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Further resolution of α‐fetoprotein glycoforms by two‐dimensional isoelectric focusing and lectin affinity electrophoresis
Author(s) -
Ichikawa Eriko,
Kuriyama Shigeki,
Yuji Jin,
Masaki Tsutomu,
Uchida Naohito,
Nishioka Mikio,
Taketa Kazuhisa
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600040
Subject(s) - affinity electrophoresis , isoelectric focusing , microbiology and biotechnology , lectin , chemistry , alpha fetoprotein , agarose gel electrophoresis , gel electrophoresis , agarose , electrophoresis , hepatocellular carcinoma , affinity chromatography , biochemistry , biology , enzyme , dna , cancer research
Human α‐fetoprotein (AFP) from serum of patients with cirrhosis and hepatocellular carcinoma (HCC) was separated into several bands by IEF and by erythroagglutinating phytohemagglutinin (E‐PHA) affinity electrophoresis. These AFP bands were directly compared in 2‐D IEF and E‐PHA affinity electrophoresis. IEF of serum AFP was run in 1% agarose IEF gel with 3% Pharmalyte 4.5–5.4. After IEF, a part of the gel was stained for AFP and another part of the gel corresponding to the area of separated AFP bands was cut in 1 mm×39 mm along the focused direction and transferred to a trough in 1% agarose gel with 0.3 mg/mL E 4 ‐PHA for second‐dimensional affinity electrophoresis. Separated 2‐D AFP spots were visualized by antibody‐affinity blotting and identified by combining the systems of Johnson et al.. (Johnson, P. J., Ho, S., Cheng, P., Chan, A. et al.., Cancer 1995, 75 , 1663–1668) for AFP‐I‐+IV and of Taketa et al.. (Taketa, K., Ichikawa, E., Taga, H., Hirai, H., Electrophoresis 1985, 6 , 492–497) for AFP‐P1–5. AFP‐P2, the major AFP glycoform, was composed of AFP‐I, AFP+I, and AFP+II; AFP‐P3, a nonspecific monosialo‐AFP, was composed of AFP+II; AFP‐P4, HCC‐specific monosialo‐AFP, was composed of AFP+II, AFP+III, and AFP+IV; and malignancy‐related AFP‐P5 was composed of AFP+I and AFP+II. Monosialo‐AFP (AFP+II) was recovered in all the glycoforms of AFP‐P2, ‐P3, ‐P4, and ‐P5; thus, AFP‐P4 is more specific to HCC than monosialylated AFP+II.