Double antibody sandwich elisa

In order to measure low concentrations of Cry1Ab, we concentrated each sample using Amicon Ultra-15 mL centrifugal filter units (30K Nominal Molecular Weight Limit, Millipore). We followed the centrifugal extraction method and subsequent Enzyme-Linked ImmunoSorbent Assay (ELISA) assay to measure Cry1Ab in stream water using field-validated techniques that have shown high recovery of Cry protein using phosphate buffered saline with tween buffer (PBST; [10 (link),11 (link)]). Briefly, to extract the protein from each water sample, we pipetted 14.5 mL of stream sample into the Amicon filter units and added 0.5 mL 1X PBST buffer. We separated the retentate from supernatant using a centrifuge with 60 mL centrifuge tube adapters at 2500 RPM for 30 minutes. The remaining concentrated Cry1Ab retentate was immediately pipetted into a sterile microcentrifuge tube.
After extraction, we quantified Cry1Ab protein in water samples using a commercial double-antibody sandwich ELISA (Agdia, Elkhart, IN, Part No: 06200/0096) as described by Strain and others [11 (link)]. For each sample, we pipetted 100 μL of the sample, standard, and blank (3 DI water, 3 PBST) retenate in triplicate onto 96-well ELISA plates. After following the manufacturer protocol, we read the plate absorbance at both 450 nm and 650 nm on a SpectraMax M5 microplate reader (Spectra Max M5, Molecular Devices, CA, USA). To correct for any turbidity in our samples, we then subtracted the resulting absorbance at 450 nm from the absorbance read at 650 nm. To account for any matrix effects of the PBST, we subtracted the mean PBST blank absorbance from the (650-450nm) estimation. Because we wanted to determine relative concentration of the protein in our sample, expressed in ng Cry1Ab L^-1 stream water, each plate included a five-point calibration curve created from a serial dilution of purified Cry1Ab (MyBioSource, Part No: MBS537737) dissolved in DI water [11 (link)]. The calibration curve ranged from 2 ng L^-1 to 400 ng L^-1Cry1Ab, and the curve was run in duplicate on each ELISA plate.

0.25 g of bark tissue from young flushes of C. macrophylla or leaf tissue from N. benthamiana plants ground in 5 mL of the extraction buffer (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 m KCL, 135 mM NaCl, 0.05% Tween^® 20, pH 7.4) per sample was used for double-antibody sandwich ELISA as per manufacturer’s instructions (Agdia, Elkhart, IN, USA) to confirm presence of viruses.

Xylem sap was collected from six 3-year-old grapevines (Vitis vinifera cv. ‘Thompson Seedless’) located at the University of California Davis (Armstrong field). Three of these plants were mechanically inoculated with Xylella fastidiosa Temecula1 12 months prior to sap collection. The presence of X. fastidiosa in the xylem sap of infected plants was confirmed using anti- X. fastidiosa antibodies in a Double Antibody Sandwich ELISA (Agdia, USA) following manufacturer’s instructions (Fig. S1). Xylem sap (30–50 mL per plant) was collected overnight in the second week of spring by drip of the cut stem of X. fastidiosa-infected and non-infected plants. To initiate sap collection, an apical segment of approximately 10 cm was cut from the stem and the vine terminal introduced into a collection tube sealed with parafilm. Xylem sap was lyophilized followed by protein precipitation using TCA/Acetone (Gorg et al., 2000 (link)). The pellets were resuspended in 300 µL of PBS (pH 7.4) and total protein was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific) following manufacturer’s instructions for subsequent SDS-PAGE and LC-MS/MS analysis.