The liposomes (25 µM phosphate) were coated in ethanol on Immuno MaxiSorp plates (NUNC, Roskilde, Denmark) and incubated overnight. The plates were blocked in 1% bovine serum albumin (BSA; Fraction V, Fatty acid free, Calbiochem, San Diego, CA, USA) in phosphate-buffered saline (PBS) and incubated with CD169 Fc or its mutant form (CD169 Fc R97A) (2 μg/mL) for 1 h at room temperature (kindly provided by Prof. Dr. P.R. Crocker, University of Dundee) [41 (link)]. Peroxidase-labelled goat anti-human IgG was added for 30 min at room temperature. After a final washing step, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma-Aldrich) was added as a substrate, and the optical density (OD) was measured in a microplate absorbance spectrophotometer (Biorad, Hercules, CA, USA) at 450 nm.
Immuno maxisorp plates
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The Immuno MaxiSorp plates are a type of laboratory equipment designed for use in immunological assays. They feature a high-binding surface that is optimized for efficient capture of target analytes. The plates are made of polystyrene and are available in various well formats to accommodate different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Microplate absorbance spectrophotometer, by Bio-Rad (2 mentions)
Tetramethylbenzidine tmb, by Merck Group (1 mentions)
Peroxidase conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (1 mentions)
3 protocols using immuno maxisorp plates
1
CD169 Fc Binding to Liposomes Assay
2
CD169-Liposome Binding Assay
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Liposomes were diluted in ethanol to a final concentration of 25 µM phospholipids and coated on Immuno MaxiSorp plates (NUNC, Roskilde, Denmark). 1% BSA (BSA; Fraction V, Fatty acid free, Calbiochem, San Diego, CA, USA) diluted in PBS was used to block coated plates. Next, samples were incubated with CD169 Fc or its mutant form (CD169 Fc R97A) (2 μg/mL) for 1 hour at room temperature (kindly provided by Prof. Dr. P.R. Crocker, University of Dundee) (57 (link)). Then, peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch, Ely, UK) was added for an additional 30 minutes and plates were washed, TMB (Sigma Aldrich, Darmstadt, Germany) was added as a substrate and the optical density (OD) measured in a microplate absorbance spectrophotometer (Biorad, Hercules, CA, USA) at 450 nm.
3
Quantitative Determination of Oxidized MIF
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For quantitative determination of oxidized MIF (oxMIF), 96-well-Immuno Maxisorp plates (Nunc) were coated with 0.5 µg/ well of 10C3 in 50 mM carbonate buffer pH 9.4 to capture total MIF species. The wells were blocked with PBS/5% BSA/ 0.1% Tween-20 for 2h at RT.
After washing with PBS-T, samples diluted in PBS/1% BSA/ 0.1% Tween-20 were added and incubated overnight at 4°C. The wells were washed 3x with PBS-T and incubated with the oxMIF specific antibody Imalumab at a 1:2,000 dilution for 4h at RT. Then, the wells were again washed 3x with PBST before a goat anti-human IgG-HrP conjugate (1:5,000 dilution) was added for 1h at RT. After 4x washing with PBS-T the ELISA was developed with 1-step Ultra TMB ELISA (Thermo Scientific). For generation of an oxMIF standard curve, recombinant wt-MIF was treated with 0.2% Proclin300 that transforms recombinant MIF into an oxMIF surrogate as described in (Thiele et al., 2015) .
After washing with PBS-T, samples diluted in PBS/1% BSA/ 0.1% Tween-20 were added and incubated overnight at 4°C. The wells were washed 3x with PBS-T and incubated with the oxMIF specific antibody Imalumab at a 1:2,000 dilution for 4h at RT. Then, the wells were again washed 3x with PBST before a goat anti-human IgG-HrP conjugate (1:5,000 dilution) was added for 1h at RT. After 4x washing with PBS-T the ELISA was developed with 1-step Ultra TMB ELISA (Thermo Scientific). For generation of an oxMIF standard curve, recombinant wt-MIF was treated with 0.2% Proclin300 that transforms recombinant MIF into an oxMIF surrogate as described in (Thiele et al., 2015) .
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