Description:FIELD OF THE INVENTION
The present invention is related to the field of novel composition/formulation for analytical and biochemical assays or techniques. More specifically, the invention relates to a reagent composition/formulation for coating and immobilization antigens, antibodies, proteins, peptides, enzymes, etc. in analytical and biochemical assays or techniques, including Enzyme-linked immunosorbent assay (ELISA).
BACKGROUND OF THE INVENTION
Immunoassays are the means of detection and diagnosis of various infectious diseases and exploits the specific interaction between an antigen and an antibody. Principally, these methods are based on a competitive binding reaction between a fixed amount of labelled form of an analyte and a variable amount of unlabelled sample analyte for a limited number of binding sites on a highly specific anti-analyte antibody.
Enzyme-linked immunosorbent assay (ELISA) is an assay technique used to detect small amounts of antibodies, antigens, peptides, proteins, glycoproteins, and viruses in biological samples. Further, ELISA may be used to detect an analyte based on antigens on the analyte that are recognisable by capture and detection agents (e.g. antibodies, aptamers, etc.), making it an important assay, especially in the life sciences. ELISA may be used to detect the presence, absence or the amount of an analyte in a sample.
ELISA coating buffers are buffer solutions used in immunoassays for the immobilization of proteins and antibodies onto plastic surfaces such as plates. The solution may contain phosphates or carbonates, and help to maximize specific binding in the solid phase, thereby increasing the stability and sensitivity of the assay.
Generally, coating of proteins is done using phosphate buffer saline (PBS) or carbonate buffer (pH-9.5-10.5) for immunoassays like ELISA where specialized polystyrene plates designed for protein binding are used. The period of coating generally ranges from 6-24 hours for coating proteins on these plates.
Chinese Patent No. CN103063830B disclose a method for studying protein-protein interaction, protein-small molecule interactions and protein-peptide interactions on specialized polystyrene plates. These plates are very expensive and increases the overall cost of the method. Immunoassays like ELISA is regarded as a gold standard because of its high sensitivity and specificity, but these assays come with a drawback of using expensive polystyrene plates and high turnaround time.
Enzyme immobilization is commonly used especially in industrial processes where matrices of different chemical nature are used to capture desired enzyme or protein for catalysis. Enzyme immobilization for catalysis has been successfully shown in lab scale, pilot scale and even in large bioreactors.
There are various types of immobilization techniques that are used which range from simple approaches like adsorption to chemically complex methods involving covalent bonding and cross-linking, encapsulation and entrapment.
U.S. Patent No. US6040182A discloses coating/Immobilization methods for the facilitation of high-protein-binding capability on tissue culture-treated ("TC") plastic assay plates such as on expensive polystyrene assay plates
U.S. Patent No. US4797354A discloses enzyme, proteins on microbial cells immobilization on in alginate and silica sol.
Japanese Publication No. JP2004170195A discloses covalent methods of immobilization.
Therefore, it is evident from the prior arts that, special treated charged plates are required for adsorption of proteins/peptides. If adsorption is possible on untreated polystyrene plates, the cost of the ELISA would be much less as the cost of these uncharged plates are affordable. These charged plates apart from being expensive have another disadvantage. The time taken for adsorption of desired protein/antigen/antibody is very long when conventional buffers like carbonate buffer, pH 9.5 or phosphate buffered saline (PBS) are used. Generally, 6-24 h incubation is required for good results.
Adsorption is the easiest way to coat or immobilize proteins as its applicability would be for wider range of proteins and the chemical complexity involved is much less compared to other approaches. However, the strength of binding is always low compared to a covalent-based immobilization. Though covalent chemistry enhances the binding efficiency of the protein, each type of chemical modification can be used only for a narrow range of proteins.
Enzyme-linked immunosorbent assay (ELISA) based methods are sensitive and reliable but requires high quality or pure antibodies or antigens or so demanding long incubation times. In addition, long turnaround times, sensitive readout formats, laborious optimizations and skilled technical manpower makes it expensive to operate. Therefore, there is a huge need for a simple solution that can address the above technical challenges.
Accordingly, it would be desirable to provide a method for detecting an analyte in a sample that takes less time and inputs to perform compared with conventional ELISA, while maintaining or improving the sensitivity of detection.
Hence, the present invention addresses these drawbacks which involves the use of a newly formulated reagent that accelerates the coating/adsorption process on any plastic plates (charged and uncharged plates) within 10-60 minutes and also aiding in eliminating blocking steps in the case of uncharged plates. This opens a whole new avenue for different application areas for antigen/antibody detection, protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, etc using non-covalent interactions.
OBJECT OF THE INVENTION
An objective of the present invention is to overcome the problems of long incubation periods, for example 6-24 hr, for coating proteins for ELISA, protein-protein interactions and protein immobilization.
Another objective of the present invention is use of inexpensive or low-cost non-ELISA plates for protein enzyme immobilization on different resins, studies involving protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, microfluidic assays, protein chips, biosensors and other assays/platforms involving coating/immobilization of proteins/enzymes/peptides during ELISA experiments or tests.
Another objective of the present invention is eliminating the blocking step when using non-ELISA plates for ELISA and other related assays.
Another objective of the present invention is using a universal agent for coating any protein/peptide on any surface.
The present invention aims to significantly reduce the turnaround time of ELISA protein-protein interactions and protein immobilization by quickly coating proteins on surfaces in less than 60 minutes, more significantly 10-30 minutes.
The present invention further aims reducing cost of the test/assay- ELISA/protein-protein interactions/protein immobilization.

SUMMARY OF THE INVENTION
Aspects of the present invention provides a novel reagent composition/formulation for coating and immobilization using antigens, antibodies, proteins, peptides, enzymes, etc. in analytical and biochemical assays or techniques, including Enzyme-linked immunosorbent assay (ELISA). Further, the present invention can also be used for protein enzyme immobilization on different resins, studies involving protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, microfluidic assays, protein chips, biosensors and other assays/platforms involving coating/immobilization of proteins/enzymes in any coated or uncoated plastic, glass, rubber, silicon, metal or polymer surfaces.
In one aspect of the present invention provides reagent buffer composition/formulation for coating and immobilization, the reagent comprising 2-20 mM Tris (pH 6.3 or pH 8.5), 0.01-2.0% Dimethyl Sulfoxide (DMSO), 0.01-2.0% butanol, 0.001 – 9% 1,6-bis (4-Chlorophenyldiguanino) hexane gluconate or digluconate, 0.001 – 8.1% Tween 20 or 80, and anionic or cationic surfactant in sufficient quantity.
The reagent composition/formulation of the present invention provides coating/immobilization proteins on different hydrophobic surfaces, glass, resins used for protein immobilization very rapidly within 10-60 minute in a non-covalent manner and enable efficient binding even on normal, cost-effective plastic or polystyrene plates circumventing the need for specialized plates.
One aspect of the present invention, the reagent significantly reduces coating proteins for ELISA, protein-protein interactions and protein immobilization to just 10-60 minutes.
One advantage of the present invention is that solve the problems of long incubation periods (6-24 h) for coating proteins for ELISA, protein-protein interactions, protein immobilization by significantly reducing to just 10-60 minutes.
One more advantage of the present invention provides use of cheap non-ELISA plates for performing ELISA is possible. Thereby significantly reducing cost of the assay.
One more advantage of the present invention provides eliminating the blocking step when using non-ELISA plates for ELISA and other related assays.
The summary of the invention is not intended to limit the key features and essential technical features of the claimed invention, and is not intended to limit the scope of protection of the claimed embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS
The above-mentioned and other features and advantages of this present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the following description of embodiments of the present disclosure taken in conjunction with the accompanying drawings. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective equivalent embodiments.
Fig. 1A is a graphical representation of Enzyme coating Assay with the enzyme Apyrase reconstituted in reagent buffer composition/formulation of the present invention, Carbonate buffer (pH 9.5), Phosphate Buffer Saline (PBS), pH 7.4 and water;
Fig. 1B is a graphical representation of Enzyme coating Assay with the enzyme alkaline phosphatase (bottom) reconstituted in reagent buffer composition/formulation of the present invention, Carbonate buffer (pH 9.5), Phosphate Buffer Saline (PBS), pH 7.4 and water;
Fig. 2 is a graphical representation of Enzyme-linked immunosorbent assay (ELISA) with His-tag peptide reconstituted in reagent buffer composition/formulation of the present invention (XIC), carbonate buffer (pH 9.5) and Phosphate Buffer Saline (PBS), pH 7.4;
Fig. 3 is a graphical representation of Enzyme-linked immunosorbent assay (ELISA) with His-tag antigen reconstituted in different batches of reagent buffer composition/formulation of the present invention (XIC) prepared to monitor consistency of performance of the reagent. Here, standard ELISA plates (Nunc) and cheap non-ELISA plates (Laxbro) were used;
Fig. 4 is a graphical representation of Enzyme-linked immunosorbent assay (ELISA) using two versions of reagent buffer composition/formulation of the present invention which differ in pH (pH 6.3 and 8.5), Nunc (charged ELISA plates) and Laxbro (non-ELISA plates) were used;
Fig. 5 is a graphical comparison Enzyme-linked immunosorbent assay (ELISA) by coating His-tag antigen using reagent buffer composition/formulation of the present invention by including and excluding the blocking step in the experiment;
Fig. 6 is a graphical comparison of Enzyme-linked immunosorbent assay (ELISA) by coating His-tag antigen using carbonate buffer with coating time of 6 h (A) and reagent buffer with coating time of just 10 minutes (B) composition/formulation of the present invention;
Fig. 7A is a graphical representation of Immobilization of Leucine dehydrogenase enzyme, in accordance with the embodiments of the present invention; and
Fig. 7B is a graphical representation of Immobilization of NADH oxidase, in accordance with the embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described by reference to more detailed embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, formulations, steps/processes, reagents, and/or components, but do not preclude the presence or addition of one or more other features, elements, integers, compositions, formulations, steps, reagents, components, and/or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.
Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.
Embodiments of the present invention provides a novel reagent composition/formulation for coating and immobilization using antigens, antibodies, proteins, enzymes, etc. in analytical and biochemical assays or techniques, including Enzyme-linked immunosorbent assay (ELISA). The present invention can also be used for protein enzyme immobilization on different resins, studies involving protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, microfluidic assays, protein chips, biosensors and other assays/platforms involving coating/immobilization of proteins/enzymes in any coated or uncoated plastic, glass, rubber, silicon, metal or polymer surfaces.
In one preferred embodiment of the present invention provides reagent buffer composition/formulation for coating and immobilization, the reagent comprising 2-20 mM Tris (PH 6.3 or pH 8.5), 0.01-2.0% Dimethyl Sulfoxide (DMSO), 0.01-2.0% butanol , 0.001 – 9% 1,6-bis (4-Chlorophenyldiguanino) hexane gluconate or digluconate, 0.001 – 8.1% Tween 20 or 80, and anionic or cationic surfactant in sufficient quantity.
The reagent is commercially made available In name of Xact InstaCoat (XIC), having unique composition/formulation to coat/immobilize proteins on different hydrophobic surfaces, glass, resins used for protein immobilization very rapidly within 10-60 min in a non-covalent manner and enable efficient binding even on normal, cost-effective plastic or polystyrene plates circumventing the need for specialized plates. In a preferred embodiment, the reagent significantly reduces coating proteins for ELISA, protein-protein interactions and protein immobilization to just 10-60 minutes.
In one embodiment, the present invention provides a very robust protein coating reagent composition/formulation that can promote interaction with any surface, even glass and resins used for immobilization of proteins. Since the interaction between proteins and surface/resin is non-covalent, it can be used as a universal reagent for coating/immobilization of proteins.
The provided composition/formulation is used for the following applications, and the results are as described below with various exemplary embodiments.

Examples
Example 1, Enzyme coating:
In one exemplary embodiment, 5-10 µg Enzymes (Apyrase and Alkaline phosphatase) are reconstituted in 1 mL of the reagent composition/formulation of the present invention. Then 100 µL of reconstituted enzyme is added to each well of the microtiter plate and incubated at 37C for 10-20 minutes. Without limiting the scope of the invention, it should be understood that any microtiter plate made of polystyrene can be used, even non-ELISA plates.
Then wells were washed with water (approx. 200 µL), and a substrate selected from 10 mM ATP for Apyrase, 10 mM para-nitro phenyl phosphate, pNPP for alkaline phosphatase are added to the wells.
After that to measure the activity of Apyrase, formation of phosphates is measured by addition of a 150 µL AMFAS (Ammonium Molybdate Ferrous Ammonium Sulphate) solution which is an equimolar mixture of Solution A (5% ammonium molybdate in 5N Hcl) and Solution B (1% ferrous ammonium sulphate). The corresponding results are shown in Fig.1A shows enzyme coating assay with Apyrase, and Fig.1B enzyme coating assay with Alkaline phosphatase. Where OD at 630 nm for Apyrase is shown in Fig.1A and OD at 405 nm for alkaline phosphatase assay is shown in Fig.1B is measured.
Fig. 1A shows Enzyme coating assay with Apyrase, where 700ng Apyrase is reconstituted in different batches of the reagent (Xact InstaCoat) of the present invention was added to wells of Nunc and Laxbro microtiter plate and incubated for 20 minutes at 37C. After washing activity was monitored by colorimetric assay using 10mM ATP. All brands of plates show similar results.
Fig.1B shows Enzyme coating assay with Alkaline phosphatase, where 700ng Alkaline phosphatase is in different batches of the reagent (Xact InstaCoat) of the present invention was added to wells of Nunc and Laxbro microtiter plate and incubated for 20 minutes at 37C. After washing activity was monitored by colorimetric assay using 10mM ATP. All brands of plates show similar results.
In Fig. 1A and 1B, activity of the enzyme after coating and washing the plates show the coating efficacy of the reagent, where the coating time is just 10 minutes. Conventional buffers like carbonate buffer (pH 9.5) and PBS (pH 7.4) do not coat in such short incubation time.
Example 2, Enzyme-linked immunosorbent assay (ELISA):
In another exemplary embodiments, antigen/antibody (1-10 µg) is reconstituted with 1 mL of the reagent composition/formulation of the present invention. Though in this example antigen is added, but antibody can also be added. Further in this example, two different pH for coating (pH 6.3 and pH8.5) are chosen, where 100 µL of reconstituted antigen/antibody is added to each well of the microtiter plate and incubated at 37C for 10-20 minutes. Without limiting the scope of the invention, it should be understood that any microtiter plate made of polystyrene can be used, even non-ELISA plates that are not charged can be used. After coating, ELISA as per the user’s protocol can be followed.
The plate is blocked only if charged/pre-treated plates are used.
Fig.2 shows Enzyme-linked immunosorbent assay (ELISA) with His-tag peptide, which means performing the ELISA by coating His-tag peptide and detection using anti-His antibody. Where 500ng of His-tag peptide reconstituted in different batches of the reagent (Xact InstaCoat) of the present invention was added to wells of non-ELISA microtiter plates (Laxbro) and incubated for 15mins at 370C. Wells were washed and probed for His-tag peptide as per standard ELISA protocol.
Fig. 3 shows Enzyme-linked immunosorbent assay (ELISA) with His-tag antigen/protein, which means performing the ELISA by coating His-tag antigen/protein and detection using anti-His antibody. Where 100ng of His-tag antigen reconstituted in different batches of the reagent (Xact InstaCoat) of the present invention was added to wells of ELISA (Nunc) and non-ELISA microtiter plates (Laxbro) and incubated for 15mins at 370C. Wells were washed and probed for His-tag peptide as per standard ELISA protocol.
Further, Fig.4 shows Enzyme-linked immunosorbent assay (ELISA) using the reagent (Xact InstaCoat) at pH 6.3 and pH 8.5 with His-tag antigen, which means performing ELISA by coating His-tag antigen and detection using anti-his antibody at different pH. Where 100ng and 400ng of His-tag antigen reconstituted in the reagent (Xact InstaCoat) (pH 6.3 and 8.5) was added to wells of microtiter plates (Nunc and Laxbro) and incubated for 15mins at 370C. Wells were washed and probed for His-tag antigen by standard ELISA methods. Both Laxbro and Nunc show similar results. This shows that the reagent enables efficient coating even on cheap non-ELISA plate, which is at par with that of Nunc. Coating is efficient at an alkaline pH of 8.5 and a slightly acidic pH of 6.3.
Example 3, (ELISA) with different brands of coating plates using the reagent buffer (Xact InstaCoat) of different pH:
Fig.4 shows Enzyme-linked immunosorbent assay (ELISA) with different brands of coating plates using the reagent buffer (Xact InstaCoat) of the present invention for different pH (i.e. pH 6.3 and pH 8.5). Fig.4 shows that the reagent buffer (Xact InstaCoat) of both pH 6.3 and pH 8.5 perform with similar efficiency in ELISA. As shown in Fig 4, the ELISA with his-tag antigen was performed and results are evaluated accordingly.
Example 4, (ELISA) with different brands of coating plates using the reagent buffer (Xact InstaCoat) of different pH:
Blocking is done with 3% BSA for 20 minutes at 370C. This step can be eliminated in the case of non-ELISA plates as shown in the figure 5. But for charged plates like Nunc, the blocking step is mandatory as it results in non-specific signal if the blocking is not performed (data not shown). Elimination of this step when ELISA is done with cheap non-ELISA plates will further reduce the turnaround time in addition to the reduction of the cost of the assay.
Example 5, Comparison of ELISA on different brands of plates (High-cost Nunc ELISA plate vs low-cost non-ELISA plate brands):
Fig.6 shows ELISA with his-tag antigen was performed on different brands of plates. It was found that when conventional buffers were used, the performance of ELISA varied on different brands of plates. Use of our reagent buffer showed no difference in performance, where even low-cost plates performed at par with that of expensive Nunc plates. As shown in Fig.6, ELISA with carbonate buffer (pH 9.5) and the reagent buffer (Xact InstaCoat) on different brands of microtiter plates. ELISA by coating His-tag antigen using carbonate buffer (A) and the reagent buffer (B) of the present invention, and detection using anti-his antibody. Where 100ng of His-tag antigen reconstituted respective buffers and was added to wells of different brands of microtiter plates and incubated for 15mins at 370C. Wells were washed and probed for His-tag antigen as mentioned above. All plates show similar results in the case of the reagent buffer (B) whereas low-cost plates like Laxbro, Tarson and HiMedia show lesser sensitivity compared to Nunc when carbonate buffer was used.
Therefore, as results shown it should be appreciated that the reagent buffer (B) of the present invention enables efficient use of low-cost plates, enabling further reduction in costs.
Example 6, Immobilization of enzymes on resins (silica and polystyrene of all mesh sizes):
In another exemplary embodiment, enzyme immobilization was performed as described herein. Where a desired amount of enzyme is mixed in the reagent (Xact InstaCoat) of the present invention, and bound to 30 mg of silica resin for 1 h at 25C. Unbound fraction is collected and kept aside for analysis for activity. The resin is washed twice with Tris Buffer Saline (TBS). Monitor the activity of the enzyme on the resin by addition of the specific substrate. It should be appreciated that Immobilization of enzymes on silica resin can be done for on all mesh sizes.
Fig.7A shows Immobilization of Leucine dehydrogenase enzyme, where Leucine dehydrogenase (Leu B) catalyzes the conversion of 3- methyl malate and NAD to its product and NADH. Activity is measured at 340 nm, indicating formation of NADH. Fig. 7A shows the activity of Leucine dehydrogenase immobilized in 30mg of the bead/matrix using NAD assay.
Fig.7B shows Immobilization of NADH oxidase, where NADH oxidase catalyzes conversion of NADH to NAD. Loss of absorbance at 340 nm indicates formation of NAD. Fig.7B shows the activity of NADH oxidase immobilized on 30mg of the bead/matrix.
Where absorbance was taken every minute after incubating substrates with the resin. The slope of the graph was calculated. The slope of NADH standard graph (2.5) was taken as reference. Therefore, the activity on the bead was calculated by dividing the slope of our reaction by the slope of the standard graph of NADH (2.5).
Example 6:
In Table 1 the efficiency of binding of each enzyme and residual activity on the resin is presented below.
Protein Protein binding (%) Activity (%)
Leucine dehydrogenase 95 95
NADH oxidase 100 75
Table 1. Efficiency of immobilization of enzyme on silica resin
The activity of the enzyme after immobilization is based on the nature of the enzyme. Leucine dehydrogenase and NADH oxidase have very good activity. In all cases, protein binding is very efficient.
Example 7:
As a control, various tests for the activity of most enzymes are performed in the presence of the reagent (Xact InstaCoat) of the present invention. As shown in Table 2, no significant activity is lost for any enzyme.
Enzyme
Residual in-solution activity after Xact InstaCoat treatment for 1 hour (%)
Leucine dehydrogenase 97
NADH oxidase 99
Apyrase 98
Table 2. Effect of Xact InstaCoat on activity of different enzymes.
With the results shown in Table 2, it is proved that treatment with the reagent (Xact InstaCoat) of the present invention does not affect the activity of the enzyme.
Example 8, Immobilization on Polystyrene resin:
Further in another exemplary embodiment of the present invention, used Apyrase enzyme to immobilize on polystyrene, since silica inhibited the activity of the enzyme. In terms of immobilization, 98% of the Apyrase reconstituted in the reagent (Xact InstaCoat) binds to silica, but Apyrase is not active on the resin. The reagent also does not affect the activity of Apyrase in solution. Therefore, we used polystyrene resin to immobilize Apyrase. Desired amount of enzyme is mixed in the reagent (Xact InstaCoat) and bound to 30 mg of polystyrene resin for 1 h at 25C. Unbound fraction is collected and kept aside for analysis for activity. The resin is washed twice with Tris Buffer Saline (TBS) and monitored the activity of the enzyme on the resin by addition of 10mM ATP. As observed in Table 3, significant binding and activity of Apyrase was observed.
Resin (30 mg) Protein binding (%) Activity (%)
Polystyrene 95 78
Table 3. Immobilization of Apyrase on Silica and Polystyrene resin
One of the advantages of the present invention is using the reagent buffer (Xact InstaCoat) in Enzyme-linked immunosorbent assay (ELISA) provides instant coating. Because the reagent buffer (Xact InstaCoat) in ELISA coats inexpensive polystyrene plates which are non-ELISA plates within 10 minutes. Because of this unique property of the reagent buffer (Xact InstaCoat), it can be used to coat antigen/antibody even on non-ELISA plates in very less time and can thereby reduce the cost and turnaround time of the experiment. The efficiency of coating in a short time on inexpensive plates is at par with that of expensive brands like Nunc. Conventional methods for coating take at least 6-24 h for effective coating. The use of the reagent buffer (Xact InstaCoat) reduces time and enables use of very cheap plastic surfaces (non-ELISA plates) for ELISA enabling to further reduce the cost.
One more advantage of the present invention, there is no blocking in non-ELISA plates. This reagent can be used to coat proteins/antigens/antibodies on non-ELISA plates. While using these plates for ELISA, the blocking step can be avoided thereby further reducing time and use of blocking reagents.
One more advantage of the present invention is in protein immobilization. Because the reagent buffer (Xact InstaCoat) also enables protein immobilization on resins in a non-covalent manner, which can be adopted as a generalized method for protein immobilization unlike most proteins requiring a lot of specific derivatizations before immobilization.
As described above in this disclosure, the invention of the reagent buffer (Xact InstaCoat) addresses the drawbacks in the prior art and opens a whole new avenue for different application areas for antigen/antibody detection, protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, etc using non-covalent interactions, which might cause weak binding but can be optimised/calibrated for better performance.
One more advantage of the present invention provides use of a universal agent for coating any protein on any surface for protein enzyme immobilization on different resins, studies involving protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, microfluidic assays, protein chips, biosensors and other assays/platforms involving coating/immobilization of proteins/enzymes.
In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results obtained.
While the invention has been illustrated by reference to specific embodiments, the invention is not intended to be limited to the details shown. Those skilled in the art will understand that variations and modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the invention. Thus, the invention is intended not to be limited by the foregoing description, but to be defined by the appended claims and their equivalents.
, C , C , C , Claims:We Claim
1. A reagent composition for rapid protein coating/immobilization, the composition comprising of:
2-20 mM Tris at pH 6.3 or pH 8.5;
0.01-2.0% Dimethyl Sulfoxide (DMSO);
0.01-2.0% butanol;
0.001 – 9% 1, 6-bis (4-Chlorophenyldiguanino) hexane gluconate or digluconate,
0.001 – 8.1% Tween 20 or Tween 80; and
anionic or cationic surfactant, and combination thereof.
2. The composition according to claim 1, wherein the reagent is used for rapid protein coating/immobilization on polystyrene, glass and other materials involved in capturing proteins.
3. The composition according to claim 1, wherein the reagent is used for coating and immobilization using antigens, antibodies, proteins, enzymes, etc. in analytical and biochemical assays or techniques, including Enzyme-linked immunosorbent assay (ELISA).
4. The composition according to claim 1, wherein the reagent is used as an important component of in vitro diagnostic kit, especially ELISA.
5. The composition according to claim 1, wherein the reagent is used in Enzyme-linked immunosorbent assay (ELISA), where even non-ELISA plates can be used.
6. The composition according to claim 1, wherein the reagent is used in Enzyme-linked immunosorbent assay (ELISA), where blocking step is not required when used for non-ELISA plates.
7. The composition according to claim 1, wherein the reagent is used for including but not limited to protein enzyme immobilization on different resins, studies involving protein-protein interactions, protein-small molecule interactions, protein-peptide interactions, microfluidic assays, protein chips, biosensors and other assays/platforms involving coating/immobilization of proteins/enzymes.
8. The composition according to claim 1, wherein the reagent enables protein immobilization on resins in a non-covalent manner.
9. The composition according to claim 1, wherein the reagent reduce time for coating proteins for ELISA, protein-protein interactions and protein immobilization to just 10-60 minutes.