Application of Citrus Tristeza Virus Antisera in Labeled Antibody, Immuno-Electron Microscopical, and Sodium Dodecyl Sulf ate-Immunodif fusion Tests's"

Citrus tristeza virus (CTV) antisera were prepared to undegraded, unfixed virus (CTV-W) , undegraded formaldehyde fixed virus (CTV-F) , and sodium dodecyl sulfate (SDS) degraded virus coat protein (CTV-S). These antisera were compared for their ability to detect CTV using in situ immunofluorescence ( ISIF) , double sandwich enzyme-linked immunosorbent assay (ELISA), serological specific electron microscopy (SSEM), and SDS immunodiffusion (SDS-ID). Gamma globulins (IgG) were separated from whole serum by sodium sulfate precipitation o r on a protein A affinity column for use in ISIF and ELISA. Unfractionated sera were used for SDS-ID and SSEM. Citrus tristeza virus could be detected in infected tissue by ISIF and SSEM with IgG prepared to CTV-W or CTV-F but not with CTV-S. In SDSID tests, CTV was detected with antisera to CTV-S or CTV-W but not with antiserum to CTV-F. Using ELISA, CTV was detected in buffered extracts with all three sera; however, nonspecific reactions interfered when IgG to CTV-S was used for both coating and conjugate steps. The nonspecific reaction was reduced when CTV-S was used as coating and CTV-W was used as the conjugate antiserum or when CTV-S IgG was isolated by a protein A affinity column. Sodium dodecyl sulfate degraded virus antigen was detected by ELISA when coating IgG to CTV-S and conjugate IgG to CTV-W was used. Presence of a t least two serological determinants for CTV was indicated. Index words. in situ immunofluorescence, serologically specific electron microscopy, enzyme-linked immunosorbent assay. Many serological methods are available for the detection and identification of plant viruses in plant tissues and extracts. Serological methods currently used for detection of citrus tristeza virus (CTV) include immunoflorescence (13), enzyme-linked immunosorbent assay (ELISA) (I), serologically specific electron microscopy (SSEM) (7) and sodium dodecyl sulfate immunodiff usion (SDSID) (8). Tsuchizaki et al. (13) used . an immunofluorescent microscopic technique for the detection of CTV in infected citrus tissues using fluorescein isothiocyanate (FITC) . An in situ immunofluorescent technique using tetramethylrhodamine isothiocyanate (TRITC) for the detection of xylem-limited bacteria in lFlorida Agricultural Experiment Stations Journal Series No. 4931. zUse of a company or product name by the U.S. Department of Agriculture does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. plant tissues was recently reported by Brlansky et al. (3). Antisera have been prepared in rabbits to undegraded, unfixed CTV (9), undegraded formaldehyde fixed CTV ( lo ) , and to sodium dodecyl sulfate degraded coat protein (8). Results reported have indicated that these sera are not equally suitable for all diagnostic procedures (1, 6, 10). In this paper we compare these antisera for detecting CTV by different serological tests. MATERIALS AND METHODS Virus isolates. The CTV isolates T-4 and T-3 from Florida were used throughout this study. The T-3 isolate produces a seedling yellows (SY) reaction whereas T-4 does not (8). Antisera. All antisera used in this study were prepared to the T-4 isolate of CTV. Antisera to undegraded, unfixed virus (CTV-W) and to undegraded formaldehyde fixed virus (CTV-F) were those prepared by Gonsalves et al. (9) Ninth ZOCV Conference and ( lo ) , respectively. Antiserum to SDS-degraded virus coat protein (CTV-S) was that prepared by Garnsey et al. (8) . Gamma globulins (IgG) separated from the whole serum by the sodium sulfate procedure of Kekwick (11) or by the protein A-Sepharose affinity chromatography procedure of Miller and Stone (12) were used for in situ immunofluorescence (ISIF) and ELISA. The concentration of IgG was estimated spectrophotometrically using E O.lojb/ 280 nm = 1.4. In some tests IgG fractionated by (NH,) ,SO,) precipitation and cellulose chromatography (1, 4) was used. Conjugation of antisera. Antisera for ISIF were conjugated with either (FITC) (Miles Laboratories, Elkhart, IN 46515) or with TRITC (Research Organics, Inc., Cleveland, OH 44125). The IgG were conjugated with FITC by the method of Blakeslee and Baines (2) and with TRITC as described by Brlansky et al. (3). The IgG for double sandwich ELISA were conjugated with alkaline phosphatase Type VII (Sigma Chemical Co., St. Louis, MO 63178) as previously described (1, 4) . I n situ immunofluorescence. Both FITCand TRITC-labeled IgG were used in this study with a modification of the technique of Brlansky et al. (3) . Sections of bark and petiole material of virusinfected and healthy citrus were cut using a Harris Model WRC cryostat (Harris Manufacturing, Inc., North Billerica, MA 01862). Longitudinal and transverse seetions 30 to 40 pm thick were immersed in labeled IgG to CTV. Labeled normal serum IgG and labeled IgG specific to Pierce's disease bacteria were used as controls. Sections were incubated a t room temperature for 1 hour or a t 37 C for 30 minutes, washed in phosphate-buffered saline for 10 to 20 minutes, and mounted on microscope slides in Aqua Mount. @ The slides were observed with a Leitz Dialuxs fluorescence microscope (E. Leitz, Inc., Rockleigh, NH 07647) equipped with a 50-V mercury lamp and viewed in the 560to 590-nm range for TRITC (Leitz filter block N2) and in the 380to 420-nm range (Leitz filter block H2) for FITC. Photographs were taken with Kodak Ektachrome EC 400 color film@ and/or Kodak Technical Pan 2415s black and white film. Serologically specific electron microscopy. The procedure for SSEM was that previously described for plant viruses by Derrick and Brlansky (5) and specifically for CTV by Garnsey et al. (7). Unfractionated antisera were used throughout a t dilutions of 1 :500 in 0.05 M Tris buffer, pH 8.0. Parlodion-coated copper grids were commonly used; although freshly prepared formvar-coated grids were also useful. In the latter case, grids were coated with 0.5% formvar in ethylene dichloride. After the IgG-sensitized grids were exposed to test extracts and rinsed, the grids were positively stained with a 5% solution of uranyl acetate in 50% ethanol for 1 minute and washed in 95% ethanol for 30 seconds. The grids were viewed with a Philips 201 transmission electron microscope. ELISA. The double sandwich ELISA procedure developed for CTV by Bar-Joseph et al. (1) was used. Immulon I1 Microtiters plates (Dynatech Laboratories, Inc., Alexandria, VA 22314) were coated with 1 pg IgG per ml for 4 hours at 37 C. Antigen preparations were prepared as previously described (1) or by grinding either healthy or CTV-infected tissue a t a ratio of 1 g fresh weight per 24 ml of 0.05 M Tris pH 8.0. Antigen preparations were then additionally diluted 1 to 50 (w/v) with either Tris buffer or Tris buffer Virus and Vroid Detection 339 containing SDS so that the final SDS concentration was 0.1%. Antigen preparations were incubated overnight a t 4 C in the microtiter plates. Conjugates were diluted 1 to 1,000 (about 1 pg IgG per ml) in a phosphate-buffered saline solution (PBS) containing 0.5% (vlv) Tween 20,2% (w/v) polyvinylpyrrolidone (40,000 molecular weight, Sigma) and 0.02% (wlv) bovine serum albumin (fraction V powder, Sigma) and incubated in the plates for 24 hours a t 4 C. After rinsing, substrate (p-nitrophenylphosphate, 1.0 mg/ml in 10% triethanolamine, pH 9.8) was added. After 30 minutes a t room temperature, the A,,, . , was read on each well using a Bio-Tek EIA Reader@, Model EL 307 (Bio-Tek Instruments, Inc., Burlington, VT 05401). Some samples were diluted with water and read in a spectrophotometer. SDS immunodiff usion. The SDSimmunodiffusion method used was that described by Garnsey et al.