Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis.
Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.
Ongay
Sara
S
Institute of Organic Chemistry (C.S.I.C.), Juan de la Cierva 3, 28006 Madrid, Spain. sara.ongay@iqog.csic.es
Lacunza
Izaskun
I
Díez-Masa
Jose Carlos
JC
Sanz
Jesús
J
de Frutos
Mercedes
M
D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods*; D002845:Chromatography; D019075:Electrophoresis, Capillary; D006801:Humans; D007118:Immunoassay; D009961:Orosomucoid / Q000737:chemistry* / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification
D015415:Biomarkers; D009961:Orosomucoid; D020033:Protein Isoforms