Total concentrations of IgM and IgG in each fraction were quantified with ELISA against ChromPure human IgM and IgG standards (Jackson ImmunoResearch). Standards and IgM- and IgG-purified fractions were coated in serial dilutions in 96-well flat bottom MaxiSorp plates (Nunc) at 4°C overnight in coating buffer (0.1 M sodium carbonate and 0.1 M sodium bicarbonate). Plates were washed in PBS-Tween 0.05% three times and blocked with 1% casein in PBS for 2 hours at 37°C, and IgG and IgM were detected with goat anti-human IgG horseradish peroxidase (HRP; Merck Millipore) and sheep anti-human IgM HRP (Chemicon Australia). Binding was detected with TMB (3, 3´,5,5´-tetramethylbenzidine) substrate (Sigma-Aldrich), and reaction was stopped using 1 M HCl. The optical density was read at 450 nm.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.