) loading an appropriate enzyme-conjugate dissolved in a suitable buffer onto the solid support obtained from step (e) followed by sonicating the said support at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C and washing with buffer; (g) adding a substrate-dye-buffer to the support as obtained from step (f) and incubating for a period ranging from 4 to 10 minutes in dark; (h) adding a stop solution to the support as obtained from step (g) followed by measuring the optical density at a suitable wavelength. In an embodiment of the present invention the solid support used is selected from the group consisting of materials such as polycarbonate, polystyrene, polypropylene, polyethylene, glass, cellulose, nitrocellulose, silica gel, polyvinyl chloride and any other solid support used for ELISA. In an embodiment of the present invention, the preferred solid support used is polystyrene. In yet another embodiment the solid support is selected from any shape, form and size such as RCR plates, EL1SA plate, microwell plate, sheets, particles such as beads and microspheres, tubes, sticks, strips, well or module. an embodiment of the present invention, the solid support used for immobilizing biomolecules is selected from any solid support capable of binding a ligand molecule either by covalent or non-covalent binding. In yet another embodiment of the present invention the active functional group may be present in the support itself or can be introduced by conventional chemical or photochemical or other methods known to prior art. In yet another embodiment of the present invention polystyrene surface is activated by coating 1-fluoro-2-nitro-4-azidobenzene (FNAB) and exposing the coated support to light source. In yet another embodiment of the present invention, the light source is selected from any light capable of carrying out the photochemical reaction such as UV lamp, laser beam, and sunlight or alike. In yet another embodiment of the present invention, time for photoreaction for activation of solid support is selected from 1 min to 30 min. In yet another preferred embodiment of the invention, the first step of SELISA, is carried out by covalently binding the antigen or antibody onto the activated plate by sonicating at a frequency ranging between 25 -130 kHz and ultrasound power output ranging from 60 W -300 W for a period of 1 -30 min at a temperature ranging from 30°C to 60°C. In yet another preferred embodiment of the invention the second step of SELISA, which is the blocking step, is carried out by sonication at a frequency ranging between 25 -130 kHz and ultrasound power output ranging from 60 W-300 W for a period of 1 -30 min at a temperature ranging from 30°C to 60°C. In yet another preferred embodiment of the invention the third step of SELISA that is corresponding antibody or antigen binding is carried out by sonication at a frequency ranging between 25 -130 kHz and ultrasound power output ranging from 60 W-300 W for a period of 1 -30 min at a temperature ranging from 30°C to 60°C. In yet another preferred embodiment of the invention, the fourth step of SELISA that is enzyme-conjugated secondry antibody binding is carried out by sonication at a frequency ranging between 25 -130 kHz, ultrasound power output ranging from 60 W-300 W for a period of 1 - 30 min at a temperature ranging from 30°C to 60°C. In yet another preferred embodiment of the invention, blocking reagent is selected from bovine serum albumin, skimmed milk powder, gelatine or any blocking agent known to prior art. In another embodiment to the present invention biomolecule is selected from antigen or antibody. In another embodiment to the present invention conjugate is selected from biomolecule having antibody or antigen conjugated with an enzyme selected from horseradish peroxides or alkaline phosphates. In yet another preferred embodiment of this invention, enzyme may be replaced by a label selected from chromophore, fluorophore and alike which facilitates its assay. In yet another preferred embodiment of this invention, the invented procedure can be used for other immunoassays like radio immunoassay, radio-immunosorbent test, radio allergosorbent test, biotin- avidin/streptavidin immunoassay, immnunoblotting, immunostaining etc. apart from different types of ELISA such as direct ELISA, indirect ELISA, sandwich ELISA and alike. In yet another preferred embodiment of this invention, an apparatus may be made which is capable of generating ultrasound waves for sonication-mediated enzyme linked immunosorbent assay (SELISA). DETAILED DESCRIPTION OF THE INVENTION The present invention provides a simple method for enzyme-linked immunosorbent assay technique on a solid surface by ultrasound waves. Any solid surface that is used for conventional ELISA can be used for sonication-mediated ELISA (sono-ELISA or SELISA). Activated surface immobilize antigen or antibody either by adsorption or by covalent binding. In the invented procedure activated surface was made which immobilizes the antigen or antibody through covalent bonding. Untreated surface is also used for SELISA, but gave low ELISA readings. In the invented procedure, all the experiments involving ultrasound waves were carried out in a sonicator bath operating at a frequency of 40 KHz. During operation 1.8 litre of water was used in the sonicator bath. During each step of the experiment initial bath temperature was 37°C. Temperature was raised during sonication in each step and was not lowered by cooling down. The sonicator bath temperature in which the each step of SELISA was carried out was in the range of 37 °C to 50 °C. Each step of SELISA was optimized by carrying out the subsequent steps by conventional ELISA. The first step of ELISA was optimized by immobilizing goat anti-human IgG onto the polystyrene microtiter plate by sonication in different time and different ultrasound output power (110 W and 120 W). In both the output power (110 W and 120 W) the maximum immobilization occurred at 10 min although 5 min immobilization also showed reasonably good ELISA readings. At higher output power that is at 120 W slightly better result was observed than 110 W. Untreated surface showed much lower ELISA values than the activated surface (Table 1 A and 1 B). In the second step of SELISA, blocking was carried out with 2% BSA in different timings by sonication at output power of 110 W and 120 W. In both the cases maximum absorbance was obtained in 10 min beyond which no appreciable increase in ELISA value was observed. (Table 2 A and 2 B). In the third step of SELISA, human IgG binding to the immobilized anti human IgG was carried out by sonicating it in different time at 110 W and 120 W. Human IgG was found to optimally bind to the solid phase in 15 min at 110 W (Table 3 A) and in 10 min at 120 W (Table 3 B). Similar results were obtained for fourth step of enzyme-conjugated antibody binding where excellent result was obtained in 15 min at 110 W (Table 4 A) and in 10 min at 120 W (Table 4 B). In all the above experiments (Table 1 -4) ultrasound wave- mediated binding at 120 W showed better result than 110 W although 110 W was also found to give comparable ELISA value. At an output power of 120 W, maximum absorbance was obtained when SELISA was carried out for 10 min in each step (Table 4 B). However, in all the above experiments (Table 1-4) 5 min sonication in each step showed fairly good ELISA value. To find out the minimum time required for SELISA at 120 W we have carried out SELISA in 4, 12, 20, 28 min respectively (for each step 1, 3, 5, 7 min respectively). To compare the results, 40 min- SELISA (10 min for each step) and control (conventional) ELISA were also carried out. Result showed that 10 min -SELISA was comparable to conventional ELISA carried out in 20 h. Gradual decline in ELISA values were observed when SELISA time was decreased from 40 min to 4 min. Nevertheless detectable ELISA values were observed even in less time (Table 5). During SELISA experiments, initial temperature of the sonicator bath was 37°C which was increased to 43°C after 10 min. To find out whether increase in ELISA value in short time in SELISA experiment is due to thermal effect, ELISA was performed by different methods in 10 min such as (i) ELISA by sonication at 37-43°C, (ii) ELISA by sonication at 37°C, (iii) ELISA by thermal incubation at 37°C, (iv) ELISA by thermal incubation at 50°C (blocking 40°C). Control experiment was carried out by conventional ELISA in 20 h. The absorbance values showed 40 min - SELISA and 20 h-conventional ELISA was very similar. Interestingly 40 min - ELISA at elevated temperature (maximum 50°C) showed much lower ELISA reading than that of SELISA even though temperature of the sonicator bath was much lower (maximum 43°C). It clearly demonstrated that ultrasound waves have non-thermal effect. It was further confirmed when sonication at 37°C gave nearly 3 times more ELISA readings than thermal incubation at 37°C (Table 6). The results clearly show that ultrasound waves exert specific or non-thermal effect which enhances the reaction rate. Although the mechanism of ultra sound effect is still debatable it is likely that ultrasound waves exert variety of non thermal bioeffects via acoustic caviation. Besides, sonication has also thermal effect. During sonication heat is generated which also play a role in sonication -mediated ELISA. Here, both the effects of sonication such as sonication thermal effect and sonication non-thermal effect have been utilized for sonication -mediated ELISA. Thus, time required in each step in SELISA may vary depending on time, output power, temperature and or frequency of sonication and excellent results can be obtained by minor modification of reaction conditions. The invented procedure can be adapted to any solid surface of any shape or size. However, polystyrene microtiter plates were used for SELISA as they were widely used for ELISA technique. Any polystyrene microtiter plates available in the market for ELISA purpose can also be used for SELISA technique. These polystyrene plates are activated by different procedures to make them suitable for ELISA. In the present invention we have activated 96-well polystyrene microtiter plates by exposing the FNAB coated wells of the plate to UV radiation or bright sunlight. This activated support was used for sonication - mediated ELISA (SEL1SA) and for control ELISA to detect human IgG. SELISA experiments were performed in an ELMA (Germany) Sonicator bath, model no. T490 DH, operating at a frequency of 40 kHz at 230 V and has a maximum capacity of about 2.6 litre. During operation 1.8 litre of water was used. Phosphate buffer saline (PBS), pH 7.2, 0.01 M was used as antibody dilution buffer. Phosphate buffer saline (PBS), pH 7.2, 0.01 M together with 0.1% Tween- 20 was used as washing buffer in all the experiments. Blocking solution was made by dissolving 2% BSA in triple distilled water Substrate solution was prepared by adding 2 mg o-phenylenediamine hydrochloride and 2 pi H2O2 in 6 mf of 0,2 M phosphate- citrate buffer, pH 5. Normal rabbit sera were taken as negative control sera (-ve sera). Horseradish peroxidase conjugated anti-human IgG was purchased from Sigma as lyophilized powder. After reconstitution, the optimum dilution was found to be 1: 2000 as determined by checkerboard titration, which was used in the experiments. In all the ELISA experiments, absorbance values are expressed as mean of three replicates. ELISA is a 5-step procedure, namely antigen binding, blocking, antibody binding, enzyme-conjugated antibody binding and colour development. Each step of SELISA was optimized by carrying out the subsequent steps by conventional ELISA procedure. Conventional ELISA was carried out by coating the wells with anti-human IgG overnight at 4°C, blocking in 1 h at 37°C followed by antibody and conjugate binding at 37°C for 3 h each. The fifth step which is common for all carried out at room temperature for 5 min and absorbance was recorded in an ELISA reader at 490 nm. The data obtained for different time and power settings clearly shows that there is a wide range of parameters where SELISA can be carried out successfully. Therefore, several embodiments of the present invention can be optimized to suit the specific need. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 Activation of polystyrene microtiter plate FNAB (1.82 mg / 50 jil of ethanol /well) was poured into the wells of polystyrene microtiter plate. Ethanol was allowed to evaporate in the dark and the wells were irradiated by UV light at 365 nm for 10 min in an UV Stratalinker (Stratagene, USA). The wells were then washed with ethanol and dried. These activated wells were used for immobilization of antigen or antibody in the invented procedure. Example 2 (A) Immobilization of goat anti-human IgG by ultrasound waves at output power of 110 W (Table 1 A) Goat anti-human IgG (0.25 ug /100 ^l PBS, pH 7.2, 0.01 M / well) was loaded in triplicate wells of microtiter plates. The plates were then sonicated for 5, 10, 15, 20, and 25 min respectively at an ultrasound output power of 110 W in a sonicator bath. The plates were then washed with washing buffer. Blocking step was done with 2% BSA solution (200 |al/well) at 37°C in 1 h. After washing, each well was loaded with (0.25 ug /100 ^l) of human IgG (dissolved in 0.01 M PBS, pH 7.4) and the plates were incubated for 3 h at 37° C. The plates were again washed and each well was loaded with 100 ^ of goat anti-human IgG-peroxidase (1:2000) solution (dissolved in 0.01 M PBS, pH 7.4) and incubated for 3 h at 37° C. After washing, 100 ^l of substrate-dye solution was loaded into each well. Color development was stopped after 5 min by adding 20 nl of 5% H2SO4. Absorbance was recorded at 490 nm. The experiments were repeated using untreated polystyrene plates. Control experiment was carried out similarly except that antihuman IgG was immobilized at 4°C in 13 h (overnight). (Table Removed) Detection of human IgG by carrying out first step by SELISA at 110 W and remaining steps by ELISA. SELISA: Step-1. Immobilization of anti-human IgG by sonication. Output power- 110 W; time-variable as in the table. Control: Step-1. 4°C, 13 h ELISA: (Step-2 to 5. conventional procedure) Blocking- 37°C,1h; Human IgG binding- 37°C,3h; Conjugate binding- 37°C, 3h. Color development - room temperature, 5 min. Example 2 (B) Immobilization of goat anti-human IgG by ultrasound waves at output power of 120 W (Table 1 B) Experiments were carried out similarly as described in example 2 (A) except that goat anti-human IgG was immobilized by sonication at an ultrasound output power of 120 W in the sonicator bath. (Table Removed) ELISA: Step-1. Immobilization of anti-human IgG by sonication. Output power- 120 W; time-variable as in the table. Control: Step-1. 4°C, 13h. ELISA: (Step-2 to 5. conventional procedure) Blocking- 37°C,1h; Human IgG binding- 37°C,3h; Conjugate binding- 37°C, 3h. Color development - room temperature, 5 min. Example 3 (A) Blocking by ultrasound waves at output power of 110 W (Table 2 A) Goat-anti human IgG (0.25 ^g/100 jJ PBS /well) was immobilized on the triplicate wells of six activated plates in 10 min by sonication at 110 W. After washing, the wells were loaded with 200 \i\ of blocking solution. The plates were then sonicated for 5, 10, 15, 20 and 25 min respectively. Incubation of both human IgG (0.25 i^g/100 ^I/well) and enzyme conjugated antibody (100 nl of 1:2000 dilution) were carried out at 37°C for 3 h each. Experiments with untreated plates were carried similarly for each corresponding time. Control experiment was carried out similarly except that blocking was carried out at 37°C for 1 h. Table 2 A. Detection of human IgG by carrying out first two steps by SELISA at 110 W and remaining steps by ELISA. SELISA: Step-1. Immobilization of anti-human IgG by sonication. Output power-110 W; time- 10 min Step-2. Blocking at 110 W in variable time as in the table. Control: Blocking- 37°C, 1h. (Table Removed) LISA: (Step-3 to 5. conventional procedure); Human IgG binding- 37°C, 3 h; Conjugate binding- 37°C, 3 h. Color development - room temperature, 5 min. Example 3 (B) Blocking by ultrasound waves at output power of 120 W (Table 2 B) Experiments were carried out similarly as described in example 3 (A) except that first two steps were carried out by sonication at an ultrasound output power of 120 W in the sonicator bath. Table 2 B. Detection of human IgG by carrying out first two steps by SELISA at 120 W and remaining steps by ELISA. SELISA: Step-1. Immobilization of anti-human IgG by sonication. Output power- 120 W; time- 10 min Step-2. Blocking at 120 W in variable time as in the table. Control: Blocking- 37°C, 1 h. ELISA: (Step-3 to 5. conventional procedure); Human IgG binding- 37°C, 3 h; Conjugate binding- 37°C, 3 h. Color development - room temperature, 5 min. Example 4 (A) Binding of human IgG by ultrasound waves at output power of 110 W (Table 3 A) The solid phases were prepared by immobilizing goat anti-human IgG (0.25 ng/100 \d PBS/ well) in activated and untreated wells of a polystyrene plate by sonicating it at 110 W for 10 min. Blocking was carried out by sonication at 110 W in 10 min. The wells were then loaded with human IgG solution (0.25 pg /100 jJ/well) and sonicated for 5, 10, 15, 20 and 25 min respectively operating at ultrasound output power of 110 W. The anti human IgG- peroxidase (100 ul/well) binding was done at 37°C for 3 h. The experiments were repeated using untreated polystyrene plates. Control experiment was carried out similarly except that human IgG binding was carried out at 37° C for 3 h. Table 3 A. Detection of human IgG by carrying out first three steps by SELISA at 110 W and remaining steps by ELISA. SELISA: Step-1: Immobilization of antihuman IgG at 110 W in 10 min. Step-2: Blocking at 110 W in 10 min. Step-3: Human IgG Binding at 110 W in variable time as in the table, Control: Human IgG Binding - 37°C, 3 h. ELISA: (Step-4 to 5. conventional procedure); Conjugate binding- 37°C, 3 h. Color development - room temperature, 5 min.. Example 4 (B) (Table Removed) inding of human IgG by ultrasound waves at output power of 120 W (Table 3 B) Experiments were carried out similarly as described in example 4 (A) except that first three steps were carried out by sonication at an ultrasound output power of 120 W in the sonicator bath. Table 3 B. Detection of human IgG by carrying out first three steps by SELISA at 120 W and remaining steps by ELISA. SELISA: Step-1: Immobilization of antihuman IgG at 120 W in 10 min. Step-2: Blocking at 120 W in 10 min. Step-3: Human IgG Binding at 120 W in variable time as in the table, Control: Human IgG Binding - 37°C, 3 h. ELISA: (Step-4 to 5. conventional procedure); Conjugate binding- 37°C, 3 h. Color development - room temperature, 5 min.. Example 5 (A) Binding of goat anti-human IgG peroxidase conjugate by ultrasound waves at output power of 110 W (Table 4 A) Preparation of solid phase and blocking were done in 10 min each at 110 W. Human IgG binding was carried out by sonication at 110 W for 15 min. Goat anti-human IgG-peroxidase conjugate (100 jjJ /well) was then sonicated at 110 W for 5, 10, 15, 20 and 25 min respectively. After colour development, the absorbance was recorded as above. Experiments were carried out with the untreated surface under similar conditions. Control experiment was carried out similarly except that anti human IgG -conjugate binding was carried out at 37° C for 3 h. Table 4 A. Detection of human IgG by carrying out first four steps by SELISA at 110 W and remaining steps by ELISA. SELISA: Step-1: Immobilization of antihuman- IgG at 110 W in 10 min. Step-2: Blocking at 110 W in 10 min. Step-3: Human IgG Binding at 110 W in 15 min. Step-4: Conjugate binding at 110 W in variable time as in the table. Control: Conjugate binding - 37°C, 3 h. Color development - room temperature, 5 min. Example 5 (B) Binding of goat anti-human IgG peroxidase conjugate by ultrasound waves at output power of 120 W (Table 4 B) Experiments were carried out similarly as described in example 5 (A) except first four steps were carried out by sonication at an ultrasound output power of 120 W in the sonicator bath. Time for first three steps was 10 min each. Table 4 B. Detection of human IgG by carrying out first four steps by SELISA at 120 W and remaining steps by ELISA. SELISA: Step-1: Immobilization of antihuman- IgG at 120 W in 10 min. Step-2: Blocking at 120 W in 10 min. Step-3: Human IgG Binding at 120 W in 10 min. Step-4: Conjugate binding at 120 W in variable time as in the table. Control: Conjugate binding - 37°C, 3 h. Color development - room temperature, 5 min. Example 6 SELISA carried out in different timings (Table 5) Human IgG was detected by carrying out SELISA at 120 W in different timings. Thus, in 5 sets of experiments SELISA was carried out in 4 min, 12 min, 20 min, 28 min and 40 min respectively with equal timings for each step (i.e. goat anti-human IgG binding, blocking, human IgG and conjugate binding). Conventional experiment was performed as described earlier. Table 5. Detection of human IgG by carrying out first four ELISA steps at ultrasound power of 120W. Time for each step: 1 min, 3 min, 5 min, 7 min and 10 min respectively in five different experiments. Initial temperature 37°C, Maximum temperature 43°C (reached after 10 min). Conventional: 4° C, 13 h ; 37° C, 1 h; 37°C, 3 h and 37°C, 3 h. Color development: room temperature, 5 minutes. (Table Removed) xample 7 ELISA in 40 minutes by different methods (Table 6) (i) SELISA was performed by immobilizing goat anti-human IgG in 10 min, blocking in 10 min, human IgG binding in 10 min and conjugate binding in 10 min by sonication at the ultrasound power output 120 W. In each step initial temperature of the sonicator bath was 37°C and final temperature was 43°C. (ii) The above experiment was repeated except that the temperature of the sonicator bath was maintained at 37°C. (iii) ELISA was carried out in a thermal incubator at 37°C. (iv) Another experiment was performed by carrying out each step of ELISA in 10 min at an elevated temperature in an incubator such as goat anti-human IgG immobilization at 50°C, blocking at 40°C, human IgG binding and conjugate binding each at 50°C respectively. (v) Conventional ELISA was performed by immobilizing goat anti-human IgG at 4°C in 13 h, blocking in 1 h at 37°C, human IgG binding in 3 h at 37°C and conjugate binding in 3 h at 37°C. All these experiments were carried out with activated and untreated plates. Table 6. Detection of human IgG by carrying out ELISA in 40 min by different methods and compared with 20 h-conventional ELISA. (i) ELISA steps 1-4: sonication at 120 W, 10 min each step, temperature 37° C to 43°C in 10 min. (ii) ELISA steps 1-4: sonication at 120 W, 10 min each step, temperature, fixed - 37° C. (iii) ELISA steps 1-4: thermal incubator, 10 min each step, temperature, fixed - 37° C. (iv) ELISA steps 1-4: thermal incubator, 10 min each step at elevated temperature (50°C, 40°C, 50° C and 50°C respectively for four steps) (v) ELISA steps 1-4: (conventional) 4°C,13 h; 37°C,1 h; 37°C, 3 h and 37°C, 3 h (Table Removed) dvantages: 1. The present invention provides an ELISA procedure, which takes 4-40 min, is much less time consuming than the conventional procedure (usually 20 h) or HELISA procedure (about 3 h) 2. The invented procedure is simple and does not require any additional expertise, reagent, or equipment. It can be carried out on common laboratory apparatus such as sonicator bath. 3. A small deviation in the incubation conditions from the optimum conditions does not affect the ELISA value significantly in the present invention in contrast to microwave- mediated ELISA (MELISA) where slight change in power or temperature gives non specific binding leading to failed ELISA We claim: 1. A novel sonication mediated enzyme-linked immunosorbent assay process characterized by sonication-mediated binding of biomolecules, wherein the said method comprising: (a) providing an activated solid support; (b) loading a biomolecule onto the activated support of step (a); (c) binding the loaded biomolecule of step (b) onto the activated solid support of step (a) by sonicating with ultrasound waves at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C followed by washing with a buffer; (d) blocking the free surface of the activated solid support having an immobilized biomolecule as obtained from step (c) by loading a blocking solution into the said support and sonicating at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C and washing with buffer; (e) loading another biomolecule complementary to that loaded in step (b) onto the activated support as obtained from step (d) followed by sonicating at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a time period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C followed by washing with buffer; (f) loading an appropriate enzyme-conjugate dissolved in a suitable buffer onto the solid support obtained from step (e) followed by sonicating the said support at a frequency ranging between 25 -130 kHz with the power output ranging from 60 W to 300 W for a period ranging from 1 to 30 minutes at a temperature ranging from 30°C to 60°C and washing with buffer; (g) adding a substrate-dye-buffer to the support as obtained from step (f) and incubating for a period ranging from 4 to 10 minutes in dark; (h) adding a stop solution to the support as obtained from step (g) followed by measuring the optical density at a suitable wavelength. 2. A process as claimed in claim 1, wherein the solid support used is selected from well, ELISA plate, microwell plate or module made of materials selected from polystyrene, polycarbonate, polypropylene, polyethylene, glass and polyvinyl chloride. 3. A process as claimed in claim 1, wherein the biomolecules are selected from the group consisting of antibody, antigen, protein and carbohydrate. 4. A process as claimed in claim 1, wherein the blocking agent used is selected from the group consisting of bovine serum albumin, skimmed milk powder and gelatin. 5. A process as claimed in claim 1, wherein the conjugate used is a biomolecule selected from the group consisting of antibody or antigen conjugated with an enzyme selected from peroxidase or alkaline phosphatase. 6. A process as claimed in claim 1, wherein the ultrasound waves for sonication-mediated enzyme linked immunosorbent assay are generated using apparatus selected from the group consisting of sonicator bath or specially designed apparatus. 7. A process as claimed in claim 1, wherein the said method is useful for assays selected from the group comprising of radio immunoassay, radio-immunosorbent, radio allergosorbent, biotin-avidin/streptavidin immunoassay, immnunoblotting, immunostaining, direct ELISA, indirect ELISA, sandwich ELISA. 8. A novel sonication mediated enzyme-linked immunosorbent assay process substantially As herein described with reference to the foregoing example.

Documents:

612-del-2007-abstract.pdf

612-del-2007-Claims-(29-07-2013).pdf

612-del-2007-claims.pdf

612-del-2007-Correspondence Others-(29-07-2013).pdf

612-DEL-2007-Correspondence-others (26-08-2008).pdf

612-del-2007-correspondence-others.pdf

612-del-2007-description (complete).pdf

612-del-2007-form-1.pdf

612-DEL-2007-Form-18 (26-08-2008).pdf

612-del-2007-form-2.pdf

612-del-2007-Form-3-(29-07-2013).pdf

612-del-2007-form-3.pdf

612-del-2007-form-5.pdf