Immunology & Immunological Disorders Poster Session
Materials and Methods
SEPARATION OF FAT FROM MILK SAMPLES
Certain portions of the defatted breast-milk were also further clarified by centrifugation twice at 20,000g for 15 mins each at 4°C.
NORMAL HUMAN SERUM
BACTERICIDAL ASSAY- VIABILITY TEST
Bacteria were cultured overnight in blood agar and suspended in sterile normal saline adjusted to 3 x 108 colony-forming units (CFU) per ml using the McFarland method (25). 20µl of the adjusted bacteria was added to round-bottom micotiter wells (Nunc A/S, Roskilde, Denmark) with 80µl of the milk oe serum sample to be tested. The trays were thoroughly mixed on an orbital mixer before and after incubation at 37°C for 2 hrs. Samples of 20µl were taken from each well before and after incubation for viable counts using poured plate method (14).
Wholemilk and defatted (or clarified) milk samples, stored at -70°C or -80°C, were tested heat-treated or not, with or without addition of Mg2+ EGTA (to support the alternative pathway of C activation), and compared with was tested undefatted and defatted, heat-treated (56°C, 30 min) and non heat-treated, with and without added normal human serum and heat-inactivated serum.
The contribution of the complement system was estimated from the difference between the bactericidal effect of fresh milk and the milk heated at 56°C for 30 minutes and fresh milk sample collected in 20mM EDTA.
COATING OF MICROTITRE PLATES WITH PATHOGENIC BACTERIA AND ACTIVATED c3
The optical density of the reaction was read within 10-30 min in a photometer (Thermostat microplate photometer, from MGW-Biotech). The level of opsonization by each milk specimen on the solid-phase bacteria was obtained by substracting the level of opsonin deposited by an heated (56°C, 30 min) sample, from that obtained from an unheated sample from the same donor. The level obtained from the heated sample constitutes the background deposition of preformed opsonins.
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|Ogundele, M.O.; (1998). Bactericidal Activity of Human Milk Complement System. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/immunology/ogundele0172/index.html|
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