Extraction of lectin and hemagglutination assay

100 mg dry seed powder defatted twice with acetone (1:2 w/v) was kept in 1000 µl of 10 mM Tris–HCl, containing 150 mM NaCl (pH 7.2). This mixture after stirring for 16 h, was centrifuged at 10,000 rpm for 20 min in cold and the supernatant was collected as a source of lectin.

The hemagglutination activity was determined using trypsinised rabbit erythrocytes. The fresh erythrocytes separated from plasma by centrifugation at 3000 rpm for 4 min at 5–10 °C and washed extensively with phosphate buffer saline (PBS). Finally, 3% suspension was prepared in PBS and the two-fold serial dilution was used to perform hemagglutination tests in microtitre plate (Liener 1962). The erythrocyte suspension was mixed with serially diluted lectin and agglutination was observed at room temperature. The reciprocal of the highest dilution of the lectin that showed complete agglutination (titre) is the unit of hemagglutination activity (U). The specific activity is measured as hemagglutination per mg of protein.