Immunology & Immunological Disorders Poster Session
Materials & Methods
Discussion & Conclusion
Activation and Deposition of Human Breast-milk Complement C3 Opsonins on a Serum Sensitive Bacteria
Contact Person: Michael O. Ogundele (firstname.lastname@example.org)
Materials and Methods
Breast-milk samples were collected from lactating mothers at various postpartum periods, by manual expression or a breast pump. The samples were placed in sterile plastic tubes and transported to the laboratory on ice and processed immediately or stored in small aliquots at -70° or -80°C until use. The samples were classified according to postpartum period of lactation as follows (3); colostrum (1-4 days PP), or transitional milk (5-30 days PP).
SEPARATION OF FAT FROM MILK SAMPLES
De-fatted milk samples were obtained by dividing whole milk samples into smaller portions and centrifuging them twice at alternate rates of 500g or 3,000g at 4°C for 15 min, aspirating the aqueous phase of the milk each time. During centrifugation, the milk separates into three layers, i.e. a cell pellet, a middle aqueous layer and an upper fat layer. The aqueous layers were collected each time and processed immediately or stored as small aliqouts at -70 or -80°C.
Samples of de-fatted and whole breast-milk, as well as serum or plasma, were heated in a water bath at 56°C for 30 min to inactivate the complement activity.
COATING OF MICROTITRE PLATES WITH PATHOGENIC BACTERIA AND ACTIVATED C3 ELISA
Flat-bottom microtiter plates (Nunc A/S, Roskilde, Denmark) were coated overnight with a suspension of killed bacteria (Escherichia coli NCTC 8007, serotype 0111 K58(B4) H2) (4), at 3 x 107/ml, in coating buffer containing 0.05M sodium bicarbonate. Unsaturated binding sites were blocked with aqueous solution of 0.5% (w/v) gelatin. Different milk samples, peroxidase-conjugated rabbit anti-human C3d antibodies and 2,2 Azino-di (3-ethylbenzthiazolinsulphuric 6 acid (ABTS) as substrate (Boehringer, Mannheim, Germany), containing 2.5mM hydrogen peroxide, were added to the plates consecutively. The incubation steps were separated by one to three washes with PBS/0.05% (w/v) Tween 20. A standard curve was included on each plate by serial dilution of purified human C3b in PBS-Tween sandwiched between a capture monoclonal antibody (I3/15) (5) and peroxidase-conjugated rabbit anti-C3d.
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 subtracting the background level of opsonin deposited by an heated (56°C, 30 min) sample, from that obtained from an unheated sample from the same donor.
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