Methods for liquid direct fluorescent antibody intracellular virus detection
View Patent ↗The present invention describes a liquid direct fluorescence antibody assay that is rapid and sensitive to detect respiratory virus in infected cells. The assay includes centrifugation of the specimen, incubation of sample and reagents in solution, and detection of the absence or presence of respiratory virus. Sapogenin is used as a detergent to permeabilize the cells for entry of the monoclonal antibodies to react with intracellular antigens. The cells are stained with fluorescently labeled monoclonal antibodies against the viral antigens along with a background stain and a fluorescent nuclear stain. This counter staining decreases background and allows co-localization of antigen and nuclear structures for enhanced detection.
1. A method for detection of an intracellular antigen comprising:
incubating a suspension comprising a biological sample, at least two differentially fluorescently labeled antibodies and a staining reagent to generate a liquid cell suspension, wherein said biological sample is suspected of comprising at least one intracellular respiratory viral antigen and wherein said liquid cell suspension comprises unfixed cells, wherein during the incubating, the at least one intracellular viral antigen binds to one of the at least two differentially fluorescently labeled antibodies to form an intracellular antigen-antibody complex, wherein said respiratory viral antigen is selected from the group consisting of a respiratory syncytial viral antigen, an influenza A viral antigen, an influenza B viral antigen, an adenovirus viral antigen, a parainfluenza viral antigen and a metapneumovirus viral antigen;
detecting the intracellular antigen-antibody complex within the liquid cell suspension by identifying the one of the at least two differentially fluorescently labeled antibodies.
2. The method of claim 1 , wherein each of the at least two differentially fluorescently labeled antibodies comprises a R-phycoerythrin fluorescent label or a fluorescein isothiocyanate fluorescent label.
3. The method of claim 1 , wherein each of the at least two differentially fluorescently labeled antibodies is a monoclonal antibody.
4. The method of claim 1 , wherein the staining reagent is detectable by fluorescent microscopy.
5. The method of claim 1 , wherein the staining reagent is selected from the group consisting of Evans blue, propidium iodide, acridine orange and combinations thereof.
6. The method of claim 1 , wherein at least one of the antibodies comprises specific affinity for said respiratory syncytial viral antigen.
7. The method of claim 1 , wherein at least one of the antibodies comprises a specific affinity for said influenza A viral antigen.
8. The method of claim 1 , wherein at least one of the antibodies comprises a specific affinity for said influenza B viral antigen.
9. The method of claim 1 , wherein at least one of the antibodies comprises a specific affinity for said adenovirus viral antigen.
10. The method of claim 1 , wherein at least one of the labeled antibodies comprises a specific affinity for said parainfluenza viral antigen.
11. The method of claim 1 , wherein at least one of the labeled antibodies comprises a specific affinity for said metapneumovirus viral antigen.
12. The method of claim 1 , wherein said suspension further comprises a permeabilization agent.
13. The method of claim 12 , wherein said permeabilizaton agent comprises a detergent.
14. The method of claim 13 , wherein said detergent is sapogenin.