Gel Zone Electrophoresis Research Articles

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Comparison of agarose gel and capillary zone electrophoresis methods with preliminary reference interval generation using capillary zone electrophoresis in bald eagles (Haliaeetus leucocephalus).

Electrophoresis can be used to aid in the diagnosis of different diseases in avian species. Capillary zone electrophoresis (CZE) is an automated method that is proposed to be superior to the dye methods used in agarose gel electrophoresis (AGE). However, reference intervals (RIs) for CZE in avian species and comparison studies between electrophoretic methods are lacking. The goals of the current study were to compare AGE and CZE methods and determine reference intervals for CZE using plasma from bald eagles (Haliaeetus leucocephalus). Heparinized plasma samples from 44 bald eagles (mean age 18.7 years) under managed care were examined by AGE and CZE. Method comparison analyses were completed, as well as the generation of preliminary RIs using the CZE method and ASVCP guidelines. Passing-Bablok regression and Bland-Altman plots demonstrate that these methods are not equivalent. All fractions were significantly correlated between the methods except for alpha 1 globulin. Inter-assay and intra-assay CVs for CZE were lower or comparable to AGE and ranged from 2.4% to 15.4%, and 0.8% to 8.3%, respectively. CZE resolved more fractions than AGE with two fractions observed in the beta and gamma region vs one for AGE in each region. CZE provided improved resolution and reproducibility for the quantitation of protein fractions in the bald eagle. Although most fraction results correlated with AGE, these methods were judged as not equivalent, necessitating method-specific Rls. Reference intervals generated from a limited number of mostly aged individuals under managed care should be considered preliminary; additional studies will aid in the production of more robust intervals.

Diagnostic accuracy of electrophoretic analysis of native or defribrinated plasma using serum as a reference sample

Electrophoretic analysis of plasma may provide inaccurate results unless plasma is defibrinated with ethanol. The aim of this study, conducted following the Standards for Reporting of Diagnostic Accuracy (STARD), was to determine whether cellulose acetate (CAE), agarose gel (AGE), and capillary zone (CZE) electrophoresis of native plasma (NP) and defibrinated plasma (DP) provided the same information as serum electrophoretograms. Serum, NP, and DP electrophoretograms obtained using the 3 methods were examined visually by 3 observers to identify changes in globulin fractions and provide diagnostic interpretations. Using serum analysis as the reference method, sensitivity, specificity, and positive likelihood ratio of NP and DP electrophoretograms to identify abnormal globulin profiles were calculated. Specimens from 46 dogs were analyzed. Albumin and α(1) - and γ-globulin fractions were lower and β(2) -globulin fractions higher in NP than in serum and DP. Interpretations of NP electrophoretograms were the same as for serum in 54.8% (CAE), 51.2% (AGE), and 51.6% (CZE), revealed an increased β-globulin fraction in 32.3-41.5%, and resulted in misinterpretation, especially for CZE analysis, in 4.7-16.1% of the dogs; all dogs with abnormal serum electrophoretograms were identified correctly using NP. Analysis of DP was similar to serum analysis in about 2/3 of the dogs. In the others, defibrination did not resolve spurious plasma findings or induced additional changes. Up to 75 and 27% of NP and DP electrophoretograms, respectively, were abnormal in dogs with normal serum electrophoretograms. Sensitivity for NP and DP analysis was high, but specificity for NP was poor. Analysis of NP provides the same information as serum analysis in > 50% of cases, and, despite low specificity, could preliminarily exclude the presence of abnormalities when only plasma is available. If electrophoretograms are not normalized by defibrination, electrophoretic abnormalities are likely present, and electrophoresis should be repeated using serum.

DNA typing of the D1S80 locus using capillary zone electrophoresis

DNA typing of the D1S80 locus was carried out using capillary polyacrylamide gel zone electrophoresis (CZE). The D1S80 allelic ladder was isolated within 40 min in polyacrylamide gel columns of 50 μm inner diameter and 30 cm length. The PCR products and ladder marker in the injection port were electrophoresed and detected by UV absorption (254 nm). The typing was determined according to migration of DNA fragments on the electropherograms, which permits simultaneous analysis of both the test sample polymerase chain reaction products and the ladder marker. The two-step sample injection method applied in this study allows us to use minute amounts of the ladder maker. This procedure provides a reliable, economical and rapid method for D1S80 typing.

Comparison of serum protein electrophoresis by agarose gel and capillary zone electrophoresis in a clinical setting

Capillary zone electrophoresis (CZE) offers the potential for automating serum protein electrophoretic analysis traditionally performed on standard thin-layer agarose gels. The following describes the use of CZE compared to agarose gel electrophoresis (AGE) for the detection of dysproteinemia and paraproteinemia in a clinical study involving 240 patients. The study includes within-run and between-run reproducibility data on the Paragon CZE 2000 Clinical Capillary Electrophoresis System, in addition to concordance data between the two methodologies. Paraprotein quantitation studies comparing AGE versus CZE were also performed. Reproducibility for the automated CZE system was superior to the AGE system. Improved reproducibility for the CZE method is largely due to measuring protein absorbance directly at 214 nm versus the traditional AGE method that measures the amount of dye adsorbed to protein. Reproducibility data as percent coefficient of variance (% CV) for the five classic bands in a normal control serum for between-run precision ranged from 1.2 to 4.5% for CZE compared to AGE, which ranged from 3.8 to 8.0% CV. Concordance studies between AGE and CZE involving dysproteinemias including hypogammaglobulinemia, polyclonal and monoclonal gammopathies, acute and chronic inflammation, nephrosis, hepatodegenerative disease, cirrhosis, and iron deficiency anemia showed 96% agreement. Paraprotein classification, which compared the CZE immunosubtraction method to immunofixation electrophoresis (IFE) on agarose, showed 100% agreement. Certain dysproteinemias involving beta lipoprotein were in partial concordance due to the inability of the CZE procedure to detect this component. Detection limits for monoclonal gammopathies, providing they were not comigrating with other proteins, were IgG 50 mg/dL, IgM 75 mg/dL, and IgA 75 mg/dL. Paraprotein quantitative studies between the two methods showed less than a +/- 0.2 g/dL variation.

Enantioseparation of N-Dansyl-dl-amino Acids by Polyacrylamide Gel Zone Electrophoresis

Enantioseparation of N-Dansyl-dl-amino Acids by Polyacrylamide Gel Zone Electrophoresis

Characterization of the Renibacterium salmoninarum haemagglutinin

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.

Monoclonal immunoglobulins: affinity blotting for low concentrations in serum.

I describe a simple, economical technique for identifying low concentrations of monoclonal immunoglobulins in the presence of excessive amounts of immunoglobulins of other classes. The technique involves binding of specific antibody to nitrocellulose, separating proteins by isoelectric focusing or zone electrophoresis in agarose gels, using capillary transfer to bind proteins to the nitrocellulose via their antibody affinity, and then detecting transferred proteins with enzyme-labeled antibody. A monoclonal immunoglobulin can be completely characterized in 2 h. No expensive equipment is required. Affinity blotting is about 10-fold as sensitive as native blotting, 100-fold as sensitive as silver staining.

A Hybrid Ampholyte Focusing Technique for Esterase D Subtyping of Evidentiary Material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).

Stability of model immune complexes during zone electrophoresis and isoelectric focusing in agarose gels.

We evaluated the stability of model immune complexes, consisting of radioiodinated follitropin (125I-FSH), bound either to a mouse monoclonal antibody to FSH or to sheep polyvalent anti-FSH antiserum, during zone electrophoresis and isoelectric focusing in agarose gels. The complexes were relatively stable during zone electrophoresis at conventional voltages (less than 40 V/cm), about 80% of the initially bound antigen migrating in the gamma region. Highter voltages resulted in a linear increase in the antigen stripped from the gamma region. By contrast, isoelectric focusing very effectively dissociated the acidic 125I-FSH from the basic sheep immunoglobulins, even if the complexes were subjected to electromotive forces alone, i.e., by loading them onto the neutral region of the gel. The monoclonal complexes were not dissociated by high voltage alone. The monoclonal complexes could be completely dissociated by loading them at the anode, where gel acidification enhances complex dissociation. These observations can form the basis of a simple technique for isolating and dissociating human immune complexes before Western immunoblot analysis. Furthermore, the ease and rapidity with which bound and free antigen can be separated during zone electrophoresis in agarose suggests that this technique may have some application in clinical immunoassays.

Pollen allergen purification by three successive preparative electrophoretic steps

AbstractWater soluble allergens of grass pollen (Dactylis glomerata) were purified by two successive steps of preparative isoelectric focusing in a flat bed of granulated gel and controlled by analytical isoelectric focusing in agarose gels. In order to separate components cofocusing in the same pH zone but having different electrophoretic mobilities, preparative zone electrophoresis in granulated gels was performed. The homogeneity of the fractions recovered was tested by isoelectric focusing and zone electrophoresis, followed by radioimmunodetection of the allergenic molecules on nitrocellulose prints. The major allergen of Dactylis glomerata pollen was purified with a yield of 7.5 mg from 10 g of pollen grains.

Polymorphism for .ALPHA.-amylase in germinating seed and malt of barley varieties detected by isoelectric-focusing gel electrophoresis.

The presence of polymorphism for α-amylase in germinating barley seeds and in malt in 130 barley varieties was examined by means of isoelectric-focusing gel electrophoresis. These varietles were classified into three types according to the banding patterns of the zymograms that were constantly obtained both in germinating seeds and in malt irrespective of pH of the buffer. Most of Japanese two-rowed varieties have specific banding patterns of the zymograms, i. e. they fall into type-1 and type-3, whereas almost all foreign two-rowed varieties fall into type-2 and type-3. This classificatlon does not agree with the classification based on agar gel zone electrophoresis.

Immune abnormalities in IgA nephropathy (Berger's disease)

Thirteen patients with IgA nephropathy confirmed by renal biopsies were studied for evidence of disorders of the immune system. Circulating immune complexes (CIC) were determined by agarose gel zone electrophoresis (AGE), the Raji cell radioimmunoassay (RIA), and the modified Raji cell (RIA) for IgA containing CIC. Serum levels of immunoglobulins (Ig) G, A, M, of C3, C4, and properdin factor B (PFB) were measured by the rate nephelometry. Tissue studies included fluorescent, light, and electron microscopy, staining for intracellular Ig, and lysozyme by immunoperoxidase (IP), and determination of glomerular C3 receptors (C3R) by EAC rosettes. IgG-CIC were detected in 69%, IgA-CIC in 50% and serum IgA was elevated (>2SD) in 46% of the patients. In the glomeruli, mesangial deposits of IgA, IgG, properdin, and C3 were present. C3R available for EAC binding to glomerular cells were decreased. IgA-producing lymphoid cells were detected in renal interstitium of our patients especially those with crescentic glomerulonephritis. Elevated serum IgA and the presence of IgA-producing cells in the renal tissue may reflect an abnormality in IgA immunoregulatory mechanisms. IgA could contribute to the pathogenesis of renal disease due to its inhibitory effect on the clearance of circulating and deposited immune complexes.

Investigation of the Heterogeneity of Heterogalactan from the Fruit Bodies of <italic>Fomitopsis pinicola</italic>, by Employing Concanavalin A-Sepharose Affinity Chromatography

A heterogalactan was isolated from the hot water extract of fruit bodies of Fomitopsis pinicola by a combination of fractionation procedures including precipitation with ethanol and with Cetavlon, and chromatography on columns of DEAE-cellulose and Sephadex G-100. Despite its apparent homogeneity on gel filtration, zone electrophoresis, sedimentation equilibration, and immunodiffusion analyses, the neutral component of heterogalactan was further fractionated into unbound, weakly bound, and strongly bound forms by affinity chromatography on a column of concanavalin A-Sepharose CL 4B. The former two polysaccharides fractions eluted with 0.1 M phosphate buffer (pH 7.0) were found to be a fucogalactan and a mannofucogalactan, respectively. A more tightly bound fraction (mannofucogalactan) was subsequently eluted with 0.1 M glucose in 1 M NaCl. The results of methylation, complete Smith degradation, and proton and 13C NMR spectroscopic analyses indicated that the three kinds of heterogalactans are all highly branched polysaccharides containing a framework of (1 leads to 6)-linked alpha-D-galactopyranosyl residues, the C-2 positions of which are substituted in different proportions with either single L-fucopyranosyl residues or disaccharide units of 3-O-alpha-D-mannopyranosyl-L-fucopyranose residues.

The apoprotein fraction of the bile lipoprotein complex: isolation, partial characterization and phospholipid binding properties

A bile apoprotein fraction (Apo BLC) was isolated by preparative isoelectric focusing (I.E.F.) from the detergent-free form of the bile lipoprotein complex (BLC). Analytical I.E.F. of Apo BLC yields a characteristic and reproducible pattern of two narrow acidic bands (pI 4,8-5,0). This apoprotein presents a strong tendency to undergo self-aggregation in aqueous buffer. A low molecular weight constituent of Apo BLC has been isolated after gel filtration, its mean Mw is estimated by SDS-PAGE at 7,500 daltons. The binding capacity of Apo BLC for phospholipids was investigated on dimyristoylphosphatidylcholine liposomes by gel filtration and zone electrophoresis. The resulting structures, larger than the original single-shelled vesicles, acquire and anodic electrophoretic mobility. Apo BLC has a weaker affinity for lysophosphatidylcholines: these phospholipids decrease the degree of aggregation of the apoprotein. These studies contribute additional data concerning the high affinity of Apo BLC for phosphatidylcholines, which are the major phospholipid constituents of bile. The discussion deals with the fact that association of Apo BLC with bile phosphatidylcholines may present some implications in the pathogeny of LpX and in the process of intestinal fat absorption.

Immunological and physicochemical properties of a highly purified allergen from Dirofilaria immitis.

Allergen in crude extract of Dirofilaria immitis was purified and separated from IgG-inducing antigens by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 gel filtration and starch gel zone electrophoresis. The purified preparation was proved to be one protein band by sodium dodecyl sulfate polyacrylamide gel (SDS-gel) electrophoresis and one precipitin arc by immunodiffusion. The molecular weight of the purified allergen was estimated to be approximately 20,000 by gel filtration and 15,000 by SDS-gel electrophoresis. The carbohydrate content of the preparation was apparently low, about 2%. The allergen was positively charged, and its determinant group was protein in nature. It was resistant to tryptic, pepsic and chymotryptic digestion, periodate oxidation and DNase and RNase digestion but very sensitive to pronase digestion. Allergen was inclined to aggregate each other in the buffered solution. It was also very resistant to vibration, heat (80 degrees C for 1 h) and acid (pH 2.5) and alkali (pH 11.0) treatments. Rats as well as mice immunized with allergen developed only a reaginic antibody and no hemagglutination antibody.

A non-barbital buffer for immunoelectrophoresis and zone electrophoresis in agarose gels.

We describe buffer for both immunoelectrophoresis and zone electrophoresis procedures in agarose gels, Tricine [N-tris(hydroxymethyl)methylglycine] and Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] being the main components. This buffer eliminates the procurement cost and inventory control associated with formulation of barbital buffers. We compare it with other common buffer formulations.

Electrophoretic Properties of Cowpea Mosaic Virus (Severe Subgroup)

SUMMARY Particles of the Puerto Rico isolate of cowpea mosaic virus (CPMV-PR), which belongs in the severe subgroup, had one electrophoretic form when analysed by 2·5% polyacrylamide gel electrophoresis and zone electrophoresis in sucrose gradients at pH 7·8, 7·5, and 5·5. The electrophoretic properties of CPMV-PR did not vary with time after inoculation but the virus could be converted to a slower migrating form by in vitro treatment with trypsin. The partial conversion by trypsin was accompanied by and was possibly the result of proteolytic conversion of the S protein subunit to a lower molecular mass form. Analysis of proteins of untreated CPMV-PR suggested that the S subunit of the virus could also be converted in vivo by plant proteases to a smaller form without any change in the particle net surface charge, while in vitro treatment with trypsin affected those S subunits not previously converted by plant proteases, by converting them to a form slightly larger and with a greater positive charge than the S subunit produced by action of the plant proteases. This hypothesis accounts both for the decrease in electrophoretic mobility and for the fact that the conversion was only partial.

The ethanol fractionation of mouse serum

A method for the ethanol fractionation of normal mouse serum has been developed which is rapid and simple to perform. The method was shown to be effective in purification of IgG1, IgG2 and γM-γA globulins. Gel filtration and zone electrophoresis proved to be effective in separating the γM-γA globulin combination into their respective components. Immunoelectrophoresis was chosen to assess the homogeneity of the fractions and the results agree with those obtained by other workers.

Characterization of contrasensitizing antibodies

This paper describes experiments on the nature of humoral antibodies which suppress induction of delayed hypersensitivity in mice to purified proteins, i.e., contrasensitizing antibodies. These antibodies may be analogous to those responsible for afferent or early central immunosuppression in immunological enhancement phenomena and for similar forms of immunosuppression sometimes referred to as immune deviation, split tolerance, or preimmunization tolerance. CF-1 mice were sensitized with either human serum albumin or ovalbumin in incomplete Freund adjuvant, were injected with contrasensitizer-rich mouse antisera or fractions thereof, and subsequently were skin-tested to measure the relative capacities of antisera or their components to interfere with sensitization. Agar gel zone electrophoresis located Contrasensitizing activity among the γ 1 globulins, and this identification was confirmed by DEAE cellulose chromatography. Fractionation on Sephadex G-200 dextran columns showed the inhibitory activity to be among molecules of approximately 170,000 molecular weight, and a combination of chromatographic procedures further indicated that this activity is due to specific antibodies closely associated with mouse IgF or 7S γ 1 globulin. Contrasensitizing antibodies also were found to be heat-stable, and although precipitin and passive hemagglutination antiserum titers often correlated well with immunosuppressive activity, neither was equivalent to it.