Fig 7. Quantities of Br-LPS interacting with PMNs.

The quantities of Br-LPS associated to PMNs were determined by Western blotting using a monoclonal antibody against Br-LPS conjugated with peroxidase enzyme. All wells were loaded with 15 μL of the respective preparation. The amounts of purified Br-LPS in the left panel were used to estimate the quantities based on a standard curve ranging from 0.06 ng to 12 ng (only wells from 1.3–6 ng are shown). The right panel corresponds to the assay: purified PMNs were incubated with Br-LPS and the associated amounts determined by Western blot (Br-LPS+PMNs). In order to have a saturating positive control, the assay was also performed in the presence of human antibodies against Br-LPS (Br-LPS+PMNs+Ac). Controls included the assay performed with Br-LPS in the absence (C₁) or presence of human antibodies (C₂) but in the absence of PMNs. PMNs alone did not show any signal. Notice that the Br-LPS molecules associated to PMNs corresponded to the lower molecular weight fraction (~30–40 MW). The amounts of Br-LPS were estimated to be in the range of 5–25 ng/10⁶ PMNs, corresponding to less than 0.25% of the original Br-LPS added. The amounts of associated Br-LPS in the presence of antibodies were between 10–50 ng/10⁶ PMNs. These estimated quantities were from four different experiments. The read-out of the corresponding bands was performed by densitometry.