Experimental infection and protection against ... - TI Pharma

86 Chapter 4
Concentration
(mg/ml, range)
Rabbit 1.31 (0.58 – 2.98)
Human
0.066 (0.007 - 0.50)
Mean ka
(1/Ms, range)
1.26x10 5
(9.1x10 3 – 3.7x10 5 )
1.50 x 10 5
(1.19x10 4 – 6.22 x 10 5 )
Mean kd
(1/s, range)
2.80x10 -3
(1.10 x10 -3 – 5.53 x10 -3 )
2.65 x 10 -3
(2.41 – 9.45 x 10 -3 )
Table 1. Mean antibody concentration, association constant (ka) and
dissociation constant (kd) for rabbit (n=38) and human (n=72) sera.
1/Ms = per Molar per second, 1/s = per second.
150 RU. The abovementioned dilution series of samples was injected at 30
µl/min. An example of a Biacore sensorgram is provided in figure 2. The
association rate constant (ka) and the dissociation rate constant (kd) were
obtained by fitting the sensorgrams with the use of BIAevaluation software 3.2
(Biacore International SA, Uppsala, Sweden). Curves were fitted according to a
simple 1:1 Langmuir binding model, with correction for a linear drifting baseline
to adjust for slow dissociation of the Pf4mH coat from anti-myc antibodies.
Rabbit sera and human sera were each measured using one chip.
Growth inhibition assay
Antibodies used for parasite inhibition assays were purified on protein A
columns (Immunopure Plus Pierce, St Louis, MO, USA) using standard protocols,
exchanged into RPMI 1640 using Amicon Ultra-15 concentrators (30 kDa cut-off,
Millipore, Ireland), filter-sterilised and stored at -20 o C until use. IgG
concentrations were determined using a Nanodrop ND-1000 spectrophotometer
(Nanodrop Technologies, Wilmington, DE, USA).
Pf strain FCR3 was cultured in vitro using standard Pf culture techniques in an
atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 (accession no. M34553)
differs by one amino acid in the pro-sequence from FVO AMA1 (accession no.
AJ277646).
The effect of purified IgG antibodies on parasite invasion was evaluated in
triplicate using 96-well flat-bottom plates (Greiner Bio-One, Alphen a/d Rijn, The
Netherlands) with synchronized cultures of Pf schizonts at a starting parasitemia
of 0.2-0.4% and a haematocrit of 2.0% in a final volume of 100 µL containing
10% control non-immune human serum, 20 µg /ml gentamicin in RPMI 1640 and
5 mg/mL purified IgG. After 40 to 42 hours, cultures were resuspended, and 50
µL was transferred into 200 µL ice-cold PBS. The cultures were then centrifuged,
the supernatant removed and the plates were frozen. Inhibition of parasite
growth was estimated using the pLDH assay as previously described [13].