Production And Use Of Polyclonal And Monoclonal Antibodies Against Glycycometus Malaysiensis For Immunological Studies

Abstract

Dust mites are major contributors of allergic diseases such as asthma and allergic rhinitis. Glycycometus malaysiensis, also known as Austroglycyphagus malaysiensis, is a domestic storage mite found in Malaysia. Though it was reported to cause sensitization in asthmatic and/or allergic rhinitis patients in South-east Asian countries such as Malaysia, Singapore and Indonesia, information on G. malaysiensis and its contribution in triggering allergic diseases are limited. The objectives of this study were to characterize and compare the protein profiles of this mite with the other five domestic house dust and storage mite species commonly found in Malaysia; to produce polyclonal and monoclonal antibodies against G. malaysiensis for the development of immunoassays to detect mite components in spiked flour and environmental samples; and to study histopathological changes and immunological responses in mouse model challenged with G. malaysiensis. G. malaysiensis was cultured, harvested, homogenized, and its proteins separated using SDS-PAGE and stained with Coomassie blue. Nineteen prominent protein bands were observed and two of them (90 and 100 kDa) were found exclusively in G. malaysiensis. A female New Zealand white rabbit and two female BALB/c mice (5-6 weeks old) were immunized with crude G. malaysiensis extract emulsified with complete or incomplete Freund’s adjuvants. Western blot analysis using the rabbit and mouse polyclonal antibodies revealed 18 and 16 prominent immuno-reactive bands respectively. Fusion was performed according to Milstein and Kohler (1975) with slight modification and all the spent culture media of the hybrid clones were screened for the presence of G. malaysiensis specific antibody using immuno-blot, enzyme-linked immunosorbent assay (ELISA) and Western blot. Three selected positive hybridoma clones (AM4A3, AM4H11 and AM1F8) were isotyped and characterized. They were all IgG1 with κ light chains. AM4A3 antibody was immuno-reactive against 12 kDa glycosylated antigenic protein that was localized at G. malaysiensis cuticle and faecal pellets. It was found to be cross-reactive against Tyrophagus putrescentiae (58%) and Dermatophagoides farinae (71%). AM4H11 and AM1F8 antibodies were found to be immuno-reactive against multiple antigenic proteins. AM4H11 antibody bound to glycosylated 98 and 100 kDa antigenic proteins localized at muscles of sectioned G. malaysiensis while AM1F8 antibody bound to 28, 34, 40 and 57 kDa antigenic proteins (all glycosylated except for 28 and 40 kDa) which are localized at the internal organs and cuticles of sectioned G. malaysiensis. AM4H11 antibody cross-reacted with Aleuroglyphus ovatus (86%), Blomia tropicalis (111%) and D. pteronyssinus (120%) but AM1F8 antibody did not cross-react with the 14 organisms tested in this study. AM4A3, AM4H11 and AM1F8 antibodies were potentially immuno-reactive against groups 2, 11 and 14 mite allergens respectively. AM4A3, AM4H11 and AM1F8 clones were propagated in peritoneal cavity of pristane primed BALB/c mice. The antibodies in spent culture supernatants and ascites fluid were purified using HiTrap Protein G HP column and fractioned protein liquid chromatography (FPLC). Sandwich ELISA was developed to detect G. malaysiensis and other mite components in single blinded spiked flour samples (n = 50) and environmental dust samples (n = 80). All three sets of sandwich ELISA developed using rabbit anti-G. malaysiensis polyclonal antibody and AM4A3, AM4H11 and AM1F8 antibodies respectively were able to differentiate spiked flour samples from the non-spiked flour samples. The sensitivities of each sandwich ELISA developed using AM4A3, AM4H11 and AM1F8 antibodies were 86.2%, 86.2% and 96.2% respectively while the specificities were 92.6%, 65.8% and 92.6% respectively. The sandwich ELISAs developed were also able to detect mite components in the environment dust samples collected from grocery shops, feed shops, store rooms, pet shops, mini markets and aquarium shops. Ten out of the 80 shops were determined to be positive for mite components and were verified under stereomicroscope examination. Female inbred BALB/c mice (n = 5) were intranasally challenged with crude G. malaysiensis mite extract. Positive and negative control groups (n = 5 each) were challenged with ovalbumin and sterile PBS respectively. Five weeks after the last intranasal challenge, the mice were re-challenged for 3 consecutive days. The mice were sacrificed via cardiac puncture a week after the re-challenge. Sera and bronchoalveolar lavage (BAL) fluid were collected. Anti-G. malaysiensis IgE was relatively low throughout the duration of study but anti-G. malaysiensis total immunoglobulins were found to increase after re-challenge with crude G. malaysiensis. Differential white blood cell count was performed on peripheral blood and BAL fluid smears. In general, the eosinophils in blood and BAL fluid were significantly higher (P = 0.000; P = < 0.001 respectively) in mice challenged with G. malaysiensis compared with those challenged with PBS. The eosinophils and monocytes in blood were increased significantly post G. malaysiensis re-challenge. Tracheae and lungs were fixed, processed, embedded, sectioned and stained with hematoxylin and eosin. Influx of inflammatory cells, hypertrophy of airway epithelial cells and multinucleated giant cells were observed in lung and trachea sections of G. malaysiensis challenged mice. The respective serum cytokine levels (GM-CSF, IFN-γ, IL-10, IL-13, IL-17a, IL-2, IL-23, IL-4, IL-5 and TNF-α) were measured using Procarta® Cytokine Assay Kit and Luminex. IL-2 and IL-23 were significantly higher (P = 0.001 and P = 0.005 respectively) in G. malaysiensis challenged mice. No significant change was seen for the other eight cytokines tested. This suggested occurance of Th17 immune response instead of Th2 immune response. Using respective monoclonal antibodies obtained, positive immuno-reactivities were observed at the air sacs and fibrocartilaginous layers of lung and trachea sections of G. malaysiensis challenged mice. In conclusion, antigenic proteins (potential allergens) of G. malaysiensis were determined and the protein profile of G. malaysiensis was compared with five other domestic storage and house dust mites; three murine monoclonal antibodies and rabbit poloclonal antibodies against G. malaysiensis were produced and exploited for the detection of G. malaysiensis and other mite allergens in dust samples; histopathological and immunological responses in mouse model challenged with G. malaysiensis were studied.