Mannan-Induced Complement Activation Assay

A functional lectin pathway activity assay was done using EDTA-plasma diluted 2.5% in barbital-buffered saline (BBS) incubated for 15′ at 37° C on 10 µg/mL mannan-coated plates.^{27 (link)} C3 deposition was revealed using a polyclonal anti-human-C3c antibody (2.4 µg/mL, Dako) and an alkaline-phosphatase labeled goat anti-rabbit IgG (1 µg/mL, Sigma). Absorption at optical density (OD) 405 nm measured using Infinite M200 spectrofluorimeter (Tecan, CH).

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Grossi C., Artusi C., Meroni P., Borghi M.O., Neglia L., Lonati P.A., Oggioni M., Tedesco F., De Simoni M.G, & Fumagalli S. (2021). β2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke. Journal of Cerebral Blood Flow & Metabolism, 41(8), 2038-2053.

Publication 2021

anti-human-C3c antibody alkaline-phosphatase labeled goat anti-rabbit IgG Infinite M200 spectrofluorimeter

Alkaline phosphatase Anti antibody Anti igg Assay Barbital Edta Goat Human Lectin M200 Mannan Optical Plasma Rabbit Saline

Corresponding Organization : Mario Negri Institute for Pharmacological Research

Other organizations : University of Milan, Istituto Ortopedico Gaetano Pini

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Variable analysis

independent variables

Dilution of EDTA-plasma (2.5% in barbital-buffered saline (BBS))
Incubation time (15 minutes)
Incubation temperature (37°C)
Mannan-coated plates (10 µg/mL)

dependent variables

C3 deposition

control variables

Polyclonal anti-human-C3c antibody (2.4 µg/mL, Dako)
Alkaline-phosphatase labeled goat anti-rabbit IgG (1 µg/mL, Sigma)
Optical density (OD) measurement at 405 nm using Infinite M200 spectrofluorimeter (Tecan, CH)

controls

No positive or negative controls were explicitly mentioned.

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