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.     

The isoelectric focusing of keratins in hair followed by silver staining

An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.     

Use of the isoelectric focusing method in examining hair proteins

Method of isoelectric focusing in thin and ultrathin layer of polyacrylamide gel followed by staining the polypeptide fractions with argentum was used to investigate human hair keratins. Method makes it possible to determine the polymorphism of keratins in hair even 1,5-2 cm long. It helps to obtain the additional information in medicolegal expertise of hair affinity.     

Determination of C3 phenotype in dried bloodstains using isoelectric focusing

Fresh whole blood and bloodstains were analyzed by isoelectric focusing (IEF) to determine the C3 phenotype of the blood donor. Three common phenotypes exist as a result of two autosomal alleles. The three phenotypes can be identified in fresh serum or in serum samples which had been stored at -20 degrees C for more than a year. Bloodstains maintained in a desiccator at 25 or at 37 degrees C retained the native form of C3 which could be detected for at least two weeks. Beyond two weeks of storage, stains became difficult to phenotype due to decreased banding intensity. Bloodstains aged longer than one month could not be phenotyped. C3 could not be detected in human semen by the serological methods employed.     

Transferrin subtyping of bloodstains and semen using isoelectric focusing and immunoblotting

Transferrin (TF) subtyping was carried out on bloodstains that had been made on cotton sheeting and stored under a variety of conditions ranging from −20°C to +37°C. The time limit of detection was longer than 54 weeks after dry storage under each condition. Moreover the correlation between isoprotein types of the TF in blood and semen samples from the same individual was determined in 103 men. All three TF common types and two rare types in all semen samples correlated with the type found in the corresponding blood sample. A combination of isoelectric focusing separation and immuno-enzyme-linked detection may prove to be very useful for forensic TF subtyping.     

The typing of α1-antitrypsin in human bloodstains by isoelectric focusing

A polyacrylamide gel isoelectric focusing (PAGIF) technique is described for the determination of α₁-antitrypsin (Pi) phenotypes in bloodstains. The time limits for Pi type determination of bloodstains kept under different storage conditions are given. The resolution of PAGIF in the typing of Pi phenotypes in human bloodstains in investigated.     

Genetic polymorphism of human parotid salivary amylase detected by isoelectric focusing electrophoresis and silver staining

In 332 samples of human parotid saliva collected at random from a Japanese population, the genetic polymorphism of salivary alpha-amylase was detected by isoelectric focusing electrophoresis in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. This polymorphism, that was tentatively designated Amy1 S, consisted of extra three isozymes of a normal pattern (Amy1 N) and isoelectric points of these three isozymes were 5.5, 5.8 and 6.1, respectively. The inheritance was controlled by a dominant allele at an autosomal locus. The frequency of the genes determining these phenotypes were studied as follows: Amy1 S = 0.014 +/- 0.004, Amy1 N = 0.986 +/- 0.004.     

Factor B subtyping in sera and bloodstains by isoelectric focusing and immunoblotting

The polymorphism of BF was investigated in 765 unrelated Japanese individuals by isoelectric focusing and immunoblotting. Besides five common subtypes three rare variants were observed. The allele frequencies were: BF*S = 0.8078, BF*FA = 0.1797, BF*FB = 0.0105, BF*Var. = 0.0020. The above method was successfully applied to subtyping BF in stored bloodstains. The determination limits were: at 4 degrees C 8 weeks, at room temperature 2 weeks and at 37 degrees C only 2 days after storage. The BF subtyping is of practical use in medicolegal individualization of unknown bloodstains.     

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.     

Transferrin subtyping of bloodstains and semen using isoelectric focusing and immunoblotting.

Transferrin (TF) subtyping was carried out on bloodstains that had been made on cotton sheeting and stored under a variety of conditions ranging from -20 degrees C to +37 degrees C. The time limit of detection was longer than 54 weeks after dry storage under each condition. Moreover the correlation between isoprotein types of the TF in blood and semen samples from the same individual was determined in 103 men. All three TF common types and two rare types in all semen samples correlated with the type found in the corresponding blood sample. A combination of isoelectric focusing separation and immuno-enzyme-linked detection may prove to be very useful for forensic TF subtyping.     

Usefulness of two methods of isoelectric focusing for the erythrocyte acid phosphatase phenotypes determination in human bloodstains

The authors tried to compare the usefulness of the isoelectric focusing of EAP in bloodstains on 0.2 mm polyacrylamide gel with their method of determination of the enzyme on 1 mm polyacrylamide gel. Both methods turned out to be useful but better results were obtained on 0.2 mm gel. Isoelectric focusing on the ultra-thin gel is more sensitive; it gives clear enzyme strips, takes less time (30 min) and demands about half the amount of material.     

C6 phenotyping in sera and bloodstains by isoelectric focusing followed by electroimmunoblotting

The genetic polymorphism of C6 was investigated in 329 unrelated Japanese individuals using isoelectric focusing in polyacrylamide gels followed by an electroimmunoblotting technique. Besides six common phenotypes C6 A, AB, B, AB2, BB2 and B2, six rare variants were observed. The allele frequencies were: C6*A = 0.4422, C6*B = 0.4757, C6*B2 = 0.0714, C6*A3 = 0.0015, C6*M1 = 0.0046 and C6*B3 = 0.0046. The population data confirmed that the C6*B2 allele is the third common allele characterizing Japanese. The present electroimmunoblotting technique was applied to demonstrate C6 types in dried bloodstains. The C6 types were determined from bloodstains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 2 weeks and at 37 degrees C for up to 4 days. The results show that this component system offers a new powerful means for the medico-legal grouping of bloodstains.     

Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing

A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*1, ESD*2 and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5.     

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 comparative study of immuno-blotting techniques for the detection of Gc-subtypes after isoelectric focusing on agarose and polyacrylamide gels

A comparison of separation and detection techniques has been carried out to determine the most suitable combination for use in Gc-subtyping. The best results (i.e., high sensitivity, distinct bands, especially with reference to the 1S and 1F separation) were achieved using isoelectric focusing in polyacrylamide gel (pH 4.5-5.4) followed by transfer to nitrocellulose membrane by electroblotting and finally detection with enzyme-linked antibody complex.     

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.     

PGD phenotyping in bloodstains, organ tissues, dental pulps and hair roots by isoelectric focusing

Polymorphism of PGD was investigated in bloodstains, organ tissues, dental pulps, hair roots and semen by isoelectric focusing. This technique provided much higher resolution of PGD isoenzymes than starch gel electrophoresis. Phenotyping was possible from bloodstains for 5 weeks, from organ tissues (except pancreas) for 1-3 weeks, from dental pulps for 2 weeks and from hair roots for 2 weeks when they were stored at room temperature. The method is simple, rapid, reliable and therefore useful in medicolegal individualization of bloodstains, organ tissues, teeth and hairs.     

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.