Robust Antibody Detection ELISA Protocols

To generate the rabbit ELISA data in Figure 1C, PfRH2, PfRH4 and PfEBA175 proteins (described above) were coated on Nunc Maxisorp plates at 100ng/well, or wells of streptavidin-coated plates (Nunc Immobilizer) were saturated with biotinylated Pf38, PfMSP2, PfMSP9, or PfRH5 in PBS and left over-night. The next day plates were washed 6x in PBS containing 0.05% Tween 20 (PBS/T). Plates were blocked with 10% skimmed milk in PBS/T for 1h, dilution series of serum samples (3-fold from 1:300) were added for 2h, before the plates were washed again. Alkaline-phosphatase-conjugated goat anti-rabbit IgG (Sigma) was used for detection. Plates were washed again and bound antibodies were detected by adding p-nitrophenylphosphate substrate (pNPP, Sigma) diluted in diethanolamine buffer (Fisher Scientific, UK). OD 405nm was read 20 minutes after addition of substrate using an ELx800 microplate reader (BioTek, UK). Endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value three standard deviations greater than the OD405 for naïve rabbit serum. Mouse ELISA was conducted using an identical method, with the exception of the use of alkaline-phosphatase-conjugated goat anti-mouse IgG (Sigma) for detection.
To generate the human ELISA data in Figure 4A-B, GST-PfMSP1₁₉ (ETSR 3D7/Mad20) or 3D7 AMA1 (described earlier) protein was coated onto 96 well Nunc-Immuno Maxisorp plates at a concentration of 2μg/mL in PBS and left over-night. The next day plates were washed 6x in PBS containing 0.05% Tween 20 (PBS/T) and blocked for 1h with Casein block solution (Pierce, UK). Plates were washed again, and then a standard, test sera, internal control and blank samples all diluted in Casein block solution were added to each plate for 2h according to published methodology^{15 ,59 (link)}. The standard was prepared from adult Kenyan immune serum and was serially diluted on every plate to make a standard curve. Plates were washed again, followed by addition for 1h of alkaline phosphatase-conjugated goat anti-human IgG (γ-chain) (Sigma) diluted 1:1000 in Casein block solution. Plates were washed and developed as above. The ELISA unit value of the standard was assigned as the reciprocal of the dilution giving an OD₄₀₅ of 1.0 in the standardized assay. The OD₄₀₅ of individual test samples was converted into ELISA units by using the standard curve and Gen5 ELISA software v1.10 (BioTek, UK). All sera tested against the GST control protein were less than the minimal detection level of the assay.
Alternatively, PfRH2, PfRH4 and PfEBA175 proteins were coated on Nunc Maxisorp plates at 100ng/well, or wells of streptavidin-coated plates (Nunc Immobilizer) were saturated with biotinylated Pf38, PfMSP2, PfMSP9, or PfRH5 in PBS and left over-night. All test sera were diluted 1:300 in Casein block solution. Washing, blocking, and detection were as for PfMSP1 and PfAMA1. OD 405nm was measured 35 minutes after substrate addition using an ELx800 microplate reader (BioTek, UK).
To generate the human ELISA data in Figure 4C, streptavidin coated plates (NUNC Immobilizer Streptavidin, Thermo Fisher Scientific Inc) were washed three times with PBS-Tween (PBS-0.1% Tween 20). Individual wells were coated with 100μL of antigen and incubated for 45min at room temperature (RT). Plates were then washed four times with Hepes-buffered saline-Tween (HBS-Tween:0.14M NaCl, 5mM KCl, 2mM CaCl₂, 1mM MgCl₂, 10mM HEPES, 0.1% Tween 20) before incubation with 100μL of test serum sample at a 1/1000 dilution (in HBS-Tween) for 1h at RT. Wells were then washed four times, with HBS replacing HBS-Tween in the final wash. Plates were then incubated for 1h at RT with 100μL of horseradish peroxidase-conjugated rabbit anti-human IgG (Dako Ltd.) at a 1/5000 dilution in HBS-Tween before the final wash and detection with H₂O₂ and o-phenylenediamine (Sigma). The reaction was stopped with 25μL of 2M H₂SO₄ per well, and absorbance was read at 492 nm. Identical positive controls (hyperimmune sera) were run in duplicate on each day of the experiment, on each plate, to allow for standardization of day-to-day and plate-to-plate variations. Twenty sera from UK residents, never exposed to malaria, were used as negative controls.