IgG1 and IgG3 antibody reactivity to recombinant ectodomain of
P. falciparum apical merozoite antigen 1(AMA-1), the 11 kDa carboxyl portion of merozoite surface antigen 1 (MSP-1
19), region II of the 175 kDa erythrocytes binding antigen (EBA-175 RII), and two recombinant proteins representing the two major allelic types of MSP-2 was assessed in plasma samples from 41 children (age range = 7 – 107 months, median = 34 months). Levels of IgM reactivity against the two allelic types of MSP-2 were also assessed. The AMA-1 antigen and EBA-175 region II were kind gifts from Sheetij Dutta and Arnoldo Barbosa (WRAIR, Maryland, USA) and have been previously described [
8,
9], while MSP-1
19 and MSP-2 proteins were provided by Jana McBride and David Cavanagh (University of Edinburgh, UK) and have also been previously described [
10,
11]. Plasma was assayed at 1:4000 dilution for antibodies to AMA-1 and at 1:500 for antibodies to the other antigens using ELISA protocols described previously [
3,
11,
12]. Briefly, the wells of 96-well plates (Immulon4; Dynatech, Chantilly, VA) were coated overnight at 4°C with either 50 ng (MSP-1
19 and MSP-2) or 100 ng (EBA-175 and AMA-1) of antigen in sodium carbonate buffer (pH 9.3), washed, and then blocked with 1% skimmed milk in coating buffer for 5 h and washed again. The wells were subsequently incubated overnight at 4°C with 100 μl of plasma diluted in 1% skimmed milk, followed by a three-hour incubation with 100 μl of horseradish peroxidase-conjugated rabbit antibodies against human antibodies (Dako Ltd, Cambridge, UK) at 1/1000 for anti-human IgG1 and IgG3 or 1/5000 for anti-human IgM, with washing between the incubations. Colour was developed with
o-phenylenediamine, the reaction stopped after 15 minutes with 20 μl of 2 M H
2SO
4, and absorbance was measured as optical density (OD) at 492 nm. All samples were simultaneously assayed in duplicate for IgG1 and IgG3 antibodies against a given antigen. Absorbance readings for responses against MSP-1
19 and MSP-2 antigens were corrected for responses to the GST fusion carrier by deducting the absorbance of the same plasma assayed against GST alone. The cut-off for absorbance readings for each antigen was set as mean plus three standard deviations of absorbance readings of sera from eight Europeans without any history of malaria infection. In order to convert absorbance to concentration (μg/ml), plates were coated with purified human myeloma proteins IgG1, and IgG3 (at threefold serial dilutions from 10 to 5.1 × 10
-4 μg/ml).
The resulting standard absorbance versus concentration curves were used for interpolation of the absorbances of the plasma samples to estimate concentrations.