Title of Invention

"A METHOD FOR THE PREPARATION OF READY-TO-USE SOLID SUPPORT FOR RAPID ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)""

Abstract The present invention provides a method for the preparation of ready-to-use solid support for ELISA for rapid identification and quantitative estimation of protein/antigen in the test samples, and performances of the assay itself. The invention also provides for a quick, accurate and stable estimation of protein/antigen in the test samples. The invention also provides an ELISA kit comprising of ready-to-use solid support along with wash buffers, chemical substrate, substrate buffer, stock solution, and positive and negative control samples.
Full Text A method for the preparation of ready-to-use solid support for rapid enzyme-linked
immunosorbent assay (ELISA)
Field of the invention
The present invention relates to a method for the preparation of ready-to-use solid support for ELISA for rapid identification and quantitative estimation of protein/antigen in the test samples, and performance of the assay itself. The invention also provides for a quick, accurate and stable estimation of protein/antigen in the test samples. The invention also provides a rapid ELISA kit comprising of ready-to-use solid support along with wash buffers, chemical substrate, substrate buffer, stock solution, and positive and negative control samples.
Background and prior art references
ELISA is a widely used method for the detection of specific proteins in a tissue sample. It involves the immobilization of an antibody (primary antibody) to a surface of substrate such as plastic, and detecting a specific antigen (protein) via binding to the immobilized antibody, followed by addition of secondary antibody or antibodies, the latter being conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase. Addition of a chemical substrate of the enzyme results in the development of a coloured reaction product, which indicates the presence of the antigen of interest in the sample.
ELISA is a time-tested and robust method, and is used for the detection of a multitude of proteins from a large number of sources. Commercial suppliers of ELISA products provide plates coated with primary antibody. The user has to then follow a procedure containing a series of steps involving addition of the sample, addition of secondary antibody or antibodies in sequence, several buffer wash steps in between each antibody addition step, and finally the detection step via substrate addition. The established procedures are often time-consuming and necessitate the formulation of various buffers and solutions. It often takes up to 24 hours to complete the protocol and obtain results.
There are a number of variations in ELISA and these determine the number of steps involved or time taken to complete the assay. For example, the secondary antibody may be conjugated to alkaline phosphatase or horseradish peroxidase, in which case the substrate for colour development can be added immediately after the secondary antibody.

This is known as direct ELISA. However, if the secondary antibody is unconjugated, then a third conjugated antibody is needed for colour detection. This type of assay is known as indirect ELISA. Antibodies (conjugated or otherwise) are either commercially available from vendors or need to be custom-produced. Thus one of the drawbacks in the established ELISA technique is to procure and maintain a stock of the necessary antibodies.
A search of the patent literature revealed one patent, WO02090983, Quantitative One-Step Immunoassay in Lyophilised Form, Inventors: Rech-Weichselbraun, I. (AT) and Staude M. (AT) directly relevant to the present invention. In this patent, proteins, antibodies and reaction enhancing agents such as biotin and streptavidin are immobilised on the plate along with coating antibody. A rehydration step is followed by sample addition and detection steps. This eliminates a number of intermediate steps. However, there are some significant differences from the present invention. These are: 1) The applications of the method described in WO02090983 are limited to detection of cytokines and related molecules used in cancer research, whereas the present invention relates to detection of proteins in plant tissues. 2) Assay times in WO02090983 vary significantly for each application, and can be as high as 250 min, whereas in the present invention assay times do not exceed 150 min. 3) WO02090983 uses a biotin-streptavidin system to enhance the sensitivity of the assay, whereas the present invention does not require this additional set of reagents.
Another related patent found was WO0214868, A rapid method for microwave mediated enzyme-linked immunosorbent assays. Publication date: 2002-02-21, Inventor(s): Sharma Gainda Lai (In); Nahar Pradeep (In); Bora Utpal (In); involving the use of a microwave oven to enhance the ELISA. However the key requirement of the microwave oven increases costs and necessitates the optimisation of protocols for each protein of interest, as each antigen to be utilised in such a method may have a different tolerance to heating by microwave radiation. Heat labile proteins would suffer adverse effects upon microwave treatment necessitating a modification in the protocol.
Objects of the invention
The main object of the present invention is to provide a method for preparing a ready-to use solid support for rapid ELISA.

Another object of the present invention is to provide a ready-to-use solid support for rapid quantification of protein/antigen in test samples.
Another object of the present invention is to provide for a quick, accurate and stable estimation of protein/antigen in the test samples.
Another object of the method is to demonstrate the rapid performance of the method.
Still another object of the present invention is to provide an ELISA kit containing ready-to-use solid support for rapid identification of protein / antigen in the test sample.
Yet another object of the invention is to provide an ELISA kit containing ready-to-use solid support for rapid quantitative estimation of protein/antigen in the test sample.
Another object of the invention is to reduce the number of steps in the procedure that an end-user has to perform in an ordinary ELISA.
Summary of the invention
In accordance to the objectives, the present invention provides a method for the preparation of ready-to-use solid support for ELISA for rapid identification and quantitative estimation of protein/antigen in the test samples and performance of the assay itself The invention also provides for a quick, accurate and stable estimation of protein/antigen in the test samples. The invention also provides an ELISA kit comprising of ready-to-use solid support along with wash buffers, chemical substrate, substrate buffer, stock solution, and positive and negative control samples.
Detailed description of the invention
Accordingly, the present invention provides a method for preparing ready-to-use solid support for rapid ELISA, wherein the said method comprises steps of:
a) adding a first monoclonal antibody dissolved in coating buffer to the wells of the solid support, incubating the solid support at about 40 °C for a period ranging between about 12 and 14 hours for binding to the solid support;
b) washing the solid support of step (a), with a washing buffer to remove the unbound monoclonal antibody;
c) adding a stabilizer solution to the wells of the solid support of step (b), incubating for a period ranging between 12 and 14 hours at about 40 °C;
d) decanting to remove the stabilizer solution of step (c), and completely drying the wells of the solid support;

e) adding to the wells of the solid support of step (d), an appropriate second antibody and an appropriate third antibody conjugated to an enzyme dissolved in a suitable buffer containing the blocking agent; and
f) freeze drying the plate of step (e), storing the plate in a sealed pack at a temperature range of about 4-8 °C for ready-to-use.
One embodiment of the present invention is a ready-to-use solid support consisting of a bound antibody, wherein said antibody is capable of forming a first antigen-antibody complex with sample antigen/protein, a second antibody forming an antigen-antibody complex with the said sample antigen/protein and a detection antibody having a label which selectively binds to the second antibody.
Another embodiment of the present invention is that the coating buffer used is selected from the group consisting of carbonate buffer and phosphate buffer, having pH in the range of 9.0-9.8.
Another embodiment of the invention is that the first monoclonal antibody used is selected from the group consisting of Cry 1 Ac, Cry2Ab and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS).
Another embodiment of the invention is that the washing buffer used is phosphate-buffered saline having a pH in the range of 6.8-7.2.
Another embodiment of the invention is that the stabilizer used is selected from a group consisting of a Phosphate-Buffered Saline, Fish Gelatin and Glycerol mixture and a Tris-buffer, Fish Gelatin and Glycerol mixture.
Another embodiment of the invention is that it provides a method, wherein the blocking agent used is selected from the group consisting of ovalbumin, bovine serum albumin, bovine nonfat milk powder, casein, fish gelatin, porcine gelatin and lambda-carrageenan.
Another embodiment of the invention is that the solid support used is selected from the group consisting of ELISA plate and microwell plate.
Another embodiment of the invention is that the material for the solid support used is either polystjTene or polypropylene.
Another embodiment of the invention is that the solid support used is polystyrene.
Another embodiment of the invention is that the second antibody is selected from the group consisting of goat polyclonal IgG raised against Cry 1 Ac, goat polyclonal IgG

raised against Cry2Ab and goat polyclonal IgG raised against 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS).
Another embodiment of the invention is that the third antibody is selected from the group consisting of polyclonal whole IgG conjugated to an enzyme. The source of this polyclonal whole IgG can be Class Mammalia or Class Aves.
Another embodiment of the invention is that the enzyme used is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.
Another embodiment of the present invention is that it provides a rapid method for performing ELISA using ready-to-use solid support, the said method comprising steps of:
a) reconstituting the ready to use plates by adding appropriate amount of distilled water;
b) adding to the plate of step (a), samples containing antigen/protein to be tested dissolved in a suitable buffer, incubating the plate at about 37 °C for about one hour for forming an immunocomplex with the bound first antibody;
c) washing the plate of step (b) with a suitable washing buffer to remove the unbound antigen;
d) adding to the plate of step (c), a buffer containing chemical substrate and incubating for about 30 minutes in dark at room temperature; and
e) detecting for the presence of the antigen by measuring absorbance in step (d) at a suitable wavelength
Another embodiment of the present invention is that the wavelength suitable for measuring the absorbance is in the range of 400-700 ran.
Another embodiment of the present invention is that it provides a method, wherein the chemical substrate is selected from the group consisting of para-nitrophenol phosphate (pNPP), Nitro Blue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate (NBT/BCIP), 2,2'-Azino-bis(3-Ethylbenz-thiazoline-6-Sulfonic Acid) (ABTS), o-Phenylenediamine (OPD), 3,3'-5,5'-Tetramethylbenzidine (TMB), o-Dianisidine, and 5-Aminosalicylic Acid (5AS).
One embodiment of the present invention is that it provides a rapid ELISA kit comprising of
a) a ready-to-use solid support with capability for detection of said protein
as described above and instructions for use of said kit,

b) wash buffers,
c) chemical substrate,
d) substrate buffer,
e) stop solution, and
f) positive and negative control samples.
Yet another embodiment of the present invention is that it provides for a quick, accurate and stable estimation of protein/antigen in the test samples.
Novelty and Inventive step
The key inventive steps and problems overcome are:
1. A novel method by which all antibodies required for the detection are made available to the assay in the wells of the ELISA plate. In order for the assay to work, a series of steps involving freeze-drying and addition of protein stabilizers in a precise, sequential manner has been devised, allowing the immobilisation or impregnation of the antibodies onto the ELISA plate.
2. Leading from the above, another key inventive step overcoming earlier problems is that of loss of viability of impregnated proteins. This occurs due to surface denaturation of the immobilised proteins possibly by means of oxidation or water loss [ref Ansari AA, Hattikudur NS, Joshi SR, Medeira MA. ELISA solid phase: stability and binding characteristics. J Immunol Methods. 84:117-24(1985)]. The present inventive steps prevent the surface denaturation of proteins immobilised to the solid support, and the procedures described herein enable the antibodies to be viable for use in the ELISA. This has been achieved by a series of coating and drying steps, alternating an antibody layer with a layer of stabiliser, and then freeze-drying or drying the material to retain its activity over a period of time.
3. A reconstitution step is incorporated in the ELISA protocol to allow the antibodies to be accessible to the protein of interest in the sample. This step is necessary to enable the immobilised, stabilised proteins to become optimally functional for the ELISA to work. No prior art was found which mentioned the stabilising of multiple proteins, i.e. "layering", on a solid support for storage and then use in an ELISA. This invention describes a novel way in which more than one protein can be successfully applied to the solid support, and subsequently used after a substantial period of time by means of a simple reconstitution step. For the end- user, this overcomes the problem of having
to obtain conjugated or unconjugated antibodies from various sources, thereby overcoming one of the main drawbacks in the conventional method.
4. The present invention obviates the need for additional equipment such as a microwave oven as described in WO0214868, A rapid method for microwave mediated enzyme-linked immunosorbent assays, Publication date: 2002-02-21, Inventor(s): Sharma Gainda Lai (In); Nahar Pradeep (In); Bora Utpal (In). Secondly, it also obviates the need to use biotin-streptavidin linked antibodies as described in WO02090983, Quantitative One-Step Immunoassay in Lyophilised Form, Inventors: Rech-Weichselbraun, I. (AT) and Staude M. (AT).
5. The invention enables a reduction in the number of steps that an end-user has to perform in an ELISA. All reagents needed to perform the assay are impregnated on to the plate by the use of particular stabihzing processes as well as particular protein stabilizers. The user oµly performs sample addition, wash and detection steps. The user adds a reconstitution buffer to the wells of the ELISA plate, followed by the samples to be tested. After an incubation period, the samples are discarded, and the plates washed. A chemical substrate is then added, which results in the appearance of a coloured reaction product in positive samples and a lack of colour in negative samples.
6. The present invention enables the user to perform a rapid ELISA in one step, as oµly samples need to be added to the ready-to-use plate prior to the detection step.
7. The present invention also provides for a quick, accurate and stable estimation of protein/antigen in the test samples.
The following examples are for understanding the invention and should not be construed to limit the scope of the invention.
Examples
Example 1 (Total time for assay: 150 min)
This method can be used for the detection of protein Cry 1 Ac in cottonseed and cotton leaf extracts in a qualitative manner, as indicated in the protocol below. Steps involved:
l)Preparation of buffers
2)ELISA plate coating with CrylAc mAb
3)Addition of Ab2 & Ab3

4)SampIe preparation
5)Assay
1) Preparation of buffers:
a) Carbonate buffer:
Sodium carbonate 1.59 g
Sodium bicarbonate 2.93 g
Sodium chloride 8.77 g
D/w IL
After preparing store at 4o C (cold room).
b) lOX PBST: (pH 7.4)
Sodium chloride 80.0 g
Sodium phosphate dibasic 11.50 g
Potassium chloride 2.0 g
Potassium dihydrogen phosphate 2.0 g
D/w IL
Add 5 ml Tween 20 to IL volume.
c) IX PBST:
Take 100 ml of lOX PBST dilute it to IL by adding D/w.
d) lOX PBSTO:
Add 0.5gm ovalbumin in 10 ml lOX PBST. Store the solution in 4o C refrigerator.
e) Substrate buffer:
Prepare 5% diethanolamine (DEA) in Milli Q, adjust the pH with concentrated HCl for 1 hr till the required pH is attained.
f) Substrate: (mg/ml cone.)
Take 25 ml substrate buffer; add 25 mg pNPP to it. Mix well.
Note: Substrate should be prepared freshly.
Remove pNPP bottle at least 20 min. before use from 4oC. After preparing
substrate buffer with substrate keep it in dark for 10 min. before use.
g) Stabilizer:
lOXPGFG/TGFG: 2.5 ml
lOXPBS : 2.5 ml
D/w : 20 ml
2) Coating ELISA Plates with CrylAc monoclonal Antibodies:
Add 250 µ\ of CrylAc monoclonal antibody per well of the Elisa plate at a concentration of 2 µ,g/ml.

Procedure:
Mix 12.8 µ1 mAb in 25 ml carbonate buffer. Using multichannel pipetter, add 250 µl in
each well of the plate. Incubate the plate 0/N at 4oC. Give two quick washes with IX PBST. Pat dry on blotting paper. Add stabilizer, 250 µl/well, and incubate 0/N at 4oC. Decant the plate & allow it to air dry completely.
3) Addition of Ab2 & Ab3:
Concentration of Ab2: 1:10,000 Concentration of Ab2: 1:5000
Procedure:
Pipette out 1.5 ^.1 of Ab2 & 3.8 µl of Ab3 stock in an eppendorf tube containing 1.5 ml
of lOX PBSTO. Mix well and add 15 |xl/well using a multichannel pipetter. Freeze-dry the plate for 15 min. Store the freeze-dried plates in sealed pack containing desiccant at 4oC, till ftirther use.
4) Sample preparation
Note: Avoid cross-contamination between samples
For seed extracts: Imbibe cotton seeds overnight in water. Remove seed coat and cut each seed to be tested in half with a clean blade. Place one half of the seed in a microcentrifuge tube and add 500 µ1 IX PBST. Crush with a pestle for 30 seconds. Spin for 30 sec in a microcentrifiige, and use 100 µl of each extract per well, taking care to avoid the pellet.
For leaf extracts: Punch out 2 leaf discs with a mcf tube by placing a leaf between the lid and the tube opening and closing the lid onto the leaf Add 500 µl X PBST. Crush with a pestle for 30 seconds. Allow to stand for few minutes, and use 100 µl of each extract per well, taking care to avoid the pellet.
5) Assay:
Reconstitute the freeze-dried plate for 30 min. by adding 150 µ,l/well Milli Q water.
After reconstitution, add samples, 100 µl/well. Incubate the plate at 37o C for Ihr. Give four quick washes with IX PBST. Pat dry. Add substrate, 250 µl/well, & incubate it for 30 min. dark at room temperature. Read the absorbance on an ELISA reader at 405 nm.
Sample Plate result For CrylAc: Results:

This method can be used for the detection of protein EPSPS in cottonseed and cotton leaf extracts in a qualitative manner, as indicated in the protocol below.
Steps involved:
l)Preparation of buffers
2)ELISA plate coating with EPSPS mAb
3)Additioii of Ab2 & Ab3
4)Sample preparation
5)Assay
1) Preparation of buffers: Please refer to Example 1
2) Coating ELISA Plates with EPSPS monoclonal Antibodies:
Add 250 µl of EPSPS monoclonal antibody per well of the Elisa plate at a concentration of 2 µg/ml.
Procedure:
Mix 13.3 (il mAb in 25 ml carbonate buffer. Using multichannel pipetter, add 250 µ1 in
each well of the plate. Incubate the plate 0/N at 4oC. Give two quick washes with IX PBST. Pat dry on blotting paper. Add stabilizer, 250 |xl/well, and incubate 0/N at 4oC. Decant the plate & allow it to air dry completely.
3) Addition of Ab2 &Ab3:
Concentration of Ab2: 1:20,000 Concentration of Ab2: 1:8000
Procedure:
Pipette out 1.0 µl of Ab2 & 3.1 \il of Ab3 stock in an eppendorf tube containing 2 ml of
lOXPBSTO.
Mix well and add 15 µl/well using a multichannel pipetter. Freeze-dry the plate for 15
min. Store the freeze-dried plates in sealed pack containing desiccant at 4o C, till further
use.
4) Sample preparation
Note: Avoid cross-contamination between samples
For seed extracts: Imbibe cotton seeds overnight in water. Remove seed coat and cut each seed to be tested in half with a clean blade. Place one half of the seed in a microcentrifuge tube and add 500 ul IX PBST. Crush with a pestle for 30 seconds. Spin

The grid below represents a 96-well ELISA plate in which CrylAc expressing cotton leaf samples have been tested using the inventive method. "Blank" refers to wells in which no cotton leaf extract has been added. This gives the baseline absorbance reading for the experiment and is subtracted from all sample readings. Absorbance values provided have blank values already subtracted (hence the blank wells read 0.0). "+ve" refers to known CrylAc expressing samples. Unmarked wells are equivalent to Blank wells, i.e.; no cotton leaf extract was added. A reading of above 0.2 is considered a positive reading. Plates prepared by the present inventive method were used in an experiment to determine whether known positive and negative samples could be accurately detected. In this example 100% (28/28) of the samples were detected accurately.
Samples added:

(TABLE REMOVED)

Absorbance values:
The plate represented above gave absorbance values as below

(TABLE REMOVED)


Example 2 (Total time: 150 min)
This method can be used for the detection of protein Cry2Ab in cottonseed and cotton leaf extracts in a qualitative manner, as indicated in the protocol below.
Steps involved:
l)Preparation of buffers
2)ELISA plate coating with CrylAb mAb
3)Addition of Ab2 & Ab3
4)Sample preparation
5)Assay
1) Preparation of buffers: Please refer to Example 1
2) Coating ELISA Plates with Cry2Ab monoclonal antibodies:
Add 250 µl of Cry2Ab monoclonal antibody per well of the Elisa plate at a concentration
of 2 µg/ml.
Procedure:
Mix 13.3 µl mAb in 25 ml carbonate buffer. Using multichannel pipetter, add 250 µ1 in
each well of the plate, iii. Incubate the plate 0/N at 4oC. Give two quick washes with IX PBST. Pat dry on blotting paper. Add stabilizer, 250 µl/well, and incubate O/N at 4oC. Decant the plate & allow it to air dry completely.
3) Addition of Ab2 & Ab3:
Concentration of Ab2: 1:4000
Concentration of Ab2: 1:5000
Procedure:
Pipette out 1.5 µ1 of Ab2 & 3.8 µl of Ab3 stock in an eppendorf tube containing 2 ml of
lOXPBSTO.
Mix well and add 15 µl/well using a multichannel pipetter. Freeze-dry the plate for 15
min. Store the freeze-dried plates in sealed pack containing desiccant at 4oC, till further
use.
4) Sample preparation
Note: Avoid cross-contamination between samples
For seed extracts: Imbibe cotton seeds overnight in water. Remove seed coat and cut each seed to be tested in half with a clean blade. Place one half of the seed in a microcentrifuge tube and add 500 µl IX PBST. Crush with a pestle for 30 seconds. Spin for 30 sec in a microcentrifuge, and use 100 µl of each extract per well, taking care to avoid the pellet.
For leaf extracts: Punch out 2 leaf discs with a mcf tube by placing a leaf between the lid and the tube opening and closing the lid onto the leaf Add 500 µl X PBST. Crush with a pestle for 30 seconds. Allow to stand for few minutes, and use 100 µl of each extract per well, taking care to avoid the pellet.
5) Assay:
Reconstitute the freeze-dried plate for 30 min. by adding 150 µl/well Milli Q. After
reconstitution, add samples, 100 µl/well. Incubate the plate at 37o C for Ihr. Give four quick washes with IX PBST. Pat dry. Add substrate, 250 µl/well, & incubate it for 30 min. dark at RT. Read the absorbance on ELISA reader at 405 nm.
Sample Plate result For CrylAb:
Results:
The grid below represents a 96-well ELISA plate in which Cry2Ab expressing cotton
leaf samples have been tested using the inventive method. "Blank" refers to wells in which no cotton leaf extract has been added. This gives the baseline absorbance reading for the experiment and is subtracted from all sample readings. Absorbance values provided have blank values already subtracted (hence the blank wells read 0.0). "+ve" refers to known Cry2Ab expressing samples. Unmarked wells are equivalent to Blank wells, i.e.; no cotton leaf extract was added. A reading of above 0.2 is considered a positive reading. Plates prepared by the present inventive method were used in an experiment to determine whether known positive and negative samples could be accurately detected. In this example 98.3% (59/60) of the samples were detected accurately.

(TABLE REMOVED)

Example 3 (Total time: 150 min)
for 30 sec in a microcentrifuge, and use 100 ul of each extract per well, taking care to avoid the pellet.
For leaf extracts: Punch out 2 leaf discs with a mcf tube by placing a leaf between the lid and the tube opening and closing the lid onto the leaf Add 500 ul X PBST. Crush with a pestle for 30 seconds, allow to stand for few minutes, and use 100 ul of each extract per well, taking care to avoid the pellet.
Assay:
Reconstitute the freeze-dried plate for 30 min by adding 150 µl/well Milli Q. After
reconstitution, add samples, 50 µl/well. Incubate the plate at 37o C for Ihr. Give four quick washes with IX PBST. Pat dry. Add substrate, 250 µl/well, & incubate it for 30 min. dark at RT. Read the absorbance on ELISA reader at 405 nm.
Sample Plate result For EPSPS:
Results:
The grid below represents a 96-well ELISA plate in which EPSPS expressing cotton leaf
samples have been tested using the inventive method. "Blank" refers to wells in which no cotton leaf extract has been added. This gives the baseline absorbance reading for the experiment and is subtracted from all sample readings. Absorbance values provided have blank values already subtracted (hence the blank wells read 0.0)."+ve" refers to known EPSPS expressing samples. Unmarked wells are equivalent to Blank wells, i.e.; no cotton leaf extract was added. A reading of above 0.1 is considered a positive reading. Plates prepared by the present inventive method were used in an experiment to determine whether known positive and negative samples could be accurately detected. In this example 100% (38/38) of the samples were detected accurately.

(TABLE REMOVED)

Absorbance values:
The plate represented above gave absorbance values as below
(TABLE REMOVED)

Advantages of the present invention
The present invention relates to a process in which ELISA plates are provided to the user
in a form in which oµly sample addition, wash and detection steps are required. The advantages are:
1. A number of steps are reduced such as sequential antibody addition, and buffer washes.
2. There is no need for the end-user to purchase any antibodies given that all reagents required for the detection are present on the plate, except the sample itself, and the substrate required for colour production.
3. The assay is equally sensitive as other, more time-consuming or cumbersome protocols.
4. The method provides for a quick, accurate and stable estimation of protein/antigen in the test samples.
References
WO02090983, Quantitative One-Step Immunoassay in Lyophilised Form, Publication date: 2002-11-14, Inventors: Rech-Weichselbraun, I. (AT) and Staude M. (AT)
WO0214868, A rapid method for microwave mediated enzyme-linked immunosorbent assays. Publication date: 2002-02-21, Inventors: Sharma, Gainda Lai (In); Nahar, Pradeep (In); Bora, Utpal (In)
Ansari AA, Hattikudur NS, Joshi SR, Medeira MA. ELISA solid phase: stability and binding characteristics. J Immunol Methods. 84:117-24(1985)








I/We Claim:
1. A method for preparing ready-to-use solid support for rapid ELISA, wherein the said
method comprises
a) applying a first monoclonal antibody to the wells of the support, wherein the first antibody binds to a target antigen and is dissolved in a coating buffer;
b) incubating the support at about 4°C for a period ranging between about 12 and 14 hours for binding to the support;
c) washing the support of step (b) with a washing buffer to remove the unbound first monoclonal antibody;
d) applying a stabilizer solution to the wells of the support of step (c) and incubating for a period ranging between about 12 and 14 hours at about 4°C;
e) removing the stabilizer solution that is unbound to the support of step (d);
f) air drying the support of step (e);
g) applying a second antibody and an enzyme linked third antibody to the said support of step (f), wherein the second antibody binds to the target antigen and the third antibody binds to the second antibody and said second and third antibodies are dissolved together in a second buffer containing a blocking agent; and
h) freeze drying the support of step (g) for about 15 minutes
2. The method as claimed in claim 1, wherein the first monoclonal antibody is selected from a group consisting of monoclonal antibodies against CrylAc, Cry2Ab and 5-enolpyruvylshikimate-3 phosphate synthase antigen.
3. The method as claimed in claim 1, wherein the coating buffer is selected from a group consisting of carbonate buffer and phosphate buffer, having pH in the range of 9.0-9.8.
4. The method as claimed in claim 1, wherein the washing buffer is phosphate buffer saline having a pH in the range of 6.8-7.2.
5. The method as claimed in claim 1, wherein the stabilizer is selected from a group consisting of Phosphate Buffered Saline, Fish Gelatin and Glycerol mixture and a Tris-buffer, Fish Gelatin and Glycerol mixture.

6. The method as claimed in claim 1, wherein the drying method of step (f) is either freeze drying or lyophilization.
7. The method as claimed in claim 1, wherein the blocking agent is selected from the group consisting of ovalbumin, bovine serum albumin, bovine non-fat milk powder, casein, fish gelatin, porcine gelatin and lambda-carrageenan.
8. The method as claimed in claim 1, wherein the second antibody is selected from a group consisting of goat polyclonal IgG raised against CrylAc, goat polyclonal IgG raised against Cry2Ab and goat polyclonal IgG raised against 5-enolpyruvylshikimate-3 phosphate synthase.
9. The method as claimed in claim 1, wherein the third antibody is selected from the group consisting of polyclonal whole IgG.
10. The method as claimed in claim 1, wherein the enzyme is selected from a group consisting of alkaline phosphatase and horse radish peroxidase.
11. A rapid method for performing ELISA using the ready-to-use solid support obtained by the method as claimed in claim 1, wherein said method comprises

a) reconstituting the ready-to-use support by adding appropriate amount of distilled water;
b) applying a sample to the support of step (a) to be tested for a target antigen/protein;
c) incubating the support of step (b) at about 37°C for about one hour to obtain an immunocomplex;
d) washing the support of step (c) with a suitable washing buffer to remove the unbound antigen/protein;
e) applying a buffer containing chemical substrate to the support of step (d) and incubating for about 30 minutes in dark at room temperature; and
f) detecting the presence of the antigen by measuring absorbance in step (e) at a suitable wavelength
12. The method as claimed in claim 11, wherein the chemical substrate is selected from
the group consisting of para-nitrophenol phosphate, Nitroblue Tetrazolium/5-Bromo-
4-Cholro-3-Indolyl Phosphate, 2,2'-Azino-bis(3-Ethylbenz-thiazoline-6-Sulfonic

acid), o-phenylenediamine, 3,3'-5,5'-Tetramethylbenzidine, o-Dianisidine and 5-Aminosalicylic Acid.
13. A ready-to-use solid support for quantitative and qualitative determination of a target antigen/protein in a sample, wherein the support has plurality of wells pre-coated with a first monoclonal antibody, a second antibody and a third antibody, wherein the first and second antibodies binds to the target antigen/protein and third antibody recognizes the second antibody, wherein the first monoclonal antibody includes monoclonal antibodies against antigen selected from a group consisting of Cry 1 Ac, Cry2Ab and 5-enolpyruvylshikimate-3 phosphate synthase antigen, wherein the support is free from a sensitivity enhancer agent.

Documents:

1600-DEL-2003-Abstract-(11-10-2010).pdf

1600-del-2003-abstract.pdf

1600-DEL-2003-Claims-(11-10-2010).pdf

1600-DEL-2003-Claims-(25-05-2011).pdf

1600-DEL-2003-Claims-(29-04-2011).pdf

1600-del-2003-claims.pdf

1600-DEL-2003-Correspondence Others-(25-05-2011).pdf

1600-DEL-2003-Correspondence Others-(29-04-2011).pdf

1600-DEL-2003-Correspondence-Others-(11-10-2010).pdf

1600-DEL-2003-Correspondence-Others-(25-02-2011).pdf

1600-DEL-2003-Correspondence-Others-(29-04-2010).pdf

1600-del-2003-correspondence-others.pdf

1600-del-2003-correspondence-po.pdf

1600-del-2003-description (complete).pdf

1600-del-2003-form-1.pdf

1600-DEL-2003-Form-13-(29-04-2011).pdf

1600-del-2003-form-18.pdf

1600-del-2003-form-2.pdf

1600-del-2003-form-26.pdf

1600-DEL-2003-Form-3-(11-10-2010).pdf

1600-DEL-2003-Form-3-(29-04-2010).pdf

1600-del-2003-form-3.pdf

1600-del-2003-form-5.pdf

1600-DEL-2003-GPA-(25-02-2011).pdf

1600-DEL-2003-Petition-137-(29-04-2010).pdf


Patent Number 248627
Indian Patent Application Number 1600/DEL/2003
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 28-Jul-2011
Date of Filing 23-Dec-2003
Name of Patentee MAHARASHTRA HYBRID SEEDS COMPANY LIMITED
Applicant Address ASHOK CENTRE, 3rd FLOOR, E-4/15, JHANDEWALA EXTENSION, PAHARGANJ, NEW DELHI-110 055, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 CHAR, BHARAT RAGHUNATH RHD-2, PHASE II, MAHYCO LIFE SCIENCE RESEARCH CENTRE, JALNA-AURANGABAD ROAD, AT; VILLAGE DAWALWADI TALUKA BADNAPUR, DISTRICT JALNA. P.O. BOX 76, JALNA-431 203, INDIA.
2 BIHANI, PANKAJ RAMESHCHANDRA B-1/D, PHASE II, MAHYCO LIFE SCIENCE RESEARCH CENTRE, JALNA-AURANGABAD ROAD, AT; VILLAGE DAWALWADI TALUKA BADNAPUR, DISTRICT JALNA. P.O. BOX 76, JALNA-431 203, INDIA.
PCT International Classification Number B10N 33/53
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA