Immunology & Immunological Disorders Poster Session
Materials & Methods
Discussion & Conclusion
New Insights into the Role of Milk Fat Globule Membrane in the Sequestration of Particulate Antigens: Interactions with the Complement System
Contact Person: Michael O. Ogundele (firstname.lastname@example.org)
Materials and Methods
Breast-milk samples were voluntarily donated by lactating mothers, with their informed consent, at various postpartum (PP) 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. One set of milk samples was collected in EDTA, to a final concentration of 20mM, while the other set of samples from the same donor was collected in an equivalent volume of PBS buffer only. The samples were classified according to postpartum period of lactation as follows (22); colostrum (1-4 days PP, 5 samples), transitional milk (5-30 days PP, 4 samples) and mature milk (>30 days PP, 4 samples).
SEPARATION OF FAT FROM MILK SAMPLES
De-fatted milk samples were obtained by dividing whole milk samples into smaller portions and centrifuging them consequtively at rates of 500g and 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 aliquots at -70 or -80°C.
Certain portions of the de-fatted breast-milk were also further clarified by centrifugation twice at 20,000g for 15 mins each at 4°C. The clarified phases were collected and stored in 1 ml aliquots at -70°C until required.
STORAGE OF MILK SAMPLES
Portions of the breast-milk were stored at 4°C and -20°C for different periods after which they were collected and either immediately processed or stored at -70 or -80°C in small aliquots until processed. Some milk samples were subjected to three cycles of rapid freeze-thawing by storing at -20°C for 6 hours, allowing them to rapidly warm to room air temperature within 2 hours, by occasionally dipping them in a warm water bath. The samples were then tested immediately or stored at -70°C until required.
BACTERIAL SEQUESTRATION ASSAY
A complement-sensitive strain of Escherichia coli NCTC 8007, serotype 0111 K58(B4) H2 was used for the assay (34).
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 (16). 20µl of the adjusted bacteria was added to round-bottom microtiter wells (Nunc A/S, Roskilde, Denmark) with 80µl of the milk sample to be tested. The trays were thoroughly mixed on an orbital mixer. Samples of 20µl were then taken from each well for bacteria counts to assess the level of sequestration from suspension by milk fat globule membranes (MFGM), using poured agar plate method (33).
The level of bacteria recovered from de-fatted milk and saline were taken as baseline, to determine the level of bacteria sequestered by MFGM in whole-milk samples.
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 (27).
BACTERICIDAL ASSAY- VIABILITY TEST
After constituting a suspension of living bacteria to 3 x 108, 20µl of it was added to round-bottom microtiter wells (Nunc A/S, Roskilde, Denmark). 80µl of milk sample, whole-milk and de-fatted (or clarified) milk samples, stored at -70°C or -80°C, heat-treated or not, to be tested was added. The trays were thoroughly mixed on an orbital mixer before and after incubation at 37°C for 2 hrs. 20µl of the suspension was taken from each well before and after incubation for viable counts using poured plate method (33).
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 or fresh milk sample collected in 20mM EDTA.
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), 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) (30) 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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