Group‐specific component subtyping: A high resolution method employing short run cathodic drift

Abstract

A high resolution polyacrylamide gel isoelectric focusing method is described for group-specific component (Gc) subtyping. With this method all the common Gc subtypes can be identified and the G c variant 1 A 1 bands are clearly resolved from the G c 1F and 1 s bands. The run time of 90 rnin represents a substantial saving in time when compared to many other methods reported in literature.Constans and Viau 111 first demonstrated in 1977 that the group-specific component (Gc) 1 allele can be subtyped into IF and 1 S alleles using polyacrylamide gel isoelectric focusing (PAGIF) with the aid of cathodic drift. Since that time many other methods have been developed, including immobilized pH gradient (IPG) gels with a narrow pH gradient 121 and separation in new, narrow-range carrier ampholytes (Pharrnacia. Westwood et al. I61 reviewed many of the isoelectric focusing methods and concluded that the IPG method provided the best resolution of Gc 1F and IS while the ultrathin PAGIF method by Edwards [61 provided a resolution that was comparable to that of the IPG method. It was also concluded that the ultrathin PAGIF methods were more sensitive than IPG methods and therefore more suitable for forensic work. Following up on the work by Edwards [61, this methods was developed, shortening the run timefrom 195 rnin [61 to 90 min whileresolvingtheGc lF, 1s andthe 1Al variant.Thisresolution is achieved by deleting sucrose, a gradient stabilizing agent, to induce cathodic drift and by decreasing the carrier ampholyte concentration to shorten the onset of cathodic drift 191. Thus, presented here is a PAGIF method using pH 4.5-5.4 Pharmalytes, MOPS and HEPES as separators and employing cathodic drift in a new fashion.Dried blood stains were extracted with 6 M urea [ 101 to alight brown color. Whole blood samples were centrifuged and the serum portions were diluted 1 :30 with 6 M urea. The standard mixture contained a mixture of two complimentary serum specimens to give Gc 2, 1F and 1s bands. Pharmalytes (3.8 % v / v), MOPS (2.64 % w / v), HEPES (0.88 % w / v) and 1.7 mg ammonium persulfate. The gels were polymerized in a 60 OC oven for 1 h and stored in the refrigerator until use. The anolyte and catholyte were 1 M phosphoric acid and 1 M sodium hydroxide, respectively. The electrode gap was 12 cm. The gels were prefocused at 10 mA limit until 1900 V was reached (about 5 min). Samples were soaked onto 3 MM Whatman filter paper stubs (3 x 7 mm) and applied 1 cm from the cathode. The samples were focused at 1900 V for a total of 90 min on a humidified cooling plate a 10 "C, and sample applicators were removed 10 rnin after starting the run.After PAGIF, the G c phenotypes were visualized using a modified immunoprinting method described by Westwood 151. G c antiserum (Atlantic Antibodies) was diluted either 1:20 or 1:30 (determined by titering a new bottle of antiserum) with distilled water and poured onto a precleaned glass plate. One side of an 8 x I5 crn cellulose acetate membrane (CAM, Sartorius) was brought in contact with the d...

Water analysis

“…The major isoelectric focusing methods that have been published for the separation of Gc phenotypes since 1978 were reviewed (354). Gc subtypes have been identified by isoelectric focusing (355)(356)(357)(358)(359). Isoelectric focusing of Gc was reported for semen, blood, and vaginal fluid (396,397).…”