Rapid ELISA processes and related compositions
The present invention provides improved and rapid detection methods for an antigen such as a chemical compound, a peptide, a nucleic acid, or a protein released from cells or virus particles in situ. The detection time for an antigen can be dramatically reduced relative to conventional technologies. The technology can particularly be used, for example, to modify and reduce the detection time significantly in traditional ELISA, and also Western blot or Dot blot assays. The improved ELISA method is rapid, economical, reproducible, simple and automatable. Also provided are compositions and kits for using the improved ELISA methods for the rapid detection of antigens.
1. A method for performing a rapid enzyme-linked immunosorbent assay (ELISA), comprising the steps of:
(a) coating a solid phase with an antigen dissolved in a quick coating buffer for between about two to twenty minutes, wherein the quick coating buffer comprises water and a metal hydroxide selected from the group consisting of sodium hydroxide, potassium hydroxide and rubidium hydroxide;
(b) blocking the solid phase with a blocking reagent dissolved in a quick blocking buffer for between about two to ten minutes, wherein the quick blocking buffer comprises a metal hydroxide selected from the group consisting of sodium hydroxide, potassium hydroxide and rubidium hydroxide; and
(c) (i) incubating the solid phase with a primary detection agent in solution;
washing the solid phase to remove any unbound primary detection agent; and
detecting the presence of an agent bound on the solid phase with a detection system that detects the bound primary detection agent; or
(ii) incubating the solid phase with a primary detection agent in solution;
washing the solid phase to remove any unbound primary detection agent;
incubating the solid phase with a secondary detection agent in solution;
washing the solid phase to remove any unbound secondary detection agent; and
detecting the presence of an agent bound on the solid phase with a detection system that detects the bound secondary detection agent; or
(iii) incubating the solid phase simultaneously with a primary detection agent and a secondary detection agent in solution;
washing the solid phase to remove any unbound primary detection agent and secondary detection agent; and
detecting the presence of an agent bound on the solid phase with a detection system that detects the bound secondary detection agent; or
(iv) incubating the solid phase with a primary detection agent in solution;
washing the solid phase to remove any unbound primary detection agent; and
incubating the solid phase with a secondary detection agent in solution;
washing the solid phase to remove any unbound secondary detection agent; and
incubating the solid phase with a tertiary detection agent in solution; and
washing the solid phase to remove any unbound tertiary detection agent; and
detecting the presence of an agent bound on the solid phase with a detection system that detects the bound tertiary detection agent; or
(v) incubating the solid phase simultaneously with a primary detection agent, a secondary detection agent and a tertiary detection agent in solution;
washing the solid phase to remove any unbound primary detection agent, secondary detection agent and tertiary detection agent; and
detecting the presence of an agent bound on the solid phase with a detection system that detects the bound tertiary detection agent.
2. The method according to claim 1 , wherein the solid phase is coated with an antigen in the quick coating buffer for about five minutes and the solid phase is blocked with the blocking reagent in the quick blocking buffer for about five minutes.
3. The method according to claim 1 , wherein the blocking reagent is selected from the group consisting of non-fat milk, casein, BSA, fish gelatin and other chemical reagents.
4. The method according to claim 1 , wherein said metal hydroxide concentration in the quick coating buffer ranges from between about 0.004 g/l to 40 g/l of buffer, and wherein the quick coating buffer has a pH ranging from between about 10.0 to 14.0.
5. The method according to claim 4 , wherein the metal hydroxide in the quick coating buffer is sodium hydroxide.
6. The method according to claim 5 , wherein the sodium hydroxide concentration is about 4 g/l of buffer, and wherein the quick coating buffer has a pH of about 13.0.
7. The method according to claim 1 , wherein said metal hydroxide a concentration in the quick blocking buffer ranges from between about 0.0056 g/l to 56 g/l of buffer, and wherein the quick blocking buffer has a pH ranging from between about 10.0 to 14.0.
8. The method according to claim 7 , wherein the metal hydroxide in the quick blocking buffer is potassium hydroxide.
9. The method according to claim 8 , wherein the potassium hydroxide concentration is about 5.6 g/l of buffer, and wherein the quick blocking buffer has a pH of about 13.0.
10. The method according to claim 1 , wherein said detection system detects the bound detection agent by measuring signals selected from the group consisting of ultraviolet light, fluorescence, luminescence, colorimetric signals and other signals capable of detecting the bound detection agent.
11. The method according to claim 1 , wherein the detection agent can be labeled or unlabeled, said detection agent selected from the group consisting of avidin, strepavidin, a chemical molecule, a peptide molecule, a protein molecule, an RNA molecule, a DNA molecule, an antibody, a fragment of an antibody, a recombinant antibody, a bacterial cell, a virus particle, a cell and a particle.
12. The method according to claim 11 , wherein the protein molecule is selected from the group consisting of protein A, protein G, protein L, protein A/G and other antibody-binding polypeptides.
13. The method according to claim 11 , wherein the detection agent is labeled, said label is selected from the group consisting of an enzyme; radioactive isotope; fluorochrome; lumichrome and biotin.
14. The method according to claim 13 , wherein the enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase and beta-galactosidase.