Quantifying GUCY2C Binding Competence

Binding competence of IT conjugates was confirmed using a GUCY2C extracellular domain (GUCY2C_1-430)-based ELISA [25 (link)]. ITs were incubated in GUCY2C_1-430-coated plates at varying concentrations. The mouse IgG component was detected with HRP-anti-mouse H + L (Jackson ImmunoResearch, #115-035-062). Ricin A chain was detected with rabbit-anti-Ricin antibody (Abcam, ab27169) followed by HRP-anti-rabbit H+L (Jackson ImmunoResearch, #111-035-003). Color was developed with Turbo-TMB substrate (Thermo Scientific, #34022) and quantified at λ480nm.

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Marszalowicz G.P., Snook A.E., Magee M.S., Merlino D., Lisa D.B, & Waldman S.A. (2014). GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer. Oncotarget, 5(19), 9460-9471.

Publication 2014

Turbo-TMB substrate

Anti antibody Elisa Gucy2c Mouse Rabbit Ricin Ricin a chain

Corresponding Organization :

Other organizations : Drexel University, Thomas Jefferson University

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Variable analysis

independent variables

Concentration of ITs

dependent variables

Binding of ITs to GUCY2C extracellular domain (GUCY2C1-430)

control variables

GUCY2C1-430-coated plates
HRP-anti-mouse H + L antibody for detection of mouse IgG component
Rabbit-anti-Ricin antibody for detection of Ricin A chain, followed by HRP-anti-rabbit H+L antibody
Turbo-TMB substrate for color development
Quantification at λ480nm

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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