Silver staining of immunofixed group specific component on cellulose acetate membranes after isoelectric focusing in narrow pH interval gels

Abstract

on cellulose acetate membranes after isoelectric focusing in narrow pH interval gels Home Office Forensic Science Service Aldermaston, Reading, BerkshireThe sensitivity of group specific component (Gc) detection by immunofixation after isoelectric focusing (IEF) has been examined by using two IEF methods: ultrathinlayer I E F and immobilized pH gradient (IPG) focusing followed by two staining methods: Ponceau S and a combination of Ponceau S with silver staining (SS). Both diluted plasma and bloodstain extracts were examined by these methods at two dilutions of immunofixation antibody -1 :6 and I :24. The cellulose acetate membranes containing the G c precipitates were washed with sodium chloride after the SS process. This greatly improved the visibility of the Gc bands. The method was found to be more sensitive than Ponceau S and its usereduced the cost ofimmunofixation by 75 %. Ultrathin-layer I E F was carried out in gels containing the pH 4.5-5.4 Pharmalytes. The separator N-2-hydroxyethylpiperazine-N' 2-ethanesulfonic acid (HEPES) was added to some gels and the effect on the resolution ofthe G c 1 subtypes was examined and compared with the separation achieved with IPG gels. It was found that ultrathin-layer IEF using narrow range Pharmalytes and the separator HEPES was the more sensitive method; however, IPG gels were easier to interpret.

GC Subtyping and HIV Infection in a Spanish Population: No Evidence of an Association between GC Subtypes and AIDS

“…In order to improve the resolution in the classification of GC subtypes, other isofocusing methods have been devel oped, including IEF in a mixture of narrow-pH-range carrier ampholytes (Pharmalyte 4.5-5.4) and zwitterionic buffers or separators [4,5], and IEF in immobilized pH gradients [6].…”

Forensic science

“…Much less bloodstained material had to be used for Gc typing (280). The typing of Gc was examined by isoelectric focusing (281,474). The importance of using 6 M urea for extracting bloodstains in order to achieve higher success rates in Gc typing has been demonstrated (282).…”