Erythrocytes agglutination assay

Some surface assays are based on whole cells and thus have the benefit that they can measure biological activity on biological surfaces. Hemagglutination assays involve observation of the agglutination of erythrocytes by bacteria, viruses. 

The agglutinating activity was assayed by using horse or mouse erythrocytes in microtiter plates. In some experiments, human erythrocytes was also used. 25 pi of 2.5 % (v/v) suspension of erythrocytes in 6.4 mM phosphate buffer saline, pH 7.2 (PBS) was added to 50 pi of serial 2-fold dilutions of the lectin fractions in PBS. The plates were incubated at room temperature for 1 hr. The results were expressed as the minimum concentration of lectin fractions (pg/ml). The agglutination inhibition was expressed as the minimum concentration of each sugar required for inhibition of the lectin fractions. 

Figure 2. Agglutinating effect of increasing dilutions of the crude extracts from the pedicellariae of T pileolus and plant lectins. The assay of agglutinating activity with mouse erythrocytes was carried out using a 2.5 % suspension in PBS. G-type, the crude extract (5 pg/ml) from the giant type pedicellariae N-type, the crude extract (5 pg/ml) from the normal type pedicellariae Control, PBS WGA, wheat germ agglutinin (50 pg/ml) Con A, concanavalin A (100 pg/ml).

The giant type extract that was dialyzed against 0.15 M NaCl solution was applied to a Sephadex G-200 column equilibrated with the same solution. Fig. 3 shows an elution pattern with three peaks. The individual three peaks were assayed for the agglutinating activity with mouse and horse erythrocytes. The agglutinating activity of third peak (the P-III fraction) was higher than that of second peak (the P-II fraction), while that of first peak (the P-I fraction) was almost insensitive. 

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