Mycobacterum avium paratuberculosis capture from milk using monoclonal and polyclonal antibodies linked to immunomagnetic beads

GILARDONI, L.; FERNÁNDEZ, B.; JAR, A. M.; MORSELLA, C.; CIRONE, K.; PAOLICCHI, F.; MUNDO, S

Rosario

Congreso; IV Reunión de La Sociedad Latinoamericana de Tuberculosis y otras Micobacterias.; 2009

La Sociedad Latinoamericana de Tuberculosis y otras Micobacterias.

Mycobacterium avium paratuberculosis (Map) is shedded by colostrum, milk and feces from symptomatic and non-symptomatic infected cattle. Map identification is difficult, as its concentration in secretions varies and its shedding is intermittent. The aim of this study was to evaluate the efficiency of immunomagnetic beads coated with specific sera (IMB) to capture Map from experimentally contaminated milk, what could be applied to a rapid and specific identification of infected animals. We developed monoclonal antibody 1A6P34 that recognizes Map and hyper-immune serum from mice vaccinated with Map (ATCC 19698) heat-inactivated and adjuvanted with mineral oil. IMB (Goat Anti-Mouse IgG Magnetic Beads, NEB, USA) were sensitized with Map-specific murine hyper-immune polyclonal serum (pAb) and/or monoclonal antibodies (mAb), or an irrelevant polyclonal serum (ipAb), following manufacturer instructions. Map (strains ATCC 19698 and INTA Balcarce M35) were adjusted at 1010 UFC/ml. 900µl of just yield milk -free of Map was inoculated with 100µl of 10 fold dilutions of bacteria. 10 µl of sensitized beads were added and incubated for 30 minutes, obtaining Map-IMBs that were recovered using a magnetic rack. The number of bacteria present in pellets and supernatants was determined and expressed as UFC/ml. Map capture capabilities of IMBs were as follows: IMB-pAb: 103 UFC/ml; IMB-mAb: 104 UFC/ml; IMB-pAb+mAb: 107 UFC/ml; IMB-ipAb: 101 UFC/ml. We could see that IMB highest efficiency to capture Map was obtained when pAb and mAb were used together to coat IMB. We are currently evaluating this methodology, associated with an IS900-PCR, for bacterial identification in field samples.