Affinity isolated antigen specific antibody is obtained from goat anti-human IgG antiserum by immunospecific purification which removes essentially all goat serum proteins, including immunoglobulins, which do not specifically bind to the γ-chain of human IgG. Goat anti-human IgG is conjugated to Sigma R-Phycoerythrin (Product No. P8912) by a modification of the method of Kronick Unbound phycoerythrin is removed by gel filtration chromatography
Specificity for the γ-chain of human IgG is determined by Ouchterlony Double Diffusion (ODD). The antibody preparation is specific for human IgG when tested against purified human IgA, IgG, IgM, Bence Jones κ and Bence Jones Lambda myeloma proteins, prior to conjugation
Identity and purity of the antibody is established by immunoelectrophoresis (IEP), prior to conjugation Electrophoresis of the antibody preparation followed by diffusion versus anti-goat IgG and anti-goat whole serum results in single arcs of precipitation.
Digestion of IgG by papain results in generation of fragment antigen binding (Fab) comprising of one complete L chain and a variable and CH1 region of H chain. Pepsin digestion of IgG results in fragment crystallisable (fc), comprises the H chain constant region.
