This study evaluated two methods for the isolation and purification of anti-DNA antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligodeoxythymidine (dT) M-280 and protein G Dynabeadsª was compared to a two step method using Melon(TM) Gel and cellulose DNA. Although Melon gel allowed for faster antibody purification and a higher recovery rate it gave a product of less purity than the magnetic bead method. Further characterization of the antibodies was done by PhastGel(TM) non-reducing SDS-PAGE and isoelectric focusing in order to analyze purity and confirm the polyclonal nature of anti-DNA antibodies. Agilent 2100, with a higher resolution then SDS-PAGE, revealed possible subclasses of different MW not detected by SDS-PAGE. ELISA showed that all four IgG antibody subclasses were present, while Western blot confirmed the presence of human IgGs. Ultraviolet spectroscopy, Agilent, and fluorescence based assays were used to demonstrate DNA hydrolytic activity of purified anti-DNA antibody. / by Anna M. Kats. / Thesis (M.S.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, FL : 2008 Mode of access: World Wide Web.

Gray mold, caused by Botrytis cinerea is the major cause of postharvest loss in strawberries. Detection of flower and fruit infections enables producers to make intelligent management decisions. A plate-trapped ELISA protocol using a Botrytis-specific monoclonal antibody (BC-12.CA4) was developed for the detection of Botrytis cinerea in strawberry flower receptacles and red fruits. Horseradish peroxidase, was chosen as enzyme conjugate because it gave lower background absorbance in disease-free samples. B. cinerea reference antigen (RAg) was isolated from strawberry. BC-12.CA4 was very sensitive to the RAg, detecting up to 6 mug/ml of RAg when mixed with strawberry extracts. The MAb did not show any reaction to Rhizopus sp., Mucor sp. and Penicillium sp. associated with strawberry. B. cinerea could be detected in receptacles two days after inoculation. Treatment of inoculated receptacles with paraquat speeded-up detection. Inoculated red fruit infection could be detected after three days of incubation. Disease in commercially-produced receptacles and red fruits were assessed visually and by ELISA. The ELISA detected B. cinerea in 95% of commercial flower samples, whereas the traditional visual method detected only 50 to 70%. No dramatic differences between methods were found for red fruits.

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