Quantifying antibody binding to Fcγ-receptors in malaria immunity

doi:10.21203/rs.3.pex-2509/v1

Method Article

Quantifying antibody binding to Fcγ-receptors in malaria immunity

https://doi.org/10.21203/rs.3.pex-2509/v1

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IgG can opsonise pathogens and mediate binding to Fcγ-receptors (FcγRs) expressed by various innate immune cells to promote antibody-mediated pathogen clearance via mechanisms including opsonic phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). High levels of IgG that bind FcγRs has been associated with protection from malaria. This assay measures FcγR-binding by antigen-specific IgG using recombinant FcγRs in a plate-based method which is suitable for high throughput. The method is suitable for using recombinant proteins or whole malaria parasites in the assays. FcγRIIa and FcγRIIIa/b are low affinity receptors and need to be used as ectodomain dimers in assays in order to achieve good activity in the assays. FcγRI can be used as a monomeric protein.

Fcγ-receptor

Fcγ receptors

cellular cytotoxicity

IgG

phagocytosis

antibody

malaria

Reagents and equipment:

High-binding flat bottom plate (e.g. 96-well flat bottom Maxisorp plates (NUNC Thermo Scientific #442404)
1% BSA in 1X PBS: dissolve 10 g of bovine serum albumin (BSA) in 1L 1X phosphate-buffered saline (PBS), stir at room temperature (RT) until completely dissolved. Filter-sterilise and store at 4°C.
Antigen
37°C incubator
PBS-Tween 0.05%: 1X PBS with 0.05% Tween-20 (v/v)
FcγRI conjugated to biotin (from commercial suppliers); FcγRIIa or FcγRIIIa conjugated to biotin (expressed and purified in-house at Burnet Institute)
Streptavidin conjugated horseradish-peroxidase (HRP) (e.g. Pierce High Sensitivity Streptavidin-HRP (Thermo Scientific #21130))
Substrate (TMB (Life technologies #00-2023) OR ABTS (Life technologies #00-2024))
Sulfuric acid (if using TMB) OR SDS (if using ABTS)
Spectrophotometer (e.g. Multiskan GO Microplate Spectrophotometer (Thermo Scientific))

Coat wells with 50 µL antigen (1 µg/mL). Incubate overnight at 4°C. (Assays can also be performed coating wells with whole parasites, including merozoites, sporozoites, or gametocytes/gametes)
Discard supernatant. Wash well 4 times with PBS-Tween 0.05%. Discard any remaining wash buffer from wells.
Block plate with 200 μL of 1% BSA in PBS and incubate at 37°C for 2 hours.
Wash 4 times with PBS-Tween 0.05%.
Add 50 µL of test serum/antibody, diluted in 1% BSA in PBS (optimisation of concentration may be necessary), to each well and incubate either for 1 hour at 37°C, for 2 hours at room temperature or overnight at 4°C.
Wash 4 times with PBS-Tween 0.05%.
Add 50 µL of FcγRIIa (0.2 ug/mL) or 50 µL of FcγRIIIa (0.1 ug/mL) or FcγRI (0.125 μg/ml), diluted in 1% BSA-PBS. Incubate at 37°C for 1 hour. NOTE – concentrations of FcγRs used may need to be optimised for the specific study purpose.
Wash 4 times with PBS-Tween 0.05%.
Add 50 µL of streptavidin-HRP, diluted 1:10,000 in 1% BSA in PBS, and incubate at 37°C for 1 hour.
Wash 4 times with PBS-Tween 0.05%.
Add 50 µL of substrate (TMB or ABTS) to each well. Incubate in the dark at room temperature while signal develops (usually 10 – 60 minutes).
Stop reaction by adding 50 µL of stopping solution (1M sulfuric acid for TMB, 1% SDS for ABTS) to each well. Read optical density immediately after stopping. TMB absorbance is measured at 450 nm, while ABTS absorbance is measured at 405 nm.

1. Feng G, Kurtovic L, Agius PA, Aitken EH, Sacarlal J, Wines BD, Hogarth PM, Rogerson SJ, Fowkes FJI, Dobaño C, Beeson JG. Induction, decay, and determinants of functional antibodies following vaccination with the RTS,S malaria vaccine in young children. BMC Med. 2022 Aug 25;20(1):289.

2. Feng G, Wines BD, Kurtovic L, Chan JA, Boeuf P, Mollard V, Cozijnsen A, Drew DR, Center RJ, Marshall DL, Chishimba S, McFadden GI, Dent AE, Chelimo K, Boyle MJ, Kazura JW, Hogarth PM, Beeson JG. Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites. Nature Commun. 2021 Mar 19;12(1):1742.

3. Wines, B.D., Vanderven, H.A., Esparon, S.E., Kristensen, A.B., Kent, S.J., and Hogarth, P.M. (2016). Dimeric FcgammaR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG. J Immunol 197, 1507-1516.

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Quantifying antibody binding to Fcγ-receptors in malaria immunity

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