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Immunology JoVE (Open Access) Citable · DOI

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

DOI: 10.3791/51590-v
What you'll learn
  • ✓ Purify viable P. falciparum merozoites from infected RBCs using high-yield methods
  • ✓ Quantify merozoite concentration and viability by flow cytometry
  • ✓ Measure opsonization-dependent phagocytosis in THP-1 cells
Protocol

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Difficulty
advanced
Total time
~4–6 hours per experiment (merozoite isolation ~2 hrs, assay setup ~2–3 hrs, flow cytometry ~1 hr)
Model organism
Plasmodium falciparum in vitro culture; THP-1 cells (human monocytic leukemia)
Biosafety
BSL-2

Steps

1
Isolate P. falciparum merozoites from infected cultures

Harvest and purify viable merozoites from synchronous P. falciparum-infected red blood cells using density gradient or selective lysis techniques to obtain high-yield, intact parasites.

▶ 01:34
2
Quantify merozoite concentration by flow cytometry

Count and assess viability of purified merozoites using flow cytometric detection of parasite-specific markers and gating strategies to determine working concentration.

▶ 04:42
3
Perform opsonization-dependent phagocytosis assay

Incubate test serum or antibodies with merozoites, then co-culture with THP-1 cells to measure antibody-mediated uptake and phagocytosis by flow cytometry.

▶ 06:03
4
Analyze phagocytosis ratio and infection outcomes

Evaluate how merozoite-to-THP-1 cell ratios affect phagocytosis efficiency and interpret results to correlate antibody opsonization with protective immunity.

▶ 07:48
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