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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)


The deposition C3 fragments, C3b, iC3b and C3dg, from HBM, identified by the antibody I3/15, were demonstrated on the killed serum susCeptible bacteria, Escherichia coli NCTC 8007, serotype 0111 K58(B4) H2.

Results showed that C-derived opsonins apparently have a greater affinity for MFGM than solid-phase killed bacteria. Diluting whole-milk in a detergent containing buffer led to an increased level of C3 fragments deposited on the killed bacteria. Whole-milk diluted in PBS buffer to deposit less C3b on the bacteria in comparison with whole-milk in 0.5% PBS-Tween-20 buffer. Tween-20 is a non-ionic detergent, capable of disrupting the MFGM through fat dissolution (Table 1). This probably led to the release of membrane-bound C fragments. The inhibition of optimal deposition of opsonin on killed bacteria by MFGM, was also observed using appropriate buffers supporting both the classical and alternative pathways of C activation, when whole-milk was compared to de-fatted milk samples. Deposition of opsonin was consistently greater in de-fatted milk compared to whole-milk samples. The inhibition of c activation was approximately equal for both the classical and alternative pathways in the colostrum, while the inhibition of the classical pathway predominated in the transitional milk (Table 2).

Table 1 Deposition of activated C3 (opsonins) on killed E. coli 0111 bacteria by human breast-milka,c (results in g/ml)

                                       Colostrum    Transitional milk
Post-partum duration of lactation      1-4 days        5-30 days
Heatedb whole-milk                     65  10*        70  5.5*
Whole-milk in PBS                      75  28         87  12.8
Whole-milk in PBS-Tw buffer           167  34        110  25
Whole-milk in dGVB2+ buffer             3  2.2        -------
Partially de-fatted milk 
(x200-1500g) in GVB2+ buffer           84  12         42  6
Whole-milk in Mg-EGTAe buffer          -------         50  6.8
Partially de-fatted milk 
(x200-1500g) in Mg-EGTA buffer         14  5.6        64  8.4
including C3b, iC3b, C3g and C3dg
* Real value of deposited opsonin with no subtraction of the background opsonization.
a All values obtained after subtraction of the background level of opsonization (except those marked by * symbol)
b Whole-milk heated to 56C for 30 minutes
c Average of two readings from each of two samples
d Supports the classical pathway of C activation
e Supports the alternative pathway of C activation

Table 2. Degree of inhibition by milk fat for the utilisation of classical and alternate pathways of C activation by whole-milk samples to deposit activated C3 samplesa, b

                                      Colostrum    Transitional milk
Combined pathway: PBS vs PBS-Tw           55              21
Classical pathway (GVB 2+ buffer)         96              100
Alternative pathway (in Mg-EGTA buffer)   100             22
a  results expressed in mean percentage 
b  calculated from results in table 1

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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