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Double Antibody Sandwich ELISA

Manufactured by Agdia
Sourced in United States

The Double Antibody Sandwich ELISA is a laboratory equipment used for the detection and quantification of target analytes in a sample. It utilizes a sandwich format with two specific antibodies that bind to the target analyte, enabling its identification and measurement.

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3 protocols using Double Antibody Sandwich ELISA

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.
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rCry1Ab was quantified in maize leaf feed by means of Agdia Cry1Ab/Cry1Ac DAS enzyme-linked immunosorbent assay (ELISA) kit (Double Antibody Sandwich ELISA) (Agdia, Elkhart, IN) following the manufacturer’s recommendations, with adaptations needed for the quantification of Cry1Ab as follows: A standard curve of serial dilutions of bacterial Cry1Ab (Abraxis, Warminster, PA) was constructed and used to estimate MON810 rCry1Ab content in maize leaf feed solutions (more detailed information in Electronic Supplementary Material 1 [file ESM1.pdf]).
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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.
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