Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to <em>Plasmodium falciparum</em> Parasites by a Flow Cytometry-Based Assay

Maria del Pilar Quintana; Nsoh  Godwin Anabire; Lars Hviid

doi:10.3791/61538

Medindo anticorpos indutores de fagocitose naturalmente adquiridos para parasitas plasmodium ...

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Immunology and Infection

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JoVE Journal Immunology and Infection

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Medindo anticorpos indutores de fagocitose naturalmente adquiridos para parasitas plasmodium falciparum por um ensaio baseado em citometria de fluxo

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09:57 min

August 6, 2020

DOI: 10.3791/61538-v

Maria del Pilar Quintana¹, Nsoh Godwin Anabire^1,2,3, Lars Hviid^1,4

¹Centre for Medical Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences,University of Copenhagen, ²West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology,University of Ghana, ³Department of Immunology,Noguchi Memorial Institute for Medical Research, ⁴Centre for Medical Parasitology, Department of Infectious Diseases,Rigshospitalet

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Overview

This protocol provides a method to measure the capacity of antibodies in sera or plasma to opsonize and induce phagocytosis of Plasmodium falciparum infected erythrocytes. It is applicable to antibodies from individuals naturally exposed to the infection as well as those induced by immunization.

Key Study Components

Area of Science

Immunology
Parasitology
Cell Biology

Background

Understanding antibody responses to Plasmodium falciparum is crucial for vaccine development.
Phagocytosis is a key immune response against infected cells.
This protocol allows for the assessment of antibody functionality in vitro.
THP-1 cells are commonly used for phagocytosis assays.

Purpose of Study

To evaluate the opsonization capacity of antibodies against infected erythrocytes.
To compare responses from naturally exposed individuals and those vaccinated.
To provide a standardized method for measuring antibody-mediated phagocytosis.

Methods Used

Culture of THP-1 cells for phagocytosis assays.
Preparation of Plasmodium falciparum infected erythrocytes.
Blocking of 96-well plates for the assay.
Measurement of phagocytosis efficiency using flow cytometry or microscopy.

Main Results

Demonstration of antibody opsonization in vitro.
Comparison of phagocytic activity between different antibody sources.
Insights into the immune response to Plasmodium falciparum.
Potential implications for vaccine efficacy evaluation.

Conclusions

This protocol is a valuable tool for studying antibody functions.
It can help identify effective vaccine candidates.
Further research can build on these findings to enhance malaria control strategies.

Frequently Asked Questions

What is the significance of opsonization?

Opsonization enhances the recognition and ingestion of pathogens by phagocytes, crucial for effective immune responses.

How are THP-1 cells used in this protocol?

THP-1 cells serve as a model for human monocytes to study phagocytosis of infected erythrocytes.

What are the expected outcomes of this study?

The study aims to quantify the ability of antibodies to promote phagocytosis, providing insights into immune responses.

Can this protocol be adapted for other pathogens?

Yes, the methodology can be modified to assess antibody functions against various pathogens.

What role do antibodies play in malaria immunity?

Antibodies can neutralize parasites and facilitate their clearance by immune cells, contributing to protective immunity.

Is this protocol suitable for clinical research?

Yes, it can be used to evaluate antibody responses in clinical samples from malaria patients or vaccine trials.

O objetivo geral deste protocolo é fornecer instruções sobre como medir a capacidade dos anticorpos presentes no soro ou plasma de indivíduos, naturalmente expostos à infecção por Plasmodium falciparum, para opsonizar e induzir fagocitose dos eritrócitos infectados por parasitas (IEs).

Este protocolo permite medir a capacidade dos anticorpos de opsonizar e induzir a fagocitose de eritrócitos infectados pelo plasmodium falciparum. Podem ser testados anticorpos presentes no soro de indivíduos naturalmente expostos à infecção por parasitas, bem como aqueles induzidos pela imunização com antígenos parasitas. Demonstrando o procedimento comigo estará Maiken Visti, um técnico do meu laboratório.

Para criar uma cultura celular THP-1 para um ensaio de fagocitose, um dia antes do experimento semeou as células em um frasco de cultura ao quadrado de 25 centímetros e uma concentração de 2,5 vezes 10 a 5 células por mililitro em THP um meio de cultura celular. No dia do experimento, pelo menos uma hora antes do bloco de experimentos para placas de fundo redondo de 96 poços com 150 microliters de FBS estéreis de 2%em PBS por poço. Para a colheita e purificação de eritrócitos infectados por trophozoitas em estágio médio e tardio em 10 mililitros de cultura parasita, com pelo menos 5% de parasitas e adicionar a uma coluna magnética.

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