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.     

Determination of phosphoglucomutase (PGM1), acid phosphatase (ACP), and esterase D (ESD) in human bloodstains by hybrid isoelectric focusing (HIEF)

PGM1, ESD, and ACP were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and HIEF. HIEF yields superior results in PGM typing from bloodstain extracts, whereas for ESD and ACP typing isoelectric focusing with carrier ampholytes seems to be the method of choice.     

Studies and observations on the use of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase

The technique of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase (PGM1) has been studied. Typing was possible without the samples attaining true equilibrium focusing conditions. The isozyme patterns so obtained were clearly defined and free from distortion. The importance of assessing relative band intensities when interpreting the isozyme patterns is discussed. Our experience of using the technique to analyse casework material is described.     

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.     

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.     

Phenotyping of alpha-2-HS glycoprotein in bloodstains by isoelectric focusing and immunoblotting

A sensitive immunoblotting procedure has been applied to the detection of alpha-2-HS-glycoprotein (A2HS) phenotypes from control and casework bloodstains. A2HS phenotypes were separated by thin layer polyacrylamide gel isoelectric focusing (PAGIEF) in gels containing Pharmalyte pH 4.2-4.9. After transfer to nitrocellulose by a rapid capillary blot, the A2HS phenotypes were developed using a double antibody enzyme-immunoassay. The evaluation of A2HS phenotyping of casework material was undertaken in parallel with phosphoglucomutase (PGM) phenotyping by PAGIEF. A total of 598 water extracts from casework bloodstains have been tested. Positive results were obtained in 84% and 75% of samples for PGM and A2HS respectively. The A2HS gene frequencies A2HS*1 = 0.6420, A2HS*2 = 0.3530, and A2HS*3 = 0.0050 were determined from a survey of 1000 people in Brisbane.     

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.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.     

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.     

The use of separator isoelectric focusing in micro-ultrathin polyacrylamide gels in the characterization of some polymorphic proteins of forensic science significance

The identification of phenotypes of erythrocyte acid phosphatase (EAP), esterase D (EsD), group specific component (Gc), and alpha-1-antitrypsin (PI) by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 45 mm) is described. The protein patterns obtained are compared favorably with the patterns seen by isoelectric focusing in conventional polyacrylamide gel dimensions (interelectrode distance: 110 to 120 mm). The technique described allows greater stability of pH gradients and is a fast and economic method.     

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.     

Transferrin (TF) typing from semen stains using isoelectric focusing and immunoblotting: correlation of TF types among blood, semen, urine, and vaginal secretion.

We describe a method for obtaining nondistorted and reproducible transferrin (TF) typing from liquid semen and semen stains. Isoelectric focusing of TF isoproteins on polyacrylamide gel (IEF-PAGE, pH 4 to 6.5) was accomplished using a 0.5 mm thick gel. The separated isoproteins were then visualized by immunoblotting with TF-specific antibody. Pretreatment of semen samples with neuraminidase enhanced the TF band resolution. The method was reliable, sensitive and simple, with a high resolution. When maintained at room temperature, laboratory-prepared semen stains were TF-typable for up to at least 50 weeks. The TF types in semen stains were correlated with the types found in the corresponding blood and urine samples. TF typing could therefore provide an additional discriminant characteristic in the forensic examination of semen stains. An evaluation of TF typing by IEF-PAGE and immunoblotting was also performed on casework samples submitted to our laboratory.     

Effect of the genetic markers Kell(K1), P1, Km(1), Tf(C

E Osterhaus  P Birkner 《Zeitschrift für Rechtsmedizin》1984,91(3):231-234

Simultaneous phenotyping of genetic markers for paternity testing

Time-and cost-saving methods for paternity testing are described. Seventeen genetic systems were divided into six groups: (1) transferrin (Tf), factor B (Bf), and phosphoglucomutase 1 (PGM1); (2) group-specific component (Gc) or alpha 1-antitrypsin (PI) and alpha 2HS-glycoprotein (HSGA); (3) complement components C6 and C7, factor 13B (F13B), and plasminogen (PLG); (4) haptoglobin (Hp), C8 alpha-gamma chain (C81), and factor I (IF); (5) red cell acid phosphatase (ACP), esterase D (ESD), and glutamic-pyruvic transaminase (GPT); and (6) 6-phosphogluconate dehydrogenase (PGD) and glyoxalase I (GLO). Each group of systems was typed simultaneously by electrophoresis or isoelectric focusing (IEF) followed by staining or immunoblotting. These methods are very practical because they afford a considerable saving of time, work and expense, and facilitate semipermanent preservation of electrophoretic patterns.     

Evaluation of a nonequilibrium isoelectric focusing (IEF) method for the simultaneous typing of esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA)

A nonequilibrium isoelectric focusing method incorporating the chemical spacers MOPS and HEPES was developed and subsequently evaluated for its ability to reliably discriminate common and rare phenotypes in the esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA) isoenzyme systems. The validation procedures used were blind testing, comparison of results to conventional methods, and evaluation of known rare variant phenotypes. This method proved to be a quick and reliable method for typing all five isoenzyme systems, while providing an excellent probability of discrimination (PD = 0.96).     

Immunological detection of human phosphoglucomutase (PGM 1) subtypes

Anti-phosphoglucomutase (PGM) antibodies have been produced by immunising a sheep with a purified preparation of rabbit skeletal muscle PGM and used to devise an immunological procedure for detecting PGM isozymes after isoelectric focusing. The anti-rabbit PGM antibodies cross react with human PGM and can be used to identify the PGM1 isozymes characteristic of this polymorphism. The patterns revealed by immunodetection are exactly comparable with those obtained by isozyme staining.     

The use of ultrathin-layer agarose gels for phenotyping erythrocyte acid phosphatase by isoelectric focusing

A method is described for the use of ultrathin-layer agarose gels in phenotyping erythrocyte acid phosphatase (EAP) by isoelectric focusing (IEF). The results obtained using ultrathin-layer agarose gels are shown to be equally reliable and reproducible in comparison to established ultrathin-layer polyacrylamide gels. IEF of EAP on 0.168-mm agarose gels took place in 90 min using the LKB Multiphor system. The technique described allows for both time and cost efficient phenotyping of EAP.     

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.     

A comparison of the isoelectric focusing patterns of red cell phosphoglucomutase separated on an ampholine, ampholine/separator and immobilised pH gradient

The isoelectric focusing patterns of red cell phosphoglucomutase (PGM1) separated on Ampholine, Ampholine/separator and rehydratable immobilised pH gradients have been compared. The sharpest and most intense zymograms have been observed on immobilised pH gradients provided that Ampholine was added during the rehydration of the gel. The addition of ampholine in the rehydration of Immobiline plates has been shown to improve the sharpness and intensity of the zymogram obtained and the potential of immobilised pH gradients for PGM typing has been demonstrated.     

The use of hybrid isoelectric focusing for the detection of polymorphic proteins in blood stains

alpha1-Antitrypsin (Pi), transferrin (Tf) and orosomucoid (ORM) were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and also with a mixture of immobilines (HIEF). HIEF yields superior results from proteins typing in bloodstain extracts, since phenotypes are better distinguished and the bands are straighter and sharper. Also the sensitivity of HIEF is similar to IEF with CA.