engleski

The novel ELISA for determination of polysaccharide specific antibodies

A novel ELISA technique using Nunc CovaLink microtiter plates has been developed to determine anti-polysaccharide antibodies in mice sera. Glucuronoxylomannan (GXM), a major capsular polysaccharide of C. Neoformans serotype B, was immobilized on CovaLink plates. The hydroxyl groups of GXM were CNBr activated and covalently coluped to free –MH2 groups of the plates. We compared the binding characteristics of GXM to CovaLink and to conventional polystyrene ELISA plate (Costar). The differences were observed in quality of standard curve with anti-GXM MoAb 2H1 (R2 velues were 0.8905 and 0.9614 for Costar and CovaLink plates, respectively) and in absorbance values of sera of immunized mice which were 2,5 times higher on CovaLink than on Costar plates. We also analyzed the influence of immunomodulatory peptidoglycan monomer on humoral immune response to GXM in mice, using the novel ELISA. We observed that the treatment with the conjugate of the immunomodulator and GXM resulted in the increase of GXM-specific IgGs in mice sera. The possible use of the method for class and subclass analysis of GXM specific antibodies is demonstrated. Finally, the method is successfully modified for determination of dextran-specific antibodies in mice sera, indicating that the described procedure could be applied for other polysaccharides that have available –OH groups for CNBr activation.

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