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.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Bibliography: leaves 129-145 / viii, 145 leaves, [50] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1985

Thesis (M. Clin. Sc.)--University of Adelaide, 1985. / Includes bibliographical references (leaves 99-106).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Surgery, Department of Surgery, University of Alberta. Title from pdf file main screen (viewed on July 28, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on August 29, 2009). Includes bibliographical references.