Serological Profiling for Malaria Surveillance Using a Standard ELISA Protocol

Abstract

The enzyme-linked immunosorbent assay (ELISA) is a reliable and relatively low-cost method for measuring soluble ligands such as antibodies and proteins in biological samples. For analysis of specific antibodies in serum, a capture antigen is immobilized onto a solid polystyrene surface from which it can capture the antibodies. The captured antibodies are subsequently detected using a secondary antibody conjugated to an enzyme. Detection is accomplished by addition of a colorimetric substrate, and the readout is absorbance (optical density). Here, we provide a detailed standardized ELISA protocol for the quantification of antibodies against malaria antigens.

Keywords: Antibodies; Antigens; ELISA; Optical density; Serum.

Fig. 1

The graph shows the optical density readings of pools of sera from malaria-exposed (red) and nonexposed donors (blue) at different concentrations of the coating antigen. The optimal coating concentration is shown by the black dotted line and corresponds to the maximum OD reading with low background and least antigen concentration

Fig. 2

A checkerboard titration of a pool of malaria-exposed sera (1:500–1:100,000) and four secondary antibody dilutions: 1:5000 (red), 1:4000 (black), 1:2500 (green), and 1:1000 (orange). Serial dilutions of a pool of malaria nonexposed sera were also tested at 1:5000 secondary antibody dilution (gray). The appropriate serum dilution is shown by the vertical dotted line and clearly differentiates between the positive and the negative sera. The optimal secondary antibody dilution (horizontal dotted line) corresponds to the OD reading where the signal is strongest and the background is low