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Griffin:Antibody Basics: Difference between revisions
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cDNA for the gene of interest is a useful tool for antibody validation. Typically, a CHO, Cos, or 293T cell may be transfected with the cDNA in order to measure antibody specificity. A western blot can be performed to compare the nontransfected lysate with the transfectant. | cDNA for the gene of interest is a useful tool for antibody validation. Typically, a CHO, Cos, or 293T cell may be transfected with the cDNA in order to measure antibody specificity. A western blot can be performed to compare the nontransfected lysate with the transfectant. | ||
=== | ===ELISA=== | ||
Microwell plates are coated with a sample containing the target antigen OR the immunizing peptide, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. This technique validates recognition or binding between antigen and antibody. | Microwell plates are coated with a sample containing the target antigen OR the immunizing peptide, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. This technique validates recognition or binding between antigen and antibody. | ||
*Direct vs. Indirect Enzyme-linked immunosorbent assay (ELISA): Indicates whether the ELISA uses secondary antibody or not (secondary used = indirect). | |||
*Sandwich vs. Conventional: Indicates what the well is coated with (and what the overall setup of ELISA assay is). In conventional ELISA, sample is coated on well, and an antibody is used for detection. In Sandwich ELISA, "capture" primary antibody is coated on well, then the well is incubated with the sample, and then a "detection" primary antibody is used for detection of antigen bound to "capture" antibody. Both sandwich and conventional ELISAs can be either direct or indirect. | |||
*Both sandwich and conventional ELISAs can be either direct or indirect. Some sources (e.g. Wikipedia) call Sandwich ELISA a "direct ELISA". This stems from the fact that Indirect Sandwich ELISA assays are rare. | |||
===Peptide neutralization=== | ===Peptide neutralization=== | ||
