Generation and validation of Aspergillus fumigatus monoclonal antibodies

Aspergillus fumigatus is a ubiquitous thermo tolerant fungus associated with a number of diseases in humans and animals. People at high risk for aspergillosis include patients with advanced AIDS, prolonged neutropenia, allogeneic hematopoietic stem cell transplant recipients, solid organ transplant recipients, and chronic granulomatous disease. The diagnosis of invasive fungal infections is difficult, and most mycology laboratories depend on the blood culture system, and the serology diagnosis of invasive aspergillosis has low sensitivity and specificity. Monoclonal antibodies (mAbs) due to their binding specificity, their homogeneity and great ability to be produced in unlimited quantities are important in diagnosis. The main objective of the present research was to develop specific monoclonal antibodies to help in the early detection of invasive aspergillosis. The hybridoma cells were obtained by fusing A. fumigatus–hyperimmunised Balb/c splenocytes with Sp 2/0-Ag14 (Sp2) myeloma cells using polyethylene glycol with molecular weight of 1450 (PEG 1450). Positive clones were screened by ELISA. Clones with high titre were selected after 4th limiting dilution, and analyzed with Western blotting. ELISA was carried out to determine the isotypes of antibodies. An ELISA based cross-reactiity test was done to examine the cross-reacts of mAbs with other species of Aspergillus and Candida. The result of isotype test showed that mAbs 1F8, 3B3, 4C6, 4E9, 5B8, 7D7 and 8G3 were IgG1; mAbs 2G10 was IgG3 and 3B2, 7B2 and 8F4 were IgM and none of the hybridoma clones produced IgA. Kappa (k) light chains were found in all of the mAbs. Both of mAb 4C6 (97 kDa) and mAb 7D7 (58 kDa) were chosen for in vivo production. Mabs 4C6, 7D7 had a strong reactivity with A. Fumigatus crude extract and did not show any cross-reactivity with other pathogenic species of Aspergillus and Candida. The above antibodies displayed a substantial amount of sensitivity and specificity and both of mAbs were able to stain and clearly identify the hyphae and conidia of A. fumigates by immunofluorescence. The study concluded that mAb 4C6 and mAb 7D7 are able to be used as an immunoprobe for recognition of epitopes responsible for shared antigenicity of fungal glycoproteins, for examining their expression during hyphal and conidial morphogenesis.

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