Malaria Immunology Targeting the Surface of Infected Erythrocytes

This volume covers a broad range of methods, technologies, and protocols on malaria. Chapters detail research on collecting parasites in the field, single molecule-level analyses of adhesive interactions, and focused studies aiming at disrupting the devastating disease. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

 Authoritative and cutting-edge, Malaria Immunology: Targeting the Surface of Infected Erythrocytes aims to be a useful and practical guide to researches to help further their study in this field. 

Chapter Analysis of var gene transcription pattern using DBLa-tags [Chapter 14] is available open access under a Creative Commons Attribution 4.0  International License via link.springer.com.

 Identification and enumeration of Plasmodium falciparum parasites by light microscopy.- Collection and cryopreservation of Plasmodium falciparum clinical isolates in the field.- Preservation of parasite RNA in the field.- Preservation and extraction of malaria parasite DNA from dried blood spots.- Establishing and maintaining in vitro cultures of asexual blood stages of Plasmodium falciparum.- Genotyping of Plasmodium falciparum isolates to assess clone composition in parasite cultures.- Immunomagnetic selection of Plasmodium falciparum-infected erythrocytes expressing particular PfEMP1 variants.- Single-cell sorting of Plasmodium falciparum-infected erythrocytes expressing particular PfEMP1 variants.- Selecting Plasmodium falciparum infected erythrocytes for adhesion to cell lines.- Synchronisation of Plasmodium falciparum and P. knowlesi in vitro cultures using a highly specific protein kinase inhibitor.- Generation of Plasmodium falciparum gametocytes in vitro with specific considerations for field isolates.- Imaging of extracellular vesicles derived from Plasmodium falciparum-infected red blood cells using atomic force microscopy.- Analysis of var gene transcript patterns by quantitative real-time PCR.- Analysis of var gene transcription pattern using DBLa-tags.- RNAseq of infected erythrocyte surface antigen-encoding genes.- Resetting var gene transcription in Plasmodium falciparum.- CRISPR/Cas9 editing of the Plasmodium falciparum genome.- CRISPR-Cas9 editing of the Plasmodium falciparum genome - Special applications.- Extraction and immunoprecipitation of VAR2CSA, the PfEMP1 associated with placental malaria.- Expression of single-domain soluble and disulfide-folded PfEMP1 antigens in the Escherichia coli SHuffle expression system.- Expression of large full-length PfEMP1 proteins in HEK293 cells.- Receptor affinity-based purification of PfEMP1 proteins.- Analysis of antibody reactivity to malaria antigens by microsphere-based multiplex immunoassay.- High-throughput BioPlex assay for the study of functionally active Plasmodium falciparum antigens that are expressed on the surface of infected erythrocytes.- Protein microarrays as a tool to analyze antibody responses to variant surface antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes.- Protocol for differential biopanning of Plasmodium falciparum phage display cDNA library to identify parasite targets of protective antibodies.- Affinity purification of PfEMP1-specific antibodies from human blood.- Production of anti-PfEMP1 polyclonal antisera in rats and mice.- Production of PfEMP1-specific mouse monoclonal antibodie.- Production of PfEMP1-specific human monoclonal antibodies from naturally immune individuals.- Visualization of infected red blood cell surface antigens by fluorescence microscop.- Analysis by flow cytometry of a2-macroglobulin and non-immune IgM-binding to Plasmodium falciparum-infected erythrocytes.- An improved method for assessing antigen presentation on the surface of Plasmodium falciparum-infected erythrocytes by immuno-electron microscopy.- Assessing antigen presentation on the surface of Plasmodium falciparum-infected erythrocytes by photoactivated localization microscopy (PALM).- Surface plasmon resonance analysis of PfEMP1 interaction with receptors.- Analysis of antibody neutralization of PfEMP1 binding by competition ELISA.- Measuring rosetting inhibition in Plasmodium falciparum parasites using a flow cytometry-based assay.- Bridging and clumping: investigating platelet interactions with Plasmodium falciparum-infected red blood cells and endothelial cells in cerebral malaria.- Assay of static adhesion of Plasmodium falciparum-infected erythrocytes to cells, including inhibition of the adhesion.- Static adhesion of Plasmodium falciparum-infected erythrocytes to purified and recombinant receptors.- Selecting Plasmodium falciparum-infected erythrocytes for adhesion to recombinant receptors under flow conditions.- Chip-based assay of adhesion of Plasmodium falciparum-infected erythrocytes to cells under flow.- Binding of Plasmodium falciparum-infected red blood cells to engineered 3D microvessels.- 3D organoid assay of the impact of Plasmodium falciparum-infected erythrocyte adhesion on the blood-brain barrier.- Receptor-functionalized lipid membranes as biomimetic surfaces for adhesion of Plasmodium falciparum-infected erythrocytes.- Antibody-dependent THP-1 cell-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes.- Assaying interactions between neutrophils and Plasmodium falciparum-infected red blood cells.- Natural killer cell antibody dependent cellular cytotoxicity (ADCC) activity against Plasmodium falciparum-infected red blood cells.- Assessing PfGARP-mediated apoptosis of blood-stage Plasmodium falciparum parasites.- Enzyme-linked immunosorbent assay for activation of the classical complement pathway by Plasmodium falciparum-infected erythrocyte surface antigen-specific antibodies.- Flow cytometry assay of Plasmodium falciparum-specific B-cell proportions.- Assessment of Plasmodium falciparum antigen-specific B cells.- Whole blood dendritic cell cytokine production assay.- Measuring the impact of malaria on the living human retina.- Ocular pathology of cerebral malaria.- Investigating interactions between endothelial cells and parasitised red blood cells in skin and subcutaneous tissue.