Further resolution of alpha-fetoprotein glycoforms by two-dimensional isoelectric focusing and lectin affinity electrophoresis.
Human alpha-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 x 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.
Ichikawa
Eriko
E
Third Department of Internal Medicine, Kagawa University School of Medicine, Kagawa, Japan.
Kuriyama
Shigeki
S
Yuji
Jin
J
Masaki
Tsutomu
T
Uchida
Naohito
N
Nishioka
Mikio
M
Taketa
Kazuhisa
K
D002240:Carbohydrate Sequence; D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D007525:Isoelectric Focusing / Q000379:methods*; D008103:Liver Cirrhosis / Q000378:metabolism; D008113:Liver Neoplasms / Q000737:chemistry; D008969:Molecular Sequence Data; D010835:Phytohemagglutinins; D020033:Protein Isoforms / Q000302:isolation & purification; D000509:alpha-Fetoproteins / Q000302:isolation & purification*
D010835:Phytohemagglutinins; D020033:Protein Isoforms; D000509:alpha-Fetoproteins; C067494:erythroagglutinating phytohemagglutinin