Sandwich ELISA for scFv Detection

Sandwich ELISA was performed following standard method, using anti-6xHis antibody (Abcam, Cambridge, UK) 1:250 in PBS + 3%BSA to reveal the scFv, followed by anti-mouse IgG-HRP conjugate (Sigma, Missouri, USA) 1:500 in PBS + 3%BSA, as described in [35 (link)]. In case the ELISA assay is performed using mAb-hERG1, the procedure differs as the 2 h incubation with the monoclonal antibody is followed by the revealing using secondary peroxidate anti-mouse antibody (no intermediate incubation with anti-tag antibodies, e.g. anti-His, occurs).

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Duranti C., Carraresi L., Sette A., Stefanini M., Lottini T., Crescioli S., Crociani O., Iamele L., De Jonge H., Gherardi E, & Arcangeli A. (2018). Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging. Oncotarget, 9(79), 34972-34989.

Publication 2018

anti-6xHis antibody anti-mouse IgG-HRP conjugate

Anti antibodies Anti antibody Assay Elisa Igg hrp Monoclonal antibody Mouse

Corresponding Organization :

Other organizations : University of Florence, King's College London, University of Pavia

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Variable analysis

independent variables

Type of ELISA assay (Sandwich ELISA vs. mAb-hERG1 ELISA)

dependent variables

Measured outcome (scFv or hERG1 protein detection)

control variables

Anti-6xHis antibody concentration (1:250 in PBS + 3%BSA)
Anti-mouse IgG-HRP conjugate concentration (1:500 in PBS + 3%BSA)
Incubation time (2 h for mAb-hERG1 ELISA)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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