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Background  Antibodies to blood stages protective against complications of  Plasmodium falciparum  infection were found to be of immunoglobulin G 1 (IgG1) and IgG3 subclasses and of high affinity to the target epitopes. These target epitopes cannot be characterized using recombinant antigens because of a lack of appropriate glycosylation, phosphorylation, methylation, and bisulfide bond formation, which determine the structure of conformational and nonlinear epitopes within the tertiary and quaternary structures of native  P. falciparum  antigens.    Objective  This study aims to develop a method for the comprehensive detection of all  P. falciparum  schizont antigens, eliciting a protective immune response.    Methods  Purified parasitophorous vacuole membrane–enclosed merozoite structures (PEMSs) containing native schizont antigens are initially generated, separated by two-dimensional (2D) gel electrophoresis and blotted onto nitrocellulose. Antigens eliciting a protective antibody response are visualized by incubation with sera from patients with clinical immunity. This is followed by the elution of low-affinity antibodies with urea and detection of protective antibody responses by incubation with anti-IgG1 and anti-IgG3 antibodies, which were conjugated to horseradish peroxidase. This is followed by visualization with a color reaction. Blot signals are normalized by relating to the intensity of blot staining with a reference antibody and housekeeping antigens. Results are corrected for intensity of exposure by the relation of antibody responses to global  P. falciparum  antibody titers. Antigens eliciting the protective responses are identified as immunorelevant from the comparison of spot positions, indicating high-affinity IgG1 or IgG3 responses on the western blot, which is unique to or consistently more intensive in clinically immune individuals compared with nonimmune individuals. The results obtained are validated by using affinity chromatography.    Results  Another group previously applied 2D western blotting to analyze antibody responses to  P. falciparum . The sera of patients allowed the detection of 42 antigenic spots on the 2D immunoblot. The spots detected were excised and subjected to mass spectrometry for identification. A total of 19 protein spots were successfully identified and corresponded to 13 distinct proteins. Another group used immunoaffinity chromatography to identify antigens bound by IgGs produced by mice with enhanced immunity to  Plasmodium yoelii . Immunorelevant antigens were isolated and identified by immobilizing immunoglobulin from immune mice to a Sephadex column and then passing a blood-stage antigen mixture through the column followed by the elution of specific bound antigens with sodium deoxycholate and the identification of those antigens by western blotting with specific antibodies.    Conclusions  2D western blotting using native antigens has the potential to identify antibody responses selective for specific defined isomeric forms of the same protein, including isoforms ( protein species ) generated by posttranscriptional modifications such as phosphorylation, glycosylation, and methylation. The process involved in 2D western blotting enables highly sensitive detection, high resolution, and preservation of antibody responses during blotting. Validation by immunoaffinity chromatography can compensate for the antigen loss associated with the blotting process. It has the potential for indirect quantification of protective antibody responses by enabling quantification of the amount of eluted antibody bound antigens through mass spectrometry.    International Registered Report Identifier (IRRID)  PRR1-10.2196/15690

jmir research protocols

The Identification of Native Epitopes Eliciting a Protective High-Affinity Immunoglobulin Subclass Response to Blood Stages of Plasmodium falciparum: Protocol for Observational Studies