Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

BREAST-MILK SAMPLES
Breast-milk samples were voluntarily donated by thirteen lactating mothers 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 20mM EDTA solution, while the other set of samples from the same mother was collected in an equivalent volume of PBS buffer only. The samples were classified as colostrum (1-4 days PP, 5 samples), transitional milk (5-30 days PP, 4 samples) and mature milk (>30 days PP, 4 samples) (10).

SEPARATION OF FAT FROM MILK SAMPLES
Defatted 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.

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
Whole blood was collected in a 10-ml syringe without anticoagulants. The blood was allowed to clot at RT and then briefly stirred with a sterile Pasteur pipette, to remove the fibrin adhering to the syringe. The blood was then centrifuged at 1,500g at 4°C without brake. The serum supernatant was collected and stored at -70°C.

COMPLEMENT INACTIVATION
Samples of defatted 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.

BACTERICIDAL ASSAY- VIABILITY TEST
A complement-sensitive strain of Escherichia coli NCTC 8007, serotype 0111 K58(B4) H2 was used, in comparison with another E coli strain 0125 (19).

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 ELISA
Flat-bottomed microtiter plates (Nunc A/S, Roskilde, Denmark) were coated overnight with a suspension of 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) (18) 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 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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