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Griffin:Antibody Basics: Difference between revisions
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==Validation of Specificity== | ==Validation of Specificity== | ||
[[Image:DirectELISA.jpg|thumb|right|Immunizing peptide conjugate to thr 96 well plate authenticates Serum titer for the immunogen]] | |||
Validation of primary antibody specificity is an ongoing topic of the utmost importance to scientists who choose to utilize immunoglobulin-based approaches. The mass of histology data, the often quite small n= number, combined with common lack of sufficient (molecular biological) controls are a compelling reason for reproducibility and disclosure of all applied compounds such as antibodies. However, the pure peptide sequence alone simply does not reveal or indicate specificity or reproducibility. Adequate control experiments are essential. | Validation of primary antibody specificity is an ongoing topic of the utmost importance to scientists who choose to utilize immunoglobulin-based approaches. The mass of histology data, the often quite small n= number, combined with common lack of sufficient (molecular biological) controls are a compelling reason for reproducibility and disclosure of all applied compounds such as antibodies. However, the pure peptide sequence alone simply does not reveal or indicate specificity or reproducibility. Adequate control experiments are essential. | ||
===cDNA=== | |||
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. | |||
===Direct ELISA=== | ===Direct 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. | |||
===Peptide neutralization=== | ===Peptide neutralization=== | ||
