|Yokomi, Raymond - Ray|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2009
Publication Date: 5/14/2011
Citation: Yokomi, R.K., Polek, M. 2011. Elevated Background in double antibody sandwich-indirect enzyme-linked immunosorbent assay for the detection of Citrus tristeza virus in mandarin cultivars. International Organization of Citrus Virologists Proceedings. p. 36-42. Interpretive Summary: Tissue extracts from mandarin cultivars occasionally induced a non-specific reaction in a serological assay for the detection of Citrus tristeza virus (CTV) resulting in a false positive test for this disease agent. This is a problem for regulatory agencies in California since CTV is a regulated pathogen and is subject to eradication by the State in certain Pest Control Districts and in citrus nurseries in central California. Immunocapture reverse transcription polymerase chain reaction tests readily and unambiguously confirmed the CTV status of all samples. The problem was associated with the high sensitivity of the reagents and the plate used in the assay. The false positive problem was resolved by several different methods including blocking with healthy mandarin extracts, using different reagents, ELISA plates and antiserum. Reactivity of mandarin samples infected with CTV was significantly higher than the false positive samples and were never in question.
Technical Abstract: Healthy tissue extracts from mandarin cultivars induced non-specific reaction in double antibody sandwich-indirect (DASI-) enzyme-linked immunosorbent assay (ELISA) for the detection of Citrus tristeza virus (CTV) by the Central California Tristeza Eradication Agency (CCTEA), Tulare, CA. This problem was seasonal occurring from March to May from field and nursery trees in central California. Problem samples were tested by immunocapture reverse transcription polymerase chain reaction and found to be negative for CTV. Several variations in the DASI-ELISA procedure were conducted and results indicated that the non-specific reaction was related to the high sensitivity of the conjugate and high binding capacity of the ELISA plate used. Two solutions to eliminate this problem were to use a different enzyme conjugate or to block incorporating extracts from uninfected mandarin tissue into the antiserum buffer. Since the elevated background in DASI-ELISA was not observed with several other CTV antisera tested, another solution was to use a different CTV antiserum. The non-specific reaction in DASI-ELISA was a problem only in healthy Mandarin cultivars. Mandarin samples infected with CTV always resulted in high absorbance values in DASI-ELISA and were never in question.