Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Ongay, Sara; Lacunza, Izaskun; Diez-Masa, Jose Carlos; Sanz, J.; Frutos, Mercedes de

doi:10.1016/j.aca.2010.01.054

Cited by 16 publications

(19 citation statements)

References 34 publications

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“…A similar pattern and signal intensity for the AGP glycoform bands was observed for serum that had undergone both acid precipitation and desalting, with up to eleven bands being noted for some of these samples, as demonstrated in Figure 5. This number of AGP glycoform bands was consistent with what has been observed by using CE and anti-AGP antibody supports for sample pretreatment [12,13]. The relative composition of the glycoform bands after both pretreatment methods did differ some from the pattern seen for commercially-prepared normal and purified AGP by giving a slight shift in intensity to bands with higher migration times (see Supplementary Materials).…”

Section: Resultssupporting

confidence: 86%

“…A number of schemes have been developed to isolate AGP from serum and related samples prior to the analysis of this glycoprotein by liquid chromatography or CE [12,13,18–20]. Previous methods for the purification of AGP have often involved multiple chromatographic steps (e.g., ion-exchange and dye-ligand affinity chromatography), which can be time-consuming and generally require relatively large sample volumes (e.g., 1–2 mL of serum or plasma) [18,19].…”

Section: Introductionmentioning

confidence: 99%

“…A method based on acidic precipitation has been described for the isolation of AGP from 50 μL of plasma [20]. However, significant interferences in the resulting samples have still been noted with this approach, which has resulted in the further use of anti-AGP antibodies in columns or on magnetic beads to purify AGP [12,13]. …”

Section: Introductionmentioning

confidence: 99%

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Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Zhang

Clarke

et al. 2017

Journal of Chromatography A

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A method based on capillary electrophoresis (CE) with electrophoretic injection and absorbance detection was developed for the direct analysis of AGP glycoforms in human serum. Electrophoretic injection of AGP was performed in the reversed-polarity mode of CE with a capillary coated with poly(ethylene oxide) and that had minimal electroosmotic flow. This situation created an essentially stationary interface between the sample and running buffer during injection and sample stacking. This approach allowed an 11,000-fold increase in sample loading for a 5 min injection versus hydrodynamic injection and without introducing any significant levels of extra band-broadening. This method was used with sample pretreatment methods based on acid precipitation and desalting to examine AGP glycoforms in only 65 μL of serum. A limit of detection of 2.1–11.3 nM was obtained for the major AGP glycoform bands in serum, and the sample pretreatment method gave a recovery of 72.3–80.9% for these glycoforms. The precision for the migration times was ± 0.08–0.13% and the precision for the peak areas was ± 0.34–1.18% when using serum samples and an internal standard. This method was used for both normal pooled serum and serum from individuals with systemic lupus erythematosus. Results were obtained in a separation time of 25 min and allowed the comparison of up to eleven glycoform bands in these samples. A similar approach may be useful in examining additional glycoproteins in serum or other types of biological samples.

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Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

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Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Zhang

Clarke

et al. 2017

Journal of Chromatography A

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“…Some limitations of these previous methods have been that they either involved tedious methods for capillary treatment or they required relatively expensive pre-treated capillaries [7–9,12]. Another approach has been to employ buffer additives such as putrescene or urea to disrupt interactions between AGP and the capillary wall and to reduce electroosmotic flow [4,5,10,11]. However, the migration times of AGP glycoforms cannot be easily used to make accurate peak assignments in such methods due to the variability in these values, the relatively large number of peaks that are present, and the similar migration times of the glycoform bands [4].…”

Section: Introductionmentioning

confidence: 99%

Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis

Zhang

Hage

2016

Journal of Chromatography A

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A relatively fast and reproducible CE separation was developed for the glycoform analysis of α1-acid glycoprotein (AGP). Factors that were considered included the pH for this separation and various techniques for coating the capillary and/or to minimize electroosmotic flow and protein adsorption. Optimum resolution of the AGP glycoforms was obtained at pH 4.2 with a running buffer containing 0.1% Brij 35 and by using static and dynamic coatings of PEO on the capillary. These conditions made it possible to separate nine AGP glycoform bands in about 20 min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38 μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240 μM. The migration times for the glycoform bands had typical within-day and day-to-day precisions of ± 0.16–0.23% or less, respectively, on a single treated capillary and the variation between capillaries was ± 0.56% or less. A charge ladder approach was employed to examine the mass or charge differences in the glycoforms that made up these bands, giving a good fit to a model in which the neighboring bands differed by one charge (e.g., from a sialic acid residue) and had an average mass difference of approximately 0.7–0.9 kDa. The approaches used to develop this separation method are not limited to AGP but could be extended to the analysis of other glycoproteins by CE.

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“…Further, more recently, purification and characterization of glycans on AGP have been done with the aid of capillary electrophoresis [9, 10], but most of these methods are unsuitable for processing a large number of plasma samples in a short period of time. The CAIE method we recently introduced can process plasma samples without purification for investigating glycoforms because AGP can be identified by means of an anti-AGP antibody but still is not applicable to large numbers of samples if rapid processing is required [3].…”

Section: Introductionmentioning

confidence: 99%

Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasmaα₁-Acid Glycoprotein

Asao

Yazawa

Nishimura

et al. 2013

BioMed Research International

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Human plasma α 1-acid glycoprotein (AGP) from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP) was significantly higher in cancer patients as compared with the healthy group (P < 0.001). Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis.

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Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Cited by 16 publications

References 34 publications

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis

Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasmaα₁-Acid Glycoprotein

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Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Cited by 16 publications

References 34 publications

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis

Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasmaα1-Acid Glycoprotein

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Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasmaα₁-Acid Glycoprotein