ELISA Assay for IgG Reactivity

IgG reactivity against recombinant proteins was measured by means of enzyme-linked immunosorbent assay (ELISA) as described elsewhere [20 (link)]. Briefly, plasma samples (1:400) followed by horseradish peroxidase–conjugated rabbit anti-human IgG (1:3000; Dako) were added to 96-well flat-bottom microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific) previously coated with recombinant proteins. Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction stopped with 0.2 mol/L sulfuric acid. The optical density was read at 450 nm (HiPo MPP-96 microplate reader; Molecular Devices), and the specific antibody levels were calculated in arbitrary units, as described elsewhere [20 (link)]. Plasma samples from Danish adults without malaria exposure and a pool of Ghanaian adults with previous P. falciparum infection were included as negative and positive controls, respectively. Negative cutoff values were calculated as the mean arbitrary unit values plus 2 standard deviations obtained with the negative control samples.

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Oleinikov A.V., Seidu Z., Oleinikov I.V., Tetteh M., Lamptey H., Ofori M.F., Hviid L, & Lopez-Perez M. (2023). Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1–Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C. The Journal of Infectious Diseases, 229(1), 203-213.

Publication 2023

horseradish peroxidase–conjugated rabbit anti-human IgG Nunc MaxiSorp

Corresponding Organization : University of Copenhagen

Other organizations : Florida Atlantic University, University of Ghana, Noguchi Memorial Institute for Medical Research, Kwame Nkrumah University of Science and Technology, Rigshospitalet

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Variable analysis

independent variables

Recombinant proteins used to coat the 96-well plates

dependent variables

IgG reactivity against recombinant proteins, measured by ELISA and calculated in arbitrary units

control variables

Plasma samples (1:400 dilution)
Horseradish peroxidase–conjugated rabbit anti-human IgG (1:3000 dilution)
TMB PLUS2 for detection
Sulfuric acid to stop the reaction
Optical density read at 450 nm

controls

Negative control: Plasma samples from Danish adults without malaria exposure
Positive control: Pool of Ghanaian adults with previous P. falciparum infection

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