Isotype-specific ELISA for Collagen II Abs

96 well MicroWell MaxiSorp flat bottom plates (Sigma-Aldrich) were coated with 2μg/ml of antigen (CII, Sigma-Aldrich)) and incubated overnight at 4°C. The following day the plates were washed and blocked. Serial serum dilutions were added to the plates and incubated overnight at 4°C. The next day, plates were incubated with anti-mouse IgG1, IgG2a, IgG2b, IgG3 and IgM isotypes coupled to Horse Radish peroxydase (Lo-Imex, Louvain, BE), revealed using a TMB kit (BD Biosciences) and read at 450nm using a ELISA plate reader [15 (link), 16 (link)].

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De Trez C., Katsandegwaza B., Caljon G, & Magez S. (2015). Experimental African Trypanosome Infection by Needle Passage or Natural Tsetse Fly Challenge Thwarts the Development of Collagen-Induced Arthritis in DBA/1 Prone Mice via an Impairment of Antigen Specific B Cell Autoantibody Titers. PLoS ONE, 10(6), e0130431.

Publication 2015

MicroWell MaxiSorp flat bottom plates TMB kit

Antigen Dilutions Elisa Horse Igg1 Igg2a Igg2b Igg3 Isotypes Mouse Radish Serum

Corresponding Organization : Instituut voor Tropische Geneeskunde

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Variable analysis

independent variables

Antigen (CII) concentration (2μg/ml)

dependent variables

Levels of mouse IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies

control variables

96 well MicroWell MaxiSorp flat bottom plates (Sigma-Aldrich)
Incubation temperature (4°C)
Incubation duration (overnight)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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