Vitamin D binding protein (Gc) subtypes by isoelectric focusing and immunoblotting

The polymorphism of the human vitamin D binding protein (Gc system) was investigated in a total of 149 sera from unrelated healthy Egyptians residing in Tanta City, Gharbiya Governorate, Nile Delta of Egypt, using isoelectric focusing (IEF) in thin-layer polyacrylamide gel followed by immunoblotting. The estimated gene frequencies were Gc1s = 0.540, Gc1f = 0.242 and Gc2 = 0.218.     

Group specific component: isoelectric focusing subtyping and immunoblot detection

A method for the detection of group specific component (Gc) by immunoblotting, following isoelectric focusing (IEF), is described. This isoelectric focusing method resolves the six common phenotypes of Gc using a narrow range pH 4.5 to 5.4 ampholyte. The Gc proteins were passively transferred from the IEF gel to nitrocellulose and detected with goat anti-Gc followed by peroxidase labeled anti-goat immunoglobulin (Ig) antibody. The increased sensitivity of this technique results in the typing of stains older than one year and also those stains with minimal concentrations of the Gc protein. The polyacrylamide gel can also be used for the subtyping of esterase D.     

Gc subtypes determined by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy)

The distribution of Gc phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 732) the six common phenotypes, Gc 1S, 1F, 1S1F, 2, 2-1S, 2-1F and a further phenotype, GC 1S1C3, were observed and the following frequencies calculated: Gc 1S = 0.560792; GC 1F = 0.159153; Gc2 = 0.277323; Gc 1C3 = 0.002732. Our gene frequencies have been compared with those found in other populations.     

Gc subtypes: demonstration using isoelectric focusing. Study of population and formal genetics

Gc-subtypes were determined by isoelectric focusing and immunfixation on samples from 492 unrelated blood donors from Berlin. The frequency of the three genes was found to be GcIF = 0.1270, GcIS = 0.6006, Gc2 = 0.2724. Analysis of 78 parents with 190 children did not show deviations from the expected mode of inheritance. Investigation of the adults from paternity cases and of their children on the other hand obtained similar results. No rare alleles were observed.     

Group specific component subtyping in bloodstains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels followed by immunoblotting

The identification of group specific component (Gc) subtypes derived from blood-stains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 50 mm) containing 4.5 to 5.4 pharmalytes is described. The separation achieved between Gc 1F and Gc 1S bands is compared favorably with that obtained using separator isoelectric focusing in conventional polyacrylamide gels dimensions (interelectrode distance: 110 to 120 mm). The technique is rapid and economical, and the immunoblotting method described is more sensitive than immunofixation followed by silver staining.     

The polymorphism of transferrin by ultrathin-layer isoelectric focusing. Tf phenotypes and TfC subtypes in the population of Padua

The distribution of Tf phenotypes in the population of Padua was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 618) nine phenotypes, Tf C₁, C₂, C₃, C_3?1, C_2?1, C_3?2, C₁B, C₂B and C₁D, were observed and the following frequencies calculated: TfC₁ = 0.77837; TfC₂ = 0.1804; TfC₃ = 0.03641; TfB = 0.0040; TfD = 0.0008. These gene frequencies have been compared to those found in other populations. Analysis of 101 mother-child pairs was in agreement with an autosomal codominant mode of inheritance.     

A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.     

Transferrin (Tf) polymorphism: An analysis by isoelectric focusing

The polymorphism of the transferrin (Tf) system was studied in a total of 300 unrelated Japanese individuals from Miyagi prefecture, the northern part of Japan, using isoelectric focusing in thin-layer polyacrylamide gel. In our population samples three common phenotypes and nine variants were observed. The calculated allele frequencies were Tf^C₁ = 0.773, Tf^C₂ = 0.212, Tf^D_chi (Chinese) = 0.008, and the combined frequencies for the Tf^B_var (variants) = 0.007. Family data (n = 44) were in accordance with an autosomal codominant fashion of inheritance. The use of isoelectric focusing procedure among Japanese will raise the probability of excluding a man falsely accused of paternity to 15.8% as compared with 1.5% when the conventional electrophoretic methods are used.     

New detection method for uropepsinogen (PGA) using isoelectric focusing and immunoblotting techniques

Uropepsinogen (PGA) was isolated and purified from human urine using a column chromatography series. The purified PGA was injected into a rabbit and a PGA-specific antibody was obtained. PGA isozymogen in human urine could be detected reproducibly by immunoblotting using this antibody after isoelectric focusing electrophoresis (IEF) on polyacrylamide gels. This technique may prove to be useful in the genetic study of PGA polymorphism.     

The detection of group specific component (Gc) in the general population of West Virginia

An isoelectric focusing method is described for the detection of group specific component (Gc) in forensic casework. Gc can be subtyped in one day using this reliable and reproducible method. The gene frequency data collected indicate that the occurrence of Gc phenotypes in the population of West Virginia is consistent with established frequencies for the system.     

Classification of alpha 2-HS-glycoprotein (alpha 2HS) types by isoelectric focusing

A sample of 300 sera from unrelated individuals from Northern Japan was examined by isoelectric focusing on polyacrylamide gels. Three common types, alpha 2HS 1-1, 2-1, and 2-2 were differentiated. The frequencies of the alpha 2HS alleles in our sample were found to be: alpha 2HS1 = 0.7250 and alpha 2HS2 = 0.2750. Analysis of 16 parents with 21 children did not show deviations from the expected mode of inheritance.     

Orosomucoid (ORM) typing by isoelectric focusing: description of two new alleles in a German population and thermostability in bloodstains

The genetic polymorphism of serum orosomucoid (ORM) was studied in 168 unrelated German individuals using isoelectric focusing followed by immunoprinting. Two new alleles, tentatively designated ORM1*14 and ORM2*13, were identified. The method was successfully applied to demonstrate ORM1 types in dried bloodstains. Each type of ORM1 was also correctly determined in bloodstains heated at 130 degrees C for 30 min. The results indicated that ORM1 is a new powerful genetic marker system for the grouping of bloodstains.     

The polymorphism of plasminogen (PLG) by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy)

The distribution of plasminogen phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 1325), the three common phenotypes PLG1, PLG2, PLG2-1 and two further phenotypes PLG1-V and PLG2-V were, observed and the following frequencies calculated: PLG1 = 0.84038; PLG2 = 0.15811; PLGV = 0.00151. These gene frequencies are compared to those found in other populations. Analysis of 41 mother-child pairs was in agreement with an autosomal codominant inheritance.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.     

Simultaneous diagnosis of coagulation factor XIIA (F13A) and plasminogen (PLG) phenotypes by polyacrylamide gel isoelectric focusing.

In this paper, we report a simple rapid method for simultaneous determination of Coagulation Factor XIIIA (F13A) and plasminogen (PLG) phenotypes by PAGIF with a nominal pH range of 3.5 to 10, followed by immunofixation and silver stain. Critical considerations concerning the conditions of molecular separation and detection strategies are also presented.