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Immunology & Immunological Disorders Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
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Activation and Deposition of Human Breast-milk Complement C3 Opsonins on a Serum Sensitive Bacteria


Contact Person: Michael O. Ogundele (mogundel@yahoo.com)


Materials and Methods

BREAST-MILK SAMPLES
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 -80C 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 4C 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 -80C.

COMPLEMENT INACTIVATION
Samples of de-fatted and whole breast-milk, as well as serum or plasma, were heated in a water bath at 56C 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 (56C, 30 min) sample, from that obtained from an unheated sample from the same donor.

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Ogundele, M.O.; (1998). Activation and Deposition of Human Breast-milk Complement C3 Opsonins on a Serum Sensitive Bacteria. 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/ogundele0177/index.html
© 1998 Author(s) Hold Copyright