Rapid C-ELISA process and related compositions
View Patent ↗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 cell-based enzyme-linked immunosorbent assay (C-ELISA), comprising the steps of:
(a) lysing cells in a quick lysis and coating buffer, wherein the quick lysis and coating buffer comprises water and a metal hydroxide selected from the group consisting of sodium hydroxide, potassium hydroxide and rubidium hydroxide;
(b) coating a solid phase with cellular proteins released in situ in the quick lysis and coating buffer;
(c) blocking the solid phase with a blocking reagent dissolved in a quick blocking buffer, wherein the quick blocking buffer comprises water and a metal hydroxide selected from the group consisting of potassium hydroxide, sodium hydroxide and rubidium hydroxide;
(d) (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;
incubating the solid phase with a secondary detection agent in solution;
washing the solid phase to remove any unbound secondary detection agent;
incubating the solid phase with a tertiary detection agent in solution;
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 the cellular proteins in the quick lysis and coating buffer for between about five minutes to ten 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 metal hydroxide has a concentration ranging from between about 0.0056 g/l to 56 g/l of quick lysis and coating buffer, and wherein the quick lysis and coating buffer has a pH ranging from between about 10.0 to 14.0.
4. The method according to claim 3 , wherein the metal hydroxide is potassium hydroxide.
5. The method according to claim 4 , wherein the potassium hydroxide concentration is about 5.6 g/l of buffer, and wherein the quick lysis and coating buffer has a pH of about 13.0.
6. The method according to claim 1 , wherein the solid phase is coated with the cellular proteins in the quick lysis and coating buffer for between about two minutes to twenty minutes and the solid phase is blocked with the blocking reagent in the quick blocking buffer for about two to twenty minutes.