Complement-fixation by antibodies to Plasmodium falciparum

doi:10.21203/rs.3.pex-2510/v1

Method Article

Complement-fixation by antibodies to Plasmodium falciparum

https://doi.org/10.21203/rs.3.pex-2510/v1

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In malaria, the fixation and activation of serum complement proteins by antibodies can support immunity by mediating parasite lysis, inhibiting host cell motility and invasion and promoting phagocytosis. Antibodies, bound to malaria antigens, may bind to C1q via their Fc region, initiating the classical complement cascade; C1q is the first step in the classical activation pathway. This protocol measures C1q-fixation by antibodies that target whole Plasmodium falciparum parasites, which can include merozoites, sporozoites, or gametocytes/gametes. The procedure is similar to an ELISA and has the advantage of having high reproducibility, good quantification, and is amenable to high throughput.

complement-fixation

complement

C1q

ELISA

merozoite

malaria

Plasmodium falciparum

antibody

IgG

Reagents and equipment:

Freshly isolated whole merozoites (1 x 106 parasites/mL), or other parasite stages (sporozoites or gametes)
96-well flat bottom Maxisorp plates (NUNC Thermo Scientific #442404)
1X phosphate-buffered saline (PBS)
PBS-Tween 0.05% (1X PBS with 0.05% Tween 20)
10% skim milk-PBS
5% skim milk-PBS
Purified human C1q (e.g. Complement C1q, Human (Millipore #204876))
Rabbit anti-C1q antibody (e.g. Beeson lab in-house reagent OR C1q complement antibody (Dako #A0136))
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (e.g. Goat Anti-Rabbit IgG Antibody (Millipore #AP307P))
Substrate (TMB (Life technologies #00-2023)
Sulphuric acid (H2SO4) 1M
Spectrophotometer (e.g. Multiskan GO Microplate Spectrophotometer (Thermo Scientific))

Coat 96-well flat bottom plate with 50 μL/well freshly isolated whole merozoites (or sporozoites, or gametocytes/gametes) diluted in 1X PBS at 1 x 106 parasites/mL (50 000 merozoites/well) and incubate at 37˚C for 2 hours.
Wash three times using PBS-Tween 0.05%.
Block plate using 10% skim milk-PBS (200 μL/well) for 2 hours at 37˚C.
Wash plate three times using PBS-Tween 0.05%.
Add 50 μL/well serum samples, diluted 1/200 in 5% skim milk-PBS, in duplicate and incubate for 2 hours at room temperature (RT).
Wash plate three times using PBS-Tween 0.05%.
Dilute purified human C1q to 10 μg/mL in 5% skim milk-PBS. Add 30 μL to each well and incubate for 30 mins at RT.
Wash plate three times using PBS-Tween 0.05%.
Dilute rabbit anti-C1q antibody at 1/2000 in 5% skim milk-PBS and add 50 μL/well. Incubate for 1 hour at RT.
Wash plate three times using PBS-Tween 0.05%.
Dilute goat anti-rabbit IgG HRP at 1/2000 in 5% skim milk-PBS and add 50 μL/well. Incubate HRP-conjugated antibody for 1 hour at RT.
Wash plate three times using PBS-Tween 0.05%.
Add 50 μL of TMB substrate into each well and incubate in the dark for 1 hour at RT. (Substrate works best at RT, remove from fridge 1 hour prior to use.)
Stop the reaction using 1M Sulphuric acid (50 μL/well).
Measure absorbance at 450 nm for TMB.

The dilution of serum samples should be optimised for each cohort. Positive serum controls and negative controls (such as malaria-naïve serum and no-serum control wells) should be included on each plate to correct for background and inter-assay variation.

We use our own in-house anti-C1q antibody. If using a C1q antibody from a commercial supplier, it will require work-up assays to ensure specificity and sensitivity.

Coating of wells with whole parasites will need to be optimised depending on the source of cells.

1. Reiling L, Boyle MJ, White MT, Wilson DW, Feng G, Weaver R, Opi DH, Persson KEM, Richards JS, Siba PM, Fowkes FJI, Takashima E, Tsuboi T, Mueller I, Beeson JG. Targets of complement-fixing antibodies in protective immunity against malaria in children. Nature Commun. 2019 Feb 5;10(1):610.

2. Boyle MJ, Reiling L, Feng G, Langer C, Osier FH, Aspeling-Jones H, Cheng YS, Stubbs J, Tetteh KK, Conway DJ, McCarthy JS, Muller I, Marsh K, Anders RF, Beeson JG. Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria. Immunity. 2015 Mar 17;42(3):580-90.

3. Kurtovic L, Behet MC, Feng G, Reiling L, Chelimo K, Dent AE, Mueller I, Kazura JW, Sauerwein RW, Fowkes FJI, Beeson JG. Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children. BMC Med. 2018 Apr 30;16(1):61.

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Complement-fixation by antibodies to Plasmodium falciparum

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Complement-fixation by antibodies to Plasmodium falciparum

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