Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins.

Immunoaffinity, capillary electrophoresis, and statistics for studying intact alpha 1-acid glycoprotein isoforms as an atherothrombosis biomarker.

Variations in the amino acid sequence, glycosylation, and/or other posttranslational modifications in glycoproteins give rise to different molecules of the glycoprotein called forms. Qualitative and/or quantitative alterations in these forms are related to pathophysiological situations in the individuals. In this study, a methodology to analyze these differences in forms of the alpha 1-acid glycoprotein (AGP) between healthy individuals and patients with two different vascular diseases is detailed. The whole methodology includes a sample preparation method based on immunochromatography, a capillary electrophoresis method for separation of AGP peaks (isoforms), and statistical methods (Linear Discriminant Analysis) for sample classification. As a result, it is shown that the methodology proposed allows studying the role of AGP isoforms as potential vascular disease biomarkers.

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein.

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.

Use of immunodotting to select the desorption agent for immunochromatography.

In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen-antibody recognition. To reuse the immunocolumn for subsequent assays, the antigen has to be eluted without causing irreversible damage of antibodies. Selection of conditions for performing immunochromatography is usually made by trial and error. This way of working is time consuming and it may ruin the column. In this article, the feasibility of using immunodotting to select the conditions to be employed in one immunochromatographic assay is shown. An immunodotting method is developed to select the best desorption agent for an enzyme-linked immunoaffinity chromatography (ELIAC) assay to determine beta-lactoglobulin (beta-LG). The effect of several factors on the immunodotting performance is studied. The way of performing solvent exchange to treat the antibody with different solutions considered as potential desorption agents to check their effect is shown. Effectiveness of the solution chosen by immunodotting (4 M MgCl(2) in 20 mM Tris, pH 5.9) as desorption agent is demonstrated by immunochromatographic assays. The immunodotting and solvent exchange methods developed should be useful to choose solvents and conditions for any other kind of assay.

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis.

Capillary zone electrophoresis of samples of recombinant human erythropoietin is performed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of alpha- and beta-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin alpha, the novel erythropoiesis-stimulating protein.

Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin.

Different profiles were obtained by capillary zone electrophoresis (CZE) of human erythropoietin (EPO) of recombinant and urinary origin. To unambiguously detect doping by EPO, direct methods able to determine the presence of the drug itself in a physiological fluid are required. Since the host cell line used for EPO production influences its glycosylation, the carbohydrate distribution of natural human EPO may be different from that of recombinant EPO. The different content in sialic acid groups between recombinant and endogenous EPO provide a basis for their distinction by CZE.