The effect of lentil lectin (LCA) on the binding of mouse monoclonal antibody (MoAb) against human α-fetoprotein (AFP) to LCA-nonreactive AFP-L1 and LCA-reactive AFP-L3 was studied on a panel of 30 MoAbs provided by the TD-2 Workshop of ISOBM for epitope mapping. LCA inhibited the binding of MoAbs 93 and 98 to AFP-L3 but not to AFP-L1, indicating that there was a competition between the MoAbs and LCA for the AFP sugar chain. With MoAbs 100, 109, 118, and 120, LCA rather increased the binding to AFP-L3 over that to AFP-L1. These modulating effects of LCA on the MoAb binding to AFP-L3 were abolished by periodate treatment of the AFP preparations without affecting the binding of MoAb to AFP. Concanavalin A had similar inhibiting and enhancing effects on MoAb binding, but equally to AFP-L1 and AFP-L3, both of which are fully reactive with concanavalin A. The results suggested that MoAbs 93 and 98 recognized epitopes closely related to sugar chain, and their binding to AFP-L3 was inhibited by the bound LCA due to steric hindrance. The enhanced binding of some MoAbs to AFP-L3 over AFP-L1 with LCA, or both glycoforms of AFP with concanavalin A, may be explained by postulating an allosteric mechanism mediated by the oligosaccharide-lectin interaction.

Lectin-gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4 degrees C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody-affinity blotting was superior for low concentrations of alpha-fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin-gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.

A newly isolated lectin Erythrina cristagalli (ECL) was tested for separation of human alpha-fetoprotein (AFP) glycoforms by affinity electrophoresis at 0.5 mg/ml and separated AFP bands were detected by antibody-affinity blotting. Three AFP bands, AFP-E1, AFP-E2 and AFP-E3 in order of increasing affinity, were obtained. Sera from control patients with chronic hepatitis and cirrhosis gave a major band of AFP-E1 and a minor or trace band of AFP-E2 (3.4 +/- 2.3%), while those from patients with mostly advanced hepatocellular carcinomas had increased proportions of AFP-E2 band (16.6 +/- 10.2%). With a cutoff level of 8% (mean + 2SD of AFP-E2 for controls), the sensitivity for hepatocellular carcinoma was 72% at a specificity of 100%. Gastrointestinal tumors had much higher percentages of AFP-E2 and occasionally positive AFP-E3. Most of the yolk sac tumors examined showed AFP-E3 in addition to AFP-E2, although AFP-E3 was a minor band. Thus, AFP-E2 is potentially a clinically useful marker for differentiation of increased AFP in hepatocellular carcinoma and other malignancies from that in precancerous chronic hepatitis or cirrhosis.