Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/1997
Publication Date: N/A
Interpretive Summary: Serological detection methods are important to control programs for citrus tristeza virus (CTV) and to continued research efforts on CTV, the most important virus pathogen of citrus. Convenient and reproducible sources of antibodies have been difficult to obtain by conventional means. Antibodies can be conveniently produced to CTV encoded proteins expressed in bacteria, but most of these antibodies are not to conformational epitopes and have not performed well as trapping antibodies for Enzyme Linked immunosorbent assays. This paper describes how adding a small amount of antibody that reacts to a conformational epitope (recognition site) of the virus coat protein greatly increases antigen trapping efficiency of antibodies to bacterially expressed CTV proteins specific to non conformational epitopes. This technique is a promising way to expand use of antibodies produced to expressed proteins as antigen trapping agents for serological assays.
Technical Abstract: Trapping properties of a panel of monoclonal antibodies (Mabs) raised against citrus tristeza virus (CTV) were analyzed in an indirect double-antibody sandwich ELISA (I-DAS-ELISA). Mabs from groups I to IV, which react to linear epitopes, performed poorly as coating antibodies, even at a 1 microgram/ml concentration of the IgGs, presumably because the respective linear epitopes were inaccessible. However, when Mabs from groups I to IV were combined with a small amount of Mabs from group V (reactive to conformational epitopes) a substantial increase in trapping of the CTV antigen occurred. In this "two antibody-binding assay" previously cryptic, linear epitopes of the CTV CP apparently became accessible to the Mabs from groups I to IV. Induced exposure of the linear epitopes of the CTV CP was revealed in "two antibody-binding assays" with pairwise combinations of different mouse Mabs, and several rabbit and chicken polyclonal antisera with different serological specificities, including antisera to bacterially expressed CP fragments. This mixed coating in I-DAS-ELISA resulted in substantially increased efficiency of the virus antigen trapping by antisera produced against bacterially expressed protein fragments, and an increased sensitivity of CTV detection after optimization of conformational and linear antibodies concentration.