Evaluation of human serological response against RBC infected by RP8 P. falciparum strain by liquid indirect immunofluorescence assay (L-IFA). Representative IgG response against iRBC from a non-exposed individual (NEI, upper row) and a briefly exposed individual (BEI, lower row) to malaria, are shown. Specific antibody response to surface proteins from live iRBC was revealed with alexa 488 goat anti-human IgG antibodies (green). P. falciparum DNA was stained with DAPI (blue), and merged images with bright field (BF) are shown (Magnification × 100).

Schematic representation of experimental workflow for an accurate identification of antigenic protein using 2-D immunoblot with a fluorescence-based method. The different steps are numbered from #1 to #6. Briefly, iRBC membrane protein extracts were pre-labelled with Cy5 cyanine (#1), separated by 2-D electrophoresis (#2), electroblotted onto membranes, probed with the pool from 5 selected BEI sera and incubated with a goat-anti-human IgG FITC-conjugate (#3). Following blot digitalization at Cy5 and FITC wavelengths (#4), two images corresponding respectively to the antigenic protein pattern and the total protein expression profile were obtained. The superimposition of these two images (#5) allowed us to excize accurately spots of interest on preparative gel run in parallel (#3'), before to submit them to mass spectrometry (MS) for identification (#6). Protein expression profile was used as "internal standard" to perform a perfect match between the blot and the preparative gel.

Analysis of immune response against iRBC membrane protein extracts using sera from non-exposed individuals (NEI), briefly exposed individuals (BEI) or highly exposed individuals (HEI) to malaria. IgG immune profiles from 4 NEI, 6 BEI and 2 HEI revealed by immunoblotting against erythrocyte membrane protein extracts from RBC (A), or iRBC (B) are presented. Two human sera, "Ctrl NEI (-)" and "Ctrl HEI (+)", loaded on each blot, were used as controls for antibody revelation and migration quality. MW: molecular weight, kDa: kiloDalton. (C) Comparative analysis of the antigenic bands number recognized by sera from NEI, BEI and HEI against RBC and iRBC membrane protein extracts. For this quantitative analysis, significant differences are indicated for each group tested between the two membrane protein extracts (p < 0.05, Mann-Whitney test). The line within the box represents the mean number of antigenic bands recognized by each sera group. The boundaries of the box indicate the 25th and 75th percentiles. Whiskers above and below the box indicate the maximum and minimum values. (D) Frequency of iRBC membrane antigenic protein bands detected by NEI and BEI. Analysis of IgG profiles using diversity database software (Biorad) allowed us to exhibit 8 antigenic bands from iRBC membrane protein extracts statistically significant (Fisher exact test, *p < 0.05; **p < 0.01, ***p < 0.001) between NEI and BEI groups. Only protein bands significantly different between NEI and BEI and detected by at least 20 percent of the individuals from a group were indicated. (E) These antigenic bands are indicated on a diagram using Roman numeral numbers, and their corresponding molecular weights are indicated in brackets.

Determination of protein spots detected by BEI sera on iRBC membrane protein extracts by 2-D immunoblotting. A 2-D immunoblot using a fluorescence-based method was performed to detect precisely and accurately antigenic protein spots. (A) Protein profile of iRBC membrane protein extracts pre-labelled with Cy5 and separated by 2-D electrophoresis. (B) Protein profile of the same protein sample used in "A" after Cy5 labelling, separation by 2-D electrophoresis and electroblotting onto nitrocellulose membrane. (C) Immunoblot pattern of a pool of 5 selected BEI sera revealed with a goat-anti-human IgG FITC-conjugate against iRBC membrane protein extracts. (D) Merged images of the electrobloted protein profile (e.i., "B") and the immunoblot pattern (e.i., "C"). Antigenic spots detected onto immunoblot (e.i., "C") are ringed and reported onto electrobloted protein profile (e.i., "B") and merged images (e.i., "D"). Antigenic spots which were excized for mass spectrometry (MS) analysis are marked with red or green circles onto gel image and those which were identified are indicated by Arabic numbers (e.i., "A"). Selected antigenic bands are indicated by Roman numbers and positioned according to their MW. Antigenic spots corresponding to selected antigenic bands are indicated by red circles, the others antigenic spots are assigned by green circles (A). A resume of the correspondence between Roman and Arabic numbers is indicated in additional file 2. MW: molecular weight, kDa: kiloDalton.