Abstract:
This invention relates to a rapid and efficient method for carrying out enzyme-linked immunosorbent assay for detection of minute quantities of biomolecules such as antigen, antibody etc. This invention particularly relates to heat-mediated immobilization of antigen or antibody on to the activated surface followed by performing subsequent steps of ELUSA by controlled temperature. The invented procedure has reduced the total time required for ELISA to around 3 h.  
     The invented ELISA procedure is rapid, economical, reproducible and simple. The invented procedure is usefull for carrying out ELISA required in clinical diagnostics, molecular biology, agriculture, food technology, environmental science etc.  
     The invented ELISA method is simple, time saving and obviates the time consuming procedure. This method has the potential for automation.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to a rapid and efficient method for carrying out enzyme-linked immunosorbent assay for detection of minute quantities of biomolecules such as antigen, antibody etc, This invention particularly relates to heat- mediated immobilization of antigen or antibody on to the activated su e followed by performing subsequent steps of ELISA by controlled temperature. The invented procedure has reduced the total time required for ELISA to around 3 h. The invented ELISA procedure is rapid, economical, reproducible and simple.  
           [0002]    The invented procedure is useful for carrying out ELISA required in clinical diagnostics, molecular biology, agriculture, food technology, environmental science etc. The invented ELISA method is simple, time saving and obviates the time co sung procedure. This method has the potential for automation.  
         BACKGROUND OF THE INVENTION  
         [0003]    Enzyme linked immunosorbent assay (ELISA) is a very sensitive technique used for detection of certain antigens and antibodies ELISA has become a useful tool in diagnosis of diseases in both animals and plants, ELISA can also be used for 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 (Ghassempour et al. 2002), detection of apoptosis in tissue culture etc. (Salgame, P. et al, 1996).  
           [0004]    Conventional ELISA procedures cared out by immobilizing antigen or antibody on a polystyrene microtiter plate through adsorption have following drawbacks: (i) ELISA values in different wells and plates are usually inconsistent (Kricka et al., 1980; Hermann and Collins, 1976); (ii) long incubation times is required; (iii) during washing some biomolecules may detach leading to nonreproducible results (Engvall and Perlmann, 1971); and (iv) usually gives lower sensitivity (Kemeny, 1997). On the other hand, covalent binding is more sensitive (Deshpande, 1996), minimizes nonspecific binding and eliminates the above shortcomings (Douglas and Monteith, 1994). Covalent binding of immunogens to grafted plastic surfaces has been reported (Larsson, P.H. et al, 1987).  
           [0005]    Aleixo et al. (1985), actived an inert polystyrene surface by nitrating the aromatic ring of the polystyrene followed by the reduction of the nitro group to an amino group. The amino polystyrene was Further activated by chemical reactions such as diazotization and the resulting activated surface was used to unimobilize antigen covalently.  
           [0006]    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, based either on adsorption or on covalent binding. In case of medical urgency, precious time is lost in diagnosis before the patient could be given medication, In agriculture, ELISA is useful for detecting pesticide residues in crop produce (Van Emon, J. M. and Lopez-Avila, V., 1992) and environmental samples (Ghassempour et al. 2002).  
           [0007]    Bora et al. (2002) have reported a rapid and simple method for a double-anti-body sandwich (DAS) ELISA and a direct antigen coating (DAC) ELISA by covalently immobilizing antigen or antibody onto the activated polystyrene surface which was developed by Nahar et al. (2001) by a rapid and simple method using 1-fluoro-2-nitro-4-azidobenzene (FNAB). Detection of antibodies at lower concentrations was also done successfully using the activated surface. The ELISA value obtained in eight hours by this method was found to be similar with the values obtained in the conventional ELISA method carried out in fifteen hours. Although this method had reduced, the time to about eight hours still there is a. need to shorten the ELISA procedure keeping in mind its enormous application in diagnostics.  
           [0008]    The applicants in the present invention have shortened the ELISA procedure by carrying the ELISA procedure on a photoactivated polymer surface at elevated temperature. More particularly, it was carried out on a photoactivated polycarbonate and polystyrene plates at a temperature ranging from 30-70° C. Although microwave-mediated rapid ELISA on an activated surface is available (P. Nahar et al. A rapid method for enzyme-linked immunosorbent assay.  
           [0009]    U.S. Pat. No. 6,498,016 (2002); PCT Application No. WO 02/14868 A), the technique is highly sensitive and slight change in energy and or time (even in seconds) can spoil the ELISA experiment. The procedure also needs microwave oven or microwave apparatus for carrying out ELISA.  
           [0010]    No literature is available for ELISA on a photoactivated surface at elevated temperature (at higher temperature than conventional ELISA usually carried out at 37° C.) by conventional heating such as heating in electric oven, incubator, water bath, thermocycler etc.  
           [0011]    The main advantage of the present invention is that a slight change in energy or time will not spoil the ELISA experiment in contrast to microwave-mediated ELISA  
           [0012]    The disadvantages of the prior art methods are given in the Table I below  
                             TABLE I                           Prior arts and their drawbacks                Prior art   Drawbacks               1.   Inert polystyrene surface is activated by   Antigen immobilization itself is a           nitration, followed by reduction (Aleixo   cumbersome, time-consuming           et al. 1985). ELISA carried out by   multistep procedure.           immobilizing antigen onto the amino   ELISA by this method is also very           polystyrene by diazotization reactions.   time consuming procedure.       2.   Using a preactivated polystyrene   The ELISA procedure is better than           microtiter plate by a simple   the conventional procedure (require           photochemical reaction, enhancement of   about 15 h). Still it requires about 8 h           the speed and sensitivity of an ELISA   when carried out on the           technique was achieved by Bora et at.   photoactivated polystyrene surface. It           (2002). Immobilization of   is a time consuming procedure           antigen)/antibody onto the photoactivated   specially when used for diagnostic           plates was done in only 45 min with   purposes           higher binding than that obtained through           Adsorption during the same period onto the           untreated surface.           ELISA was carried out with the solid           phase prepared on the photoactivated           surface and nearly 1.5-2-folds higher           readings were obtained.       3.   PCT patent no. WO 02/14868 A1   Procedure needs microwave oven or           discloses a method for carrying out   apparatus for carrying ELISA.           enzyme-linked immunosorbent assay by   This ELISA procedure is very           controlled microwave irradiation in less   delicate. Slight variation in optimum           than 10 min.   condition may hamper the ELISA               procedure.                  
 
         OBJECTS OF THE INVENTION  
         [0013]    The main object of the present invention is to provide a heat-mediated ELISA procedure on an activated surface in short time.  
           [0014]    Another object of the present invention is to provide a rapid technique, which can be carried out at high temperature using my temperature source.  
           [0015]    Another object of the present invention is to provide a rapid technique, which has the potential for automation.  
         SUMMARY OF THE INVENTION  
         [0016]    With a view to achieve the objectives and overcoming the disadvantage of known ELISA method, a rapid and efficient method is provided for heat-mediated ELISA (HELISA) which comprises the steps of (i) covalent immobilization of the antigen or antibody on to the activated solid surface by heat, (ii) blocking the free surface with blocking agent by controlled heat, (iii) binding of antibody or antigen by controlled heat (iv) binding of conjugate by controlled heat, (v) adding dye-substrate to the wells and (vi) recording the absorbance value.  
           [0017]    Heat-mediated ELISA (HELISA) procedure is carried out on an activated surface in less than 3 h and with the same efficacy as in the conventional ELISA carried out at 37° C., in about 15 h. To overcome the problem of detachment of the solid phase prepared by adsorbtion, the applicants activate the surface of the wells of a PCR plate prior to use, The activated surface immobilizes the antigen through a covalent bond by controlled temperature. This covalently immobilized antigen is stable enough to withstand repeated but brief heat exposure, which were needed for performing the subsequent steps of ELISA.  
           [0018]    Subsequent steps of biomolecule binding in the ELISA procedure are through non covalent binding which are susceptible to heat energy. These problems are overcome by controlling time and temperature in each step.  
           [0019]    Novelty of the present invention is that the ELISA is carried out on an activated surface capable of forming covalent linkage with the biomolecule.  
           [0020]    In the invented procedure, all steps of ELISA such as antigen binding, blocking, antibody and conjugate binding are carried out by thermal activation in a thermocycler and only enzyme-substrate reaction is performed out side thermocycler at room temperature.  
           [0021]    The invented procedure of ELISA is rapid with comparable or even better ELISA value than the conventional method.  
           [0022]    Another novelty of the present invention is that the invented ELISA procedure can be filly or partially automated with the use of a specially designed device.  
           [0023]    Another novelty of the present invention is that the invented procedure can be used for other immunoassays like radio immunoassay, radio-immunosorbent test, radio allergosorbent test, biotin-avidin/streptavidin immunoassay, immunoblotting, immunostaining etc. apart from different types of ELISA such as direct ELISA, indirect ELISA, sandwich ELISA and like.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0024]    The present invention provides a simple approach for enzyme-linked immunosorbent assay technique on all activated polycarbonate or polystyrene surface by heat exposure. Activated surface immobilizes antigen through covalent bonding by thermochemical reaction. This covalently immobilized antigen is stable enough to withstand repeated heat exposure, which are needed for performing the subsequent steps of ELISA. ELISA is a multistep and delicate process where improper condition in any step may hamper the whole result.  
           [0025]    In the invented procedure, the first step of ELISA was performed by covalent immobilization of the goat anti-human IgG onto the activated polycarbonate PCR plate by thermal incubation for 1 h in different temperatures. The optimum temperature for antigen immobilization was found as 50° C. In experiments carried out on binding of goat anti-human IgG for the same duration in same incubation temperature on untreated surface showed lower ELISA value (Table 1).  
           [0026]    Immobilization of the Goat anti-human IgG onto the activated polycarbonate PCR plate by thermal incubation at 50° C. was detectable even in 20 min, which increases with the increase in time of incubation. At 60 min, the antigen binding becomes more or less same as 40 min thermal incubation. Hence, optimum time for antigen immobilization was taken as 40 min (Table 2).  
           [0027]    In the second step of HELISA, blocking was carried out with 2% BSA in 1 h at different temperatures in the thermocycler. The optimum temperature for blocking was 40° C. Further, increase in incubation temperature showed non-specific blinding (Table 3) The optimum time for blocking at 40° C. with 2% BSA was found to be 40 min. Further, decrease in incubation time showed inadequate blocking (Table 4).  
           [0028]    In the third step of HELISA, antibody binding to the immobilized antigen was carried out in 1 h at different temperatures. Human IgG was found to optimally bind to the solid phase at 50° C. (Table 5).  
           [0029]    The optimum time for antibody binding to the immobilized antigen at 50° C. was found to be 45 min (Table 6).  
           [0030]    In the fourth step of HELISA, conjugate binding was carried out in 1 h at different temperatures. Excellent result was obtained at 50° C. of incubation (Table 7).  
           [0031]    The optimization of conjugate binding time was found in the thermocycler at 50° C. by incubating at different duration of time. The best result was obtained in 40 min (Table 8).  
           [0032]    In all the experiments, lower ELISA values were obtained when they were carried out on untreated surface.  
           [0033]    The applicants have also compared ELISA on activated polycarbonate and polystyrene plates at 37° C. Thus, when ELISA steps such as antigen binding, blocking, antibody binding and antibody-conjugate binding were carried out in 45 min, 1 h, 3 h and 3 h respectively the ELISA values on both the surfaces were found similar (Table 9).  
           [0034]    The applicants also compared ELISA values, obtained on activated polycarbonate and polystyrene plates by carrying out ELISA steps at 37° C. with HELISA timings such as in 40 min, 40 min, 45 min and 40 min respectively. In both the plates, the results were much lower than conventional ELISA or HELISA (Table 9). It showed that the temperature is responsible for enhanced ELISA.  
           [0035]    In another experiment HELISA steps (50° C., 40 min; 40° C., 40 min; 50° C., 45 min and 50° C., 40 min) were carried out on both the activated plates. For PCR polycarbonate plate thermocycler was used for high temperature incubation and for polystyrene microtiter plate, ordinary incubator was used, as normal polystyrene ELISA plates cannot fit into conventional thermocycler. However, incubator temperature for HELISA on polystyrene plate was kept 2° C. higher than the thermocycler temperature in each step of ELISA. In incubator a little higher temperature is required as heat transfer is little slow here than in the thermocycler. HELISA can also be coed out on polystyrene plate in an incubator without increasing its temperature but increasing 5 min for each step of ELISA to compensate delay in heat transfer in incubator. In all cases, surfaces without activation showed much lower absorbance value. Results obtained in both these plates were very similar which suggests that heat mediated ELISA (HELISA) can be carried out on activated polystyrene microtiter plate also.  
           [0036]    IgE was also detected with same efficacy as IgG by the invented method. The standard curve prepared for quantitative assay of human IgE (Table 10 A) and the amount of IgE present m patient sera (Table 10 B) by HELISA (in 2 h 50 min) and ELISA (in 7 h 50 min) methods showed comparable results (Table 10). Thus, the invented method can reduce assay time to 2 h 50 min from 7 h 50 min without compromising the ELISA value.  
           [0037]    The invented HELISA procedure is also very sensitive as seen from Table 11, where it detected IgE in 1/8 dilution of patient sera. Thus, the present invention is rapid, sensitive and simple and can be carried out in any normal laboratory set up.  
           [0038]    The total time required for HELISA is less than 3 h. However, time required in each step may vary depending on the biomolecules where excellent results can be obtained by minor modification of reaction conditions that is minor alteration in duration and temperature of incubation. Instead of thermocycler HELISA can also be carried out in an incubator maintaining he temperature of 52° C. for antigen binding, antibody binding and conjugate binding, and 42° C. for blocking for a time period of 40, 45, 40 and 40 min respectively.  
           [0039]    Accordingly, the present invention provides a rapid method for enzyme-linked immunosorbent assay characterized in using an activated solid support wherein the said method comprises:  
           [0040]    (a) providing an activated solid support,  
           [0041]    (b) loading a biomolecule selected from an antigen or antibody by dissolving the said biomolecule in a coating buffer into the activated well of the said solid support and heating the said well at a temperature ranging from 40-80° C. for a period ranging from 10-70 min followed by washing the well thoroughly with an appropriate washing buffer,  
           [0042]    (c) blocking the well having an immobilized biomolecule as obtained from step (b) as above by loading blocking solution into the said well and heating the said well at a temperature ranging from 40-70°C. for a period ranging from 10-60 min and washing the said well with an appropriate washing buffer,  
           [0043]    (d) loading the corresponding antibody or antigen dissolved in a buffer into the well immobilized with antigen or antibody as obtained from step (c) as above followed by heating the said well at a temperature ranging from 40-80° C. for a period ranging from 10-70 min followed by washing with washing buffer,  
           [0044]    (e) loading an appropriate enzyme- conjugate dissolved in a suitable buffer into the above said well obtained from step (d) and heating the said well at a temperature ranging from 40-80 ° C. for a period ranging from 20-70 min followed by washing with a washing buffer,  
           [0045]    (f) adding a substrate-dye-buffer to the above well as obtained from step (e) as above and keeping it for a period ranging from 4 to 10 min in dark followed by addition of stop solution and measuring optical density of the solution by spectrophotometer at a suitable wavelength.  
           [0046]    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 silicagel, polyvinyl chloride and like.  
           [0047]    In an embodiment of the present invention, the preferred solid supports used are polycarbonate and polystyrene.  
           [0048]    In yet another embodiment the solid support is selected from any shape, form and size such as PCR plates, ELISA plate, microwell plate, sheets, test particles such as beads and microspheres, test tubes, test sticks, test strips, well or module.  
           [0049]    In an embodiment of the present invention, the activated solid support used for immobilizing biomolecules is selected from any solid support capable of binding ligand molecules particularly by covalent binding.  
           [0050]    In yet another embodiment of the present invention, the activated solid support may have active functional group for covalent binding which is selected from halide, aldehyde, acetyl, epoxide, succinimide, isothiocyanate, acylazide and like.  
           [0051]    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.  
           [0052]    In yet another embodiment of the present invention the functional group is introduced on to the solid support by photochemical reaction using a photoactive compound which is selected from 4-azido-1-fluoro-2-nitrobenzene (1-fluoro-2-nitro-4-azidobenzene), N -hydroxysulfo-succimdyl 4-azidobenzoate, N-hydroxysulfo-succinimidyl 4-azidosalicyclic acid, quinone or its derivatives, and like.  
           [0053]    In yet another embodiment of the present invention, polycarbonate and polystyrene surface are activated by coating 4-azido-1-fluoro-2-nitrobenzene and exposing the coated support to UV radiation at 365 nm.  
           [0054]    In yet another embodiment of the present invention, the light source for photochemical reaction is selected from UV lamp, laser beam, bright sunlight or like  
           [0055]    In yet another embodiment of the present invention, time for photoreaction for activation of solid support is selected from 10 seconds to 10 hours.  
           [0056]    In yet another embodiment of the invention, incubation is performed in an apparatus selected from laboratory polymerase chain reaction (PCR) thermocycler, specially designed thermocycler, incubator, water bath or any apparatus or chamber in which heat is generated and like.  
           [0057]    In yet another preferred embodiment of the invention, the first step of ELISA, is carried out by covalent binding of antigen or antibody onto the activated plate by heating at a temperature ranging from 40-80° C. for a period ranging from 10-70 min.  
           [0058]    In yet another preferred embodiment of the invention the second step of ELISA, that is the blocking step is carried out by heating at a temperature ranging from 40-70° C. for a period ranging from 10-60 min.  
           [0059]    In yet another preferred embodiment of the invention the third step of ELISA, that is corresponding antibody or antigen binding is carried out by heating at a temperature ranging from 40-80° C. for a period ranging from 10-70 min.  
           [0060]    In yet another preferred embodiment of the invention, the fourth step of ELISA that is enzyme-conjugate binding is carried out by heating at a temperature ranging from 40-80° C. for a period ranging from 10-70 min.  
           [0061]    In yet another preferred embodiment of the invention the total time for antigen binding, blocking, antibody binding and conjugate binding is ranging from 2-4 h wherein the total time for conventional ELISA method usually ranges from 8 h to 24 h,  
           [0062]    In another embodiment to the present invention, the antigen can be dissolved in a coating buffer such as carbonate buffer, phosphate buffer and like of suitable composition having a pH, in the range of from 6.5 to 11 with molarity ranging from 0.005 M to 0.1 M and should be compatible with the antigen.  
           [0063]    In yet another preferred embodiment of the invention, washing buffer used is a mixture of phosphate buffer having a pH, in the range of from 6.5 to 11, with molarity ranging from 0.005 M to 0.1 M and tween 20 in the range of between 0.05% to 3%.  
           [0064]    In yet another preferred embodiment of the invention, blocking reagent is selected from bovine serum albumin, skimmed milk powder, and gelatin and like.  
           [0065]    In another embodiment to the present invention biomolecule is selected from antigen or antibody. Antigen may be any biomolecule, microorganis, ect that elicits or has the potential to elicit an immune response.  
           [0066]    In another embodiment to the present invention conjugate is selected from biomolecule having antibody or antigen conjugated with an enzyme selected from horseradish peroxidase or alkaline phosphatase. In yet another preferred embodiment of this invention, enzyme may be replaced by a label selected from chromophore, fluorophore and like 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, immunoblotting, immunostaining etc. apart from different types of ELISA such as direct ELISA, indirect ELISA, sandwich ELISA and like.  
           [0067]    In yet another preferred embodiment of this invention, the invented procedure can be automated or semiautomated, An apparatus may be made to fit microtiter ELISA plates for heat-mediated enzyme linked immunosorbent assay (HELISA) comprising a reaction chamber consisting of rapid heating system for temperature ranging from 40-70° C. and a cooling system such as exhaust fan for carrying out the steps of antigen binding, blocking, antibody binding and antibody enzyme conjugate binding.  
           [0068]    In the invented procedure, inert solid surface such as polycarbonate PCR plate (usually used for polymerase chain reaction) and 96-well polystyrene microtiter plate were activated by a photochemical reaction carried out in dry condition using FNAB. The solid support was activated by exposing the FNAB coated support to TV radiation or bright sunlight. This activated support was used for heat-mediated ELISA (HELISA) and for control ELISA to detect antibodies, to example goat anti-human IgG and goat anti-human IgE.  
           [0069]    Thermal reaction was performed inside a thermocycler (Perkin Elmer, Gene Amp PCR System 2400) or in a BOD incubator. Carbonate buffer, pH 9.6, 0.1 M was used as coating buffer.  
           [0070]    Phosphate buffer saline (PBS), pH 7.2, 0.01 M was used as antibody dilution buffer.  
           [0071]    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 unless mentioned otherwise. Blocking solution was made by dissolving 2% BSA in 0.01 M PBS, pH 7.2. Substrate solution was prepared by adding 0.067% of o-phenylenediamine and 0.043% of H 2 O 2  in 0.1 M phosphate citrate buffer, pH 4.5). Normal goat sera were taken as negative control sera (-ve sera). 100 μl of diluted (1:300)-ve sera was used for each well.  
           [0072]    Horse radish peroxidase conjugated anti-rabbit IgG was purchased from Sigma as lyophilized powder.  
           [0073]    After reconstitution, the optimum dilution was found to be 1:5000 as determined by checkerboard titration, which was used in the experiments.  
           [0074]    In all the ELISA experiments, absorbance values are expressed as mean of three replicates.  
           [0075]    ELISA is a 5-step procedure, namely antigen binding, blocking, antibody binding, conjugate binding and color development. Each step of HELISA was optimized by carrying out the subsequent steps by conventional ELISA procedure. Conventional ELUSA was carried out by coating the wells with antigen overnight at 4° C., (45 min at 37° C. for activated wells), blocking in 1 h at 37° C. followed by antibody and conjugate binding at 37° C. for 3 h each and color development that is enzyme-substrate reaction at room temperature for 5 min followed by reading absorbance.  
           [0076]    The present Invention and its Advantages  
                                           Present invention   Advantages                   1.   Present invention provides a method for   1. Present invention provides an           Heat-mediated ELISA (HELISA)   ELISA procedure, which takes around           procedure by doing it on an activated   3 h, less than required in conventional           polymer surface capable of forming a   procedure (usually 10-15 h).           covalent bond with antigen or antibody.   2. The procedure even takes less than               the recently published procedure of               ELISA (about 8 h) (Bora et al. 2002)               on the photoactivated surface               3. The invented procedure is sensitive               and requires minute quantities of               precious antigen or antibody.               4. 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               thermocycler, incubator or even on               water bath.               5. The procedure can be easily automated.               6. A small deviation in the               incubation conditions from the               optimum conditions does not effect               the ELISA value significantly in the               present invention.                  
 
           [0077]    This invention is further explained with the help of the following examples and should not be construed to limit the scope of the invention. 
       
    
    
     EXAMPLE 1  
       [0078]    Activation of Polycarbonate and Polystyrene Surfaces  
         [0079]    Wells of a PCR plate (polycarbonate plate, Greiner, Germany) were loaded with 6.0 μmol of 1-flouro-2-nitro-4-azidobenzene (FNAB), dissolved in 50 μl of methanol per well and dried completely in the dark. FNAB coated wells were then irradiated for 7.5 min with UV irradiation of 365 nm wavelength in a UV Stratainker 2400 (Stratagene®, USA) or kept under bright sunlight for 15 min. Wells of polystyrene microtiter plate were activated similarly using 10 μmol of FNAB, dissolved in 50 μl methanol dried and irradiated for 10 min at 365 nm or sunlight for 15 min.  
         [0080]    The wells were then washed several times with methanol to remove the unbound linker and dried at room temperature. These activated wells were used for immobilization of antigen or antibody in the invented procedure.  
       EXAMPLE 2  
       [0081]    Determination of Goat Anti-human IgG Concentration for the Preparation of Solid Phase on Activated Polycarbonate Surface  
         [0082]    A double dilution series (2000−0.061 g/ml) of goat anti-human IgG (dissolved 0.1 M carbonate/bicarbonate buffer, pH 9.6) was loaded in triplicate wells (90 μl/well for each dilution) of an activated and untreated polycarbonate plates. The plates were then incubated at 50° C. for 1 h in a thermocycler The plates were then washed for six times with washing buffer (0.05% Tween 20 with 0.01 M PBS) and blocking step was done with 2% BSA solution (100 μl/well) at 37° C. in 1 h. After washing, each well was loaded with 90 μl of a 250 ng/ml of a human IgG solution (dissolved in 0.01 M PBS, pH 7.4) and the plates were incubated at 37° for 3 h. The plates were again washed for six times and each well was loaded with 90 μl of a 1/5000 (v/v), goat anti-human IgG-peroxidase conjugate solution (dissolved in 0.01 M PBS, pH 7.4) and incubated at 37° C. for 3 h, After washing, 90 μl of substrate-dye solution (0.4 mg o-phenylenediamine hydrochloride and 2 μI H 2 O 2  in 10 ml of 0.2 M citrate buffer, pH 5) was loaded into each well. Colour development was stopped after 5 min by adding 10 μl of 5% H2 SO 4 . The solutions were transferred into wells of a polystyrene microtiter plate and absorbance was recorded at 490 nm.  
       EXAMPLE 3  
       [0083]    Optimization of Temperature for Immobilization of Goat Anti-human IgG on the Activated and Untreated Polycarbonate Plates (Table 1)  
         [0084]    Triplicate wells of eight activated polycarbonate plates, were loaded with goat anti-human IgG solution (90 μl/well of a 250 ng/ml solution) and incubated for 60 min at 35, 40, 45, 50, 55, 60, 65 and 70° C. respectively in a thermocycler. Goat anti-human IgG was also similarly immobilized in untreated polycarbonate plates. The remaining steps of ELISA were then carried out with these solid phases by conventional ELISA method as described in example 2.  
       EXAMPLE 4  
       [0085]    Optimization of Goat Anti-human IgG Incubation Time for the Preparation of Solid Phase Sable 2)  
         [0086]    Triplicate wells of six activated and six untreated polycarbonate plates were loaded with 90 μl/well of goat anti-human IgG (250 ng/ml) and plates were incubated at 50° C. for 10 20, 30, 40, 50 and 60 min respectively in a thermocycler. Remaining steps of ELISA were carried out with these solid phases by conventional ELISA method described in example 2.  
       EXAMPLE 5  
       [0087]    Studies on the Incubation Temperature of Blocking Step (Table 3)  
         [0088]    Goat-anti human IgG was immobilized as described above on the triplicate wells of six activated plates at 50° C. in 40 min incubation. After washing, the wells were loaded with 100 μl of a 2% (w/v) BSA solution. The plates were then incubated for 1 h at 40, 45, 50, 55, 60 and 65° C. respectively. Incubation of human IgG and second antibody-enzyme conjugate were done at 37° C. for 3 h each. Control experiments with untreated plates were carried for each corresponding temperature similarly.  
       EXAMPLE 6  
       [0089]    Optimization of Incubation Time for Blocking Step (Table 4)  
         [0090]    Goat-anti human IgG was immobilized as described in example 5 on the triplicate wells of six activated plates. After washing, the wells were loaded with 100 μl of a 2% (w/v) BSA solution. The plates were then incubated at 40° C. for 10, 20, 30, 40, 50 and 60 min respectively. Human IgG and second antibody-enzyme conjugate were incubated at 37° C. for 3 h each Control experiments with untreated plates were carried for each corresponding time.  
       EXAMPLE 7  
       [0091]    Optimum Dose of Human IgG Required for the Assay  
         [0092]    The solid phases were prepared by immobilizing goat anti-human IgG (250 ng/ml) in activated and untreated wells of a polycarbonate plate at 50° C. for 40 min. Washing and blocking (40° C., 40 min) was done as described previously. The wells were then loaded with a two fold dilution series (1000-0.122 ng/ml) of a human IgG solution and incubated for 3 h at 37° C. The remaining steps of ELISA was carried as previously described.  
       EXAMPLE 8  
       [0093]    Determination of Optimum Temperature for the Binding of Human IgG with the Solid Phase (Table 5)  
         [0094]    The solid phase was prepared on an activated polycarbonate plate as described in example 5. Blocking was done under optimal conditions (40° C., 40 min) as above. After washing, 90 μl of a human IgG (from a solution of 125 ng/ml) was incubated with the solid phase for 60 min at 40° C. The plate was washed and a 90 μl of 1/5000 dilution of goat anti-human IgG peroxidase conjugate was incubated for 3 h at 37° C. The absorbance was recorded as described. Similarly, five more experiments were done by incrementing the temperature of incubation of human IgG by 5° C. up to 65° C. Control experiments were carried out with untreated polycarbonate plates for corresponding temperature.  
       EXAMPLE 9  
       [0095]    Binding of Human IgG onto the Solid Phase in Different Incubation Time (Table 6)  
         [0096]    90 μl of human IgG solution (from a solution of 125 ng/ml) was incubated at 50° C. for 15 min with solid phase prepared as in example 8. The washed wells were then incubated with a 1/5000 dilution of goat anti-human IgG-peroxidase at 37° C. for 3 h and assayed as previously described. Four more experiments were carried out by incubating human IgG for 30, 45, 60 and 90 min respectively. Control experiments were carried out with untreated polycarbonate surface under similar conditions.  
       EXAMPLE 10  
       [0097]    Optimum Temperature for Binding of Goat Anti-human IgG Peroxidase Conjugate with Human IgG Bound to Solid Phase (Table 7)  
         [0098]    Preparation of solid phase and blocking were carried out as in example 8. Incubation of human IgG was carried out at 50° C. for 45 min.as above. 1/5000 v/v dilution of goat anti-human IgG-peroxidase (90 μl/well) was then incubated for 60 min at 40° C. After washing, absorbance was recorded as above. Five more experiments were done similarly at different incubation temperatures (45, 50, 55, 60 and 65° C.) of second antibody-enzyme conjugate. Control experiments were carried out with untreated surface under similar conditions.  
       EXAMPLE 11  
       [0099]    Optimization of Incubation Time for Binding of Goat Anti-human IgG Peroxidase Conjugate to Solid Phase-bound Human IgG (Table 8)  
         [0100]    Immobilization of goat anti-human IgG, blocking and human IgG were incubated as in example 10. Second antibody-enzyme conjugate (1/5000, v/v) was incubated at 50° C. for 10 min and assayed. Five more experiments were carried out by incubating the conjugate at 20, 30, 40, 50 and 60 m respectively. Control experiments were carried out in corresponding time with untreated surface.  
       EXAMPLE 12  
       [0101]    Comparative Studies of HELISA and Conventional ELISA on Polycarbonate and Polystyrene Surfaces for the Detection of Human IgG (Table 9)  
         [0102]    In polycarbonate (PC 1) and polystyrene (PS 1) plates, ELISAs were performed by immobilizing goat anti-human IgG in 45 min at 37° C., 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. In plate PC 2 and PS 2 ELISAs were performed by immobilizing goat anti-human IgG in 40 min, blocking in 40 min, binding human IgG in 45 min and conjugate binding in 40 min at 37° C. In plate PC 3 ELISA was performed by immobilizing goat anti-human IgG in 40 min at 50° C., blocking in 40 min at 40° C., binding human IgG in 45 min at 50° C. and conjugate binding in 40 min at 50° C. HELISA on PS 3 plate were carried out in an incubator similarly as in PC 3 except that the temperature of the incubator in each step was 2° C. higher then PC 3 experiment carried out in a thermocycler. All these experiments were carried out with activated and untreated plates.  
       EXAMPLE 13  
       [0103]    Quantitative Determination of IgE in Patient Sera by HELISA and Conventional ELISA Procedures (Table 10)  
         [0104]    A. Preparation of Standard Curves by HELISA and Conventional ELISA Procedures (Table 10 A)  
         [0105]    In HELISA procedure, goat anti-human IgE (0.75 μl of a 1 μg/ml solution) was immobilized into activated polycarbonate wells in 40 min at 50° C. The plate was washed for six times with washing buffer (50 mM trig, 0.14 NaCl, pH 8.0 and 0.05% tween 20). Blocking was done with 2% BSA by incubating the plate at 40° C. for 40 min. The wells were tend loaded with 0.75 μl of IgE standards (Methyl Laboratories Inc., USA) in a double dilution series (1000, 500, 250, 125, 62.5, 31.25 and 15.34 ng/ml) and incubated for 45 min at 50° C. After washing, the wells were loaded with 0.75 μl of goat anti-human IgE peroxidase solution and incubated for 40 min at 50° C. Plate was again washed and assayed for enzyme.  
         [0106]    The conventional ELISA was carried out by immobilizing the capture antibody (goat anti-human IgE) on activated surface by incubating 45 min. at 37° C. or overnight at 4° C. Blocking was done in 1 h at 37° C. The standard IgE, and conjugate were incubated for 3 h each at 37° C.  
         [0107]    B. Determination of IgE in Patient&#39;s Sera (Table 10 B)  
         [0108]    HELISA and conventional ELISA were carried out similarly except that 1/4 (v/v, diluted) patient&#39;s sera were used instead of IgE standard for determining IgE in patient&#39;s sera.  
       Example 14  
       [0109]    Sensitivity of the Solid Phase in Detecting IgE in Patients Sera Table 11)  
         [0110]    The capture antibody was immobilized in 40 min at 50° C. in both untreated and activated polycarbonate wells. After blocking in 40 min at 40° C., the wells were loaded with human blood sera in different dilutions (1/4, 1/8, 1/16 and 1/32) and incubated for 45 min at 50° C. Conjugate incubation was at 50° C. in 40 min. Colour development was at ambient temperature for 5 min. Absorbance recorded as described earlier.  
                                                                                                         TABLE 1                           Detection of human IgG in by carrying out first step       by HELISA and remaining steps by ELISA procedures.                Activated plate   Untreated plate            Temperature   +ve       −ve       +ve       −ve           ° C.   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    35   0.442   0.011   0.117   0.005   0.350   0.007   0.105   0.006       40   0.510   0.006   0.115   0.011   0.348   0.016   0.114   0.006       45   0.633   0.010   0.104   0.024   0.350   0.009   0.104   0.012       50   0.675   0.014   0.112   0.019   0.333   0.012   0.101   0.018       55   0.642   0.005   0.099   0.025   0.305   0.006   0.097   0.023       60   0.560   0.008   0.115   0.003   0.279   0.008   0.095   0.022       65   0.411   0.006   0.094   0.021   0.261   0.007   0.095   0.021       70   0.343   0.008   0.076   0.009   0.209   0.004   0.068   0.007                                  
 
         [0111]    [0111]                                                                                                         TABLE 2                           Detection of human IgG by carrying out first step       by HELISA and remaining steps by ELISA procedures.                Activated plate   Untreated plate                +ve       −ve       +ve       −ve           Time (min)   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    10   0.226   0.009   0.089   0.011   0.172   0.003   0.102   0.005       20   0.295   0.020   0.106   0.011   0.197   0.009   0.112   0.005       30   0.478   0.006   0.111   0.01   0.267   0.008   0.101   0.013       40   0.623   0.01   0.108   0.004   0.319   0.004   0.108   0.014       50   0.624   0.013   0.118   0.004   0.346   0.013   0.098   0.008       60   0.622   0.006   0.123   0.004   0.378   0.010   0.114   0.002                                    
         [0112]    [0112]                                                                                                         TABLE 3                           Detection of human IgG by carrying out first two steps       by HELISA and remaining steps by ELISA procedure.                Activated plate   Untreated plate            Temperature   +ve       −ve       +ve       −ve           ° C.   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    40   0.576   0.009   0.076   0.003   0.324   0.003   0.069   0.003       45   0.563   0.010   0.075   0.001   0.318   0.005   0.081   0.014       50   0.549   0.008   0.172   0.006   0.319   0.006   0.123   0.010       55   0.616   0.006   0.183   0.005   0.336   0.009   0.135   0.010       60   0.618   0.006   0.200   0.008   0.353   0.005   0.173   0.005       65   0.633   0.005   0.208   0.005   0.358   0.005   0.194   0.008                                    
         [0113]    [0113]                                                                                                         TABLE 4                           Detection of human IgG by carrying out first two steps       by HELISA and remaining steps by ELISA procedures.                Activated plate   Untreated plate                +ve       −ve       +ve       −ve           Time (min)   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    10   0.66   0.006   0.228   0.004   0.322   0.007   0.12   0.008       20   0.655   0.007   0.149   0.007   0.302   0.007   0.113   0.007       30   0.620   0.005   0.097   0.013   0.278   0.007   0.079   0.006       40   0.608   0.006   0.075   0.011   0.28   0.004   0.057   0.01       50   0.613   0.002   0.067   0.013   0.274   0.005   0.048   0.006       60   0.609   0.003   0.069   0.008   0.281   0.008   0.048   0.005                                    
         [0114]    [0114]                                                                                                         TABLE 5                           Detection of human IgG by carrying out first three steps       by HELISA and remaining steps by ELISA procedures.                Activated plate   Untreated plate            Temperature   +ve       −ve       +ve       −ve           ° C.   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    40   0.419   0.004   0.063   0.015   0.318   0.005   0.066   0.005       45   0.531   0.013   0.085   0.003   0.309   0.003   0.073   0.012       50   0.613   0.006   0.105   0.005   0.281   0.006   0.090   0.009       55   0.607   0.006   0.078   0.017   0.271   0.008   0.086   0.004       60   0.576   0.009   0.098   0.008   0.268   0.006   0.078   0.012       65   0.534   0.006   0.080   0.004   0.264   0.003   0.081   0.011                                    
         [0115]    [0115]                                                                                                         TABLE 6                           Detection of human IgG by carrying out first three steps       by HELISA and remaining steps by ELISA procedure.                Activated plate   Untreated plate                +ve       −ve       +ve       −ve           Time (min)   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    15   0.234   0.008   0.088   0.006   0.164   0.009   0.068   0.011       30   0.423   0.006   0.104   0.003   0.236   0.009   0.070   0.011       45   0.620   0.007   0.091   0.011   0.281   0.008   0.071   0.027       60   0.623   0.015   0.081   0.006   0.309   0.008   0.080   0.003       90   0.615   0.012   0.098   0.008   0.337   0.006   0.075   0.008                                    
         [0116]    [0116]                                                                                                         TABLE 7                           Detection of human IgG by carrying out first four steps       by HELISA and last step by conventional procedure.                Activated plate   Untreated plate            Temperature   +ve       −ve       +ve       −ve           ° C.   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    40   0.407   0.012   0.108   0.012   0.335   0.007   0.073   0.008       45   0.585   0.010   0.081   0.010   0.331   0.008   0.056   0.011       50   0.614   0.010   0.084   0.005   0.299   0.010   0.067   0.005       55   0.607   0.005   0.088   0.009   0.282   0.007   0.073   0.003       60   0.565   0.012   0.077   0.004   0.250   0.006   0.077   0.016       65   0.524   0.009   0.057   0.011   0.238   0.004   0.055   0.011                                    
         [0117]    [0117]                                                                                                         TABLE 8                           Detection of human IgG by carrying out first four steps       by HELISA and last step by conventional procedure.                Activated plate   Untreated plate                +ve       −ve       +ve       −ve           Time (min)   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    10   0.302   0.013   0.05   0.007   0.183   0.006   0.063   0.01       20   0.403   0.012   0.055   0.013   0.223   0.004   0.056   0.009       30   0.543   0.011   0.07   0.006   0.247   0.008   0.057   0.005       40   0.618   0.008   0.083   0.009   0.281   0.004   0.074   0.008       50   0.615   0.007   0.078   0.012   0.307   0.005   0.081   0.008       60   0.623   0.008   0.111   0.007   0.321   0.004   0.091   0.005                                    
         [0118]    [0118]                                                                                                         TABLE 9                           Detection of human IgG: Comparison of HELISA and       ELISA in FNAB activated and untreated polycarbonate       (PC) and polystyrene (PS) plates.                Activated plate   Untreated plate            Treatments   +ve       −ve       +ve       −ve           ° C.   sera   ±sd   sera   ±sd   sera   ±sd   sera   ±sd                    PC1   0.552   0.017   0.082   0.01   0.276   0.003   0.083   0.003       PS1   0.545   0.007   0.099   0.014   0.261   0.008   0.079   0.004       PC2   0.376   0.007   0.073   0.006   0.258   0.005   0.077   0.005       PS2   0.359   0.004   0.071   0.007   0.219   0.006   0.067   0.022       PC3   0.626   0.009   0.113   0.005   0.280   0.009   0.074   0.007       PS3   0.615   0.005   0.071   0.007   0.246   0.005   0.063   0.02                                                            
         [0119]    [0119]                                                                                                             TABLE 10                       Quantitative assay of human IgE antibodies: Comparision of HELISA       and ELISA procedures on activated polycarbonate plates.                   TABLE 10 A            Standard   HELISA       ELISA                IgE (ng/ml)   Mean   ±sd   Mean   ±sd               15.625   0.067   0.006   0.041   0.004       31.25   0.149   0.007   0.125   0.003       62.5   0.266   0.009   0.247   0.006       125   0.357   0.009   0.362   0.005       250   0.444   0.010   0.460   0.005       500   0.535   0.007   0.549   0.006       1000   0.675   0.008   0.653   0.010                    TABLE 10 B                HELISA       ELISA                Sample No.   Mean   ±sd   Mean   ±sd               1   0.376   0.005   0.369   0.004       2   0.340   0.007   0.342   0.005                                                                                                                    
         [0120]    [0120]                                                               TABLE 11                           Comparison of sensitivity of the activated and untreated polycarbonate       plate towards the detection of IgE in diluted sera.                Activated       Untreated               plate       plate                Sera   +ve       +ve               dilution   sera   ±sd   sera   ±sd                       ¼   0.377   0.007   0.223   0.005           ⅛   0.230   0.024   0.150   0.006           {fraction (1/16)}   0.094   0.007   0.075   0.012           {fraction (1/32)}   0.078   0.012   0.075   0.003                                                                                    
         [0121]    Advantages  
         [0122]    Conventional methods of ELISA usually take 8-15 h for completion, which is the major drawback for a procedure used worldwide in different fields apart from clinical diagnostics. In case of medical urgency, precious time is lost in diagnosis before the patient could be given medication. Therefore, a rapid ELISA procedure invented herein (HELISA) will be beneficial and useful for diagnosis of diseases, biomedical research and other related fields. Main advantages of the invented ELISA procedure are:  
         [0123]    1. The invented procedure is rapid than the conventional method of ELISA.  
         [0124]    2. The total time required in the invented method is less than 3 h. Thus, it obviates the time consuming procedure.  
         [0125]    3. The invented procedure is sensitive and requires minute quantities of precious antigen or antibody.  
         [0126]    4. The procedure is simple and does not require any additional expertise or reagent to do it.  
         [0127]    5. The invented procedure is cost effective and does not require any additional equipment except a heating apparatus, which is common in most of the laboratories.  
         [0128]    6. The invented procedure is reproducible which is an important criterion for ELISA.  
         [0129]    7. The procedure gives minimal or negligible non-specific binding.  
         [0130]    8. The procedure has the potential for automation, which can minimize human error.  
         [0131]    References.  
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         [0137]    6. Hermann, J. E. and Collins, M. F. (1976)  J Immunol. Methods  10, 363-6.  
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         [0139]    8. Kemeny, D. M., 1997. In: Johnstone, A. P., Turner, M. W. (Eds.), Immunochemistry I: A Practical Approach. IRL Press at Oxford Univ. Press, New York, and pp.-147-175.  
         [0140]    9. Deshpande, S. S. (1996) Enzyme Immunoassays from Concept to Product Development. Chapman &amp; Hall, New York.  
         [0141]    10. Douglas, A. S. and Monteith, C. A. (1994)  Clin. Chem . 40, 1833-37.  
         [0142]    11. Larsson, P. H., Johansson, S. G. O., Hult, A. and Grothe, S. (1987)  J Immunol. Methods  98, 129-35.  
         [0143]    12. Aleixo, J. A. G., Swaminathan, B., Minnich, S. A. and Wallshein, V. A. (1985)  J. Immunoassay  6, 391-401.  
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         [0146]    Other References  
         [0147]    Patent Documents  
         [0148]    1. P. Nahar. A method for photochemical activation of polymer surface and immobilization of biomolecules onto the activated surface US patent filed June, 2002, NF 304/02  
         [0149]    2. P. Nahar, U. Bora and G. L. Sharma. (2002) A rapid method for enzyme-linked immunosorbent assay. U.S. Pat. No. 6,498,016  
         [0150]    3. P. Nahar, U. Bora and G. L. Sharma. A rapid method for enzyme-linked immunosorbent assay. PCT patent no. WO 02/14868 A1