Comparative analysis of prostate-specific antigen by two-dimensional gel electrophoresis and capillary electrophoresis.

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

On-capillary fluorescent labeling and capillary electrophoresis laser-induced fluorescence analysis of glycoforms of intact prostate-specific antigen.

The test used in clinics as prostate cancer (PCa) biomarker, based on the concentration of the glycoprotein prostate-specific antigen (PSA) in serum, leads to an elevated number of false positives. In the search for new PCa biomarkers, analysis of the proportions of different groups of glycoforms of PSA is promising. Peaks of PSA, called isoforms and containing one or several glycoforms of the glycoprotein, can be separated by CE. For those samples in which PSA concentration is very low, a very sensitive detection technique, such as LIF, would be required. However, CE separation of fluorescently labeled isoforms of glycoproteins is challenging. In this work, three different methods of fluorescent derivatization of PSA were assayed with the aim of finding conditions allowing labeling of the glycoprotein compatible with CE resolution of its isoforms. NanoOrange, as a noncovalent label; 5-(iodoacetamide) fluorescein and BODIPY® FL C1 -IA, as covalent tags of thiol groups; and Chromeo™ P503, as a covalent tag of amino groups, were tried. Only the derivatization with the P503 fluorogenic dye led to the resolution by CE-LIF of several isoforms of labeled PSA. Adapting this derivatization method to be performed on-column leads to a reduction in labeling time from 4 h to 45 s. Automation of the whole analysis permitted to carry out fluorescent labeling and CE separation of PSA isoforms in less than 12 min.

Capillary gel electrophoresis of very high molecular weight glycoproteins. Commercial and tailor-made gels for analysis of human monomeric and secretory immunoglobulin A.

Capillary gel electrophoresis (CGE) has been widely used for analysis of proteins according to their size. However, to our knowledge, this technique has not been optimized to immunoglobulin A (IgA) analysis, a protein of current and emerging high interest in several fields. IgA is the first barrier of human body against pathogens. This protein in human milk and colostrum is essential for immune protection of newborns and treatment of milk for storage in Human Milk Banks may alter IgA. The emerging use of IgA as therapeutic treatment also encourages the development of analysis methods for this class of immunoglobulins. IgA is far more heterogeneously glycosylated and complex than the well-studied IgG molecules. IgA in serum is mainly monomeric (mIgA) with about 160 kDa, while in secretions such as saliva, milk, colostrum, etc, secretory immunoglobulin A (sIgA) is the predominant form. This is a dimer where both monomers are linked by the J-chain and the secretory component accounting all together for a MW higher than 400 kDa including the glycans. This size is far from the 225 kDa MW for which commercial CGE kits are intended. The general rules governing CGE behavior of analytes cannot be directly applied to every protein. Addressing studies directed specifically to target proteins is specially needed for the large size and highly complex target analytes of this study. In this work the effect of several factors on CGE analysis of human serum and colostrum IgA is studied. The feasibility of performing analysis of both IgA classes using a commercial CGE kit is shown. In addition, this work introduces another novelty by preparing tailor-made reproducible gel buffers and to characterize them in terms of dynamic viscosity, conductivity, and electroosmotic flow mobility in bare fused silica capillaries. The possibility of analyzing mIgA and sIgA in less than 10 min using these tailor-made gels is demonstrated. Inter-day variation (RSD) for the main peak of sIgA is 0.25% for migration time (tm) and 0.27% for percentage corrected peak area (Acorr).

Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization.

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection.

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in < 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.

Study of the capillary electrophoresis profile of intact α-1-acid glycoprotein isoforms as a biomarker of atherothrombosis.

α-1-Acid glycoprotein (AGP) is a serum glycoprotein that presents several isoforms. Changes in the isoforms of AGP have been related to different pathological states including cardiovascular diseases (CVDs) such as acute myocardial infarction. However, to our knowledge, the role of variations of AGP isoforms as a potential biomarker of atherothrombosis has not been addressed. In this work, a preliminary study about differences in the capillary zone electrophoresis (CZE) profile of intact (non-hydrolyzed) AGP isoforms between healthy individuals and patients with atherothrombosis, specifically abdominal aortic aneurysm (AAA) and carotid atherosclerosis (CTA), has been performed. Biological samples (plasmas and sera) were analyzed by CZE after immunoaffinity chromatography purification. Up to 13 peaks corresponding to groups of isoforms of intact AGP from plasma samples were detected by CZE-UV. Electrophoretic profiles were aligned, peaks assigned, and linear discriminant analysis (LDA) of percentage of the corrected areas of AGP peaks was employed to discriminate and classify the CZE profiles of AGP samples. LDA enabled to accomplish 92.9% of correct classification of the AGP samples when the three groups of samples were considered. Besides, the LDA model showed high predictive power in the groups healthy vs. sick, healthy vs. AAA, and healthy vs. CTA. The described method was a successful approach to study the potential of AGP isoforms profile as a biomarker of atherothrombosis. To the best of our knowledge this has been the first time that a possible role of the CZE profile of intact AGP isoforms as a biomarker of vascular diseases has been demonstrated.

Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer.

α-1-acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE-ESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.

Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact alpha-1-acid glycoprotein from human serum.

Differences in alpha-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situations. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient purification methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the determination of isoforms of the intact glycoprotein by CZE-UV and by a developed CZE-ESI-TOF-MS method. Both preparation methods, which differ in the pre-treatment of the sample prior to an anti-AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE-UV and CZE-ESI-TOF-MS indicates that serum AGP purified without acidic precipitation as pre-treatment is more adequate due to AGP higher yield, which leads to better CZE-Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF 165.

The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF(165)) is the main component. In spite of the potential as biomarker that this protein has, information about its physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS methods for intact recombinant human VEGF(165) are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glycosylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF(165) expressed in insect cells and up to three in VEGF(165) expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF(165) expressed in insect cells to be in a range of 6.8-8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial characterization of VEGF(165) based on its MS fingerprint. MALDI-MS analysis of VEGF(165) expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF(165) as a result of the different PTMs of the protein. According to the MALDI-MS analysis, VEGF(165) expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF(165) of both origins obtained by CIEF and MALDI-MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF(165) as biomarkers. Finally, to the best of our knowledge, this is the first time that intact VEGF(165) has been analyzed by CIEF and MALDI-TOF-MS.

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165.

The vascular endothelial growth factor 165 (VEGF(165)) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF(165) arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF(165) are developed. Using a computer program previously developed in-house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF(165) expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF(165), mean a starting point that permits the study of the role of VEGF(165) as a potential biomarker of different diseases and physiological processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF(165) have been developed.

Monitorization of α1-Acid Glycoprotein Deglycosylation Using SU-8 Microchips Electrophoresis with LIF Detection.

In the last few years, biopharmaceuticals-therapeutic drugs which are generally obtained by using molecular biology techniques-have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

Capillary Electrophoresis Analysis of Prostate-Specific Antigen (PSA).

The Capillary Electrophoresis (CE) profile of isoforms (peaks) of a glycoprotein can be useful to show alterations in its posttranslational modifications (PTMs) linked to diseases. These changes can modify the electrophoretic mobility of these isoforms in a minor extent and, therefore, very reproducible CE methods are needed to detect them. In this chapter, a method for the analysis of prostate-specific antigen (PSA) by Capillary Zone Electrophoresis (CZE) with UV detection is detailed. High reproducibility in the separation of a large number of PSA isoforms is achieved by performing capillary conditioning in acid media and by using a background electrolyte (BGE) at pH 8.0 formulated with decamethonium bromide and urea.

Immunochromatographic removal of albumin in erythropoietin biopharmaceutical formulations for its analysis by capillary electrophoresis.

Human serum albumin (HSA) is added to some pharmaceutical preparations as an excipient. This is the case for some of the commercial preparations of recombinant erythropoietin (rEPO). Differences in the number of the sialic acid moieties in the different rEPO glycoforms confer to these forms different net charges and different bioactivity. Knowledge of the isoforms present in each pharmaceutical product is then of interest. Differences in net charge of the rEPO forms make possible their separation by electrophoretical methods. However it has been observed in our laboratory that the amount of HSA usually present in these drug formulations interferes or even precludes separation of rEPO bands by capillary zone electrophoresis (CZE). In this work, an immunochromatographic method to remove HSA from rEPO biopharmaceutical formulations and a procedure to concentrate the sample that is needed to be performed prior to the analysis by CZE are developed. A home-made computer program to compare the percentage of correct assignments of electrophoretical bands provided by different migration parameters is used to study the effect of HSA remaining in samples on the accuracy of assignment of rEPO bands. When there exists a residual concentration of HSA in the sample (<2mg/ml) only the effective electrophoretic mobility is a reliable migration parameter to assign rEPO bands with a 100% of correct assignment. This parameter allows the correct assignment of bands of rEPO from pharmaceutical products formulated with HSA after immunochromatographic removal of HSA. Electrophoretical bands found in epoetin alpha, one of the commercial formulations of rEPO, are independent of the molecular mass of the excipients. The methodology used in this work for the analysis by CZE and the assignment of rEPO isoforms, as well as for the immunochromatographic HSA removal in the pharmaceutical products could be of high interest for the health authorities to control the quality of the product in marketing surveillance studies and for the quality control laboratories of the manufacturers.

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.

Frontal analysis for characterizing the adsorption-desorption behavior of beta-lactoglobulin on immunoadsorbents.

High-performance frontal affinity chromatography was employed to study the adsorption-desorption kinetics characterizing the retention of beta-lactoglobulin (beta-LG) onto polyclonal anti-beta-lactoglobulin (anti-beta-LG) chromatographic supports. The adsorption and desorption processes were studied by analyzing two different elution fronts separated by a relatively long rinsing step. The method consists in performing two successive frontal injections of the protein. In between, the column was rinsed with a given volume of mobile phase (buffer alone). During this rinsing stage, a partial desorption may occur and a novel amount of protein could be adsorbed in the second frontal injection step. The whole process (first adsorption, possible desorption, and second adsorption) was simulated by a numerical procedure, in which the column was divided into a large number of slices. A model based on bi-Langmuir type kinetics was used to describe the adsorption of the protein on the support. The model assumes a non-uniform adsorbent with two types of binding sites. At equilibrium the adsorption isotherm is of the bi-Langmuir type. A global adsorption effect was considered which includes the effective binding process and the mass transfer resistances due to the transport to the binding site. Therefore, the column capacity and the kinetic parameters of the model (apparent adsorption and desorption rate constants) were determined from the best fit of the first and second adsorption fronts to the experimental ones. The other parameters of the model are the saturation capacities for the adsorption on each type of sites. The equilibrium affinity constants were estimated in a single experiment from the ratio of the apparent adsorption and desorption rate constants. The high values found (around 10(8) M(-1)) reveal a strong interaction of beta-LG with the immunoadsorbent. Kinetic measurements were carried out at different flow rates. Both the apparent adsorption and desorption kinetics were faster at larger flow rates, indicating an important contribution of the mass transfer resistance in the stagnant fluid at the particle boundary. However, as expected, close values were found for the resulting equilibrium constants calculated from the ratio of the apparent adsorption and desorption rate constant determined at various flow rates.

Impact of capillary conditioning and background electrolyte composition on capillary electrophoresis analysis of prostate specific antigen isoforms.

Glycoproteins expressed in the human body can experience modifications as result of pathological situations. Detection of those changes can be useful as disease biomarkers. As a result of these modifications, size and/or electrical charge of the glycoprotein can be altered. Migration in capillary zone electrophoresis (CZE) is governed by the size to charge ratio of the analyte and therefore this separation technique can be used to monitor those modifications. At its turn, the alteration of the electrophoretical pattern of a given glycoprotein could be used as disease biomarker. To this aim, high repeatability for separation of a large number of peaks for a given glycoprotein is desirable. For prostate cancer, new markers are needed to decrease the high number of false positive results provided by the biomarkers currently used in clinics. In this sense, CZE methods for analysis of the several prostate specific antigen (PSA) peaks which this glycoprotein exhibit, called isoforms and containing one or more glycoforms, could be useful to study the PSA pattern as prostate cancer marker. In this study two complementary strategies to achieve both lot-to-lot capillary repeatability and high resolution of a large number of PSA isoforms are developed. Better performance and precision have been obtained for capillaries conditioned with HCl than for those conditioned with NaOH. Optimization of the background electrolyte (BGE) pH value to 8.0 and inclusion of 3M urea on its composition were the two factors of highest impact for enhancing resolution of the highest number of PSA peaks. Under the optimized conditions for capillary conditioning and BGE pH and composition, long-term resolution of 10 isoforms of PSA was achieved. Inter-day (n=3) %RSD was 0.55 for the ratio tm/tEOF, 1.15 for µeff, and 5.02 for % Acorr of the PSA peaks.

Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (µZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker.

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.

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.

Improved capillary isoelectric focusing method for recombinant erythropoietin analysis.

Human erythropoietin (EPO) is an endogenously produced glycoprotein, which plays a key role in the erythropoiesis process. Production of erythropoietin by recombinant DNA techniques has made possible its therapeutical use besides its misuse in sport competitions. The link between glycosylated form and protein activity makes necessary a method to analyze the glycoforms' distribution in the recombinant products. In this work, a capillary isoelectric focusing (cIEF) method is presented that allows the analysis of erythropoietin glycoforms. Besides, the cIEF method can be easily implemented in different laboratories. In order to get a feasible and precise cIEF method the following factors have been studied and optimized: (i) neutral coated capillaries, 27 cm long are employed, (ii) ampholytes in the pH range 2 to 10 are used, (iii) bovine beta-lactoglobulin A is chosen as internal standard, (iv) anolyte consisting of 91 mM H3PO4 in cIEF gel is made by weight and catholyte is prepared by titrating 20 mM NaOH with H3PO4 to pH 11.85-11.90, (v) sample is completely depleted of excipients and sodium chloride 10 mM final concentration is added, and (v) t(n)/t(I.S.) and (A(n) - A(I.S.))/A(I.S.), n being the recombinant EPO glycoform considered and I.S. the internal standard, are chosen as indexes to express migration time and area. As a result, a precise method to analyze erythropoietin by capillary isoelectric focusing is achieved with intra-assay RSD < or = 0.5% for index time and < or = 1.5% for index area and inter-sample, inter-anolyte, and inter-catholyte precision better than 3.4% for index time and RSD lower than 2.2% for index area.