Production Of Monoclonal Antibodies Against Aleuroglyphus Ovatus And Their Use In Immunological Studies Of A. Ovatus Sensitisation In A Mouse Model

Abstract

Aleuroglyphus ovatus (Troupeau) is a brown-legged storage mite. It is a common pest of various stored products and was reported to be associated with occupational allergy and food anaphylaxis. A. ovatus has a shorter life cycle and can multiply and colonise more rapidly compared with other mites. It takes approximately 2-3 weeks to mature from eggs to adult stages. A. ovatus was first reported in peninsular Malaysia in year 1980. However, the prevalence, sensitization rate and the allergenicity of A. ovatus have not been investigated. No detection kit for A. ovatus is available. This study was conducted to compare the antigenicity of A. ovatus with other species of mites in Malaysia, to determine the prevalence of allergic sensitisation to A. ovatus in patients from a Penang private hospital, to produce and characterise polyclonal and monoclonal antibodies against A. ovatus, and to study histopathological and immunological responses in a mouse model challenged with A. ovatus. A. ovatus was cultured, harvested, hormogenised, the extracted proteins quantified and used for the immunisation of rabbit and mice, sandwich ELISA and intranasal challenges. The crude extracts of A. ovatus, Blomia tropicalis, Dermatophagoides pteronyssinus, D. farinae, Glycycometus malaysiensis and Tyrophagus putrescentiae were compared. Twenty-four healthy and fifty-six allergic individuals were recruited for A. ovatus-specific IgE quantification. The monoclonal antibodies were produced using hybridoma technology with slight modification. The antibodies were isotyped, characterised and used for sandwich ELISA and for the localisation of mite antigen/allergen on whole and sectioned mites and lung sections using immunoperoxidase staining. The cross-reactivity and inhibition levels of other common organisms towards the selected antibodies were determined. Hundred flour samples were randomly spiked with known amount of A. ovatus. Fifty environmental dust samples were collected. Both spiked flour and environmental dust samples were used for the determination of sensitivity and specificity of the developed sandwich ELISA. Eighteen Balb/c mice were challenged intranasally with either A. ovatus, ovalbumin or PBS. The sera were collected and the serum cytokine levels were quantified. Peripheral blood smears were prepared. Bronchoalveolar lavage was obtained and the lung and trachea were collected for immunohistochemistry staining. Out of the 80 recruited subjects, 57.5% of them had high A. ovatus-specific IgE levels. Clone Ao3B8 (45 kDa; IgM), Ao2F2 (53 kDa; IgM) and Ao1B6 (60, 100 and 170 kDa; IgG1) were characterised. Ao3B8, Ao2F2 and Ao1B6 were reactive against the mite gut, gut and muscles, and the integument respectively. The sandwich ELISA developed using the three selected antibodies were able to detect A. ovatus in spiked flour and dust samples. Significant differences were observed in the number of neutrophils, basophils and macrophages in BAL smears of A. ovatus treated and negative control. Significant increase of IL-2 and IL- 5 levels was observed in A. ovatus challenged mice compared with negative control. Inflammation with infiltration of polymorphs was observed in the lung of A. ovatus challenged mice. The three antibodies were able to localise A. ovatus antigens/allergens in the lung and trachea sections. In conclusion, the three antibodies were able to detect the presence of A. ovatus in the spiked flour and dust samples, and tissue sections. These antibodies are of potential use for the detection of A. ovatus in food and dust samples, and allergy testing against A. ovatus.