A novel ELISA for determination of polysaccharide specific immunoglobulins.
A novel ELISA using Nunc CovaLink microtiter plates has been developed for the determination of polysaccharide specific antibodies in mice sera. Glucuronoxylomannan (GXM), a major capsular polysaccharide of Cryptococcus neoformans serotype B, was immobilised on CovaLink plates by covalent linkage of CNBr activated hydroxyl groups of the sugar and free bnd NH(2) groups of the plates. The binding characteristics of GXM to CovaLink and to conventional polystyrene ELISA plate (Costar) were compared. The differences were observed in quality of standard curve with anti-GXM MoAb 2H1 (R(2) values were 0.9468 and 0.9872 for Costar and CovaLink plates, respectively) and in absorbance values of sera of immunised mice which were 2.5 times higher on CovaLink than on Costar plates. Negative control was low and having the same value on both the plates. Using the novel ELISA we have analysed the influence of immunomodulatory peptidoglycan monomer on humoral immune response to GXM in mice. The treatment with the conjugate of the immunomodulator and GXM resulted in the increase of GXM-specific IgGs in mice sera. Finally, the method has been successfully modified for the determination of dextran-specific antibodies in mice sera, indicating that the described procedure could be applied for other polysaccharides that have &z.sbnd;OH groups available for CNBr activation.
Spoljar
B H
BH
Section of Radioimmunology and Chemistry, Institute of Immunology, Rockefellerova 10, HR-10000, Zagreb, Croatia. bspoljar@inet.hr
Tomasic
J
J
D000818:Animals; D000908:Antibodies, Fungal / Q000097:blood*; D000918:Antibody Specificity; D000946:Antigens, Fungal; D003453:Cryptococcosis / Q000276:immunology; D003455:Cryptococcus neoformans / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005657:Fungal Vaccines / Q000276:immunology; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood*; D008297:Male; D051379:Mice; D011134:Polysaccharides / Q000276:immunology*
D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D005657:Fungal Vaccines; D007074:Immunoglobulin G; D011134:Polysaccharides; C027478:glucuronoxylomannan