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Currently submitted to: JMIR Research Protocols

Date Submitted: Jul 30, 2019
Open Peer Review Period: Aug 2, 2019 - Sep 27, 2019
(currently open for review)

Identification of native epitopes eliciting a protective high affinity immunoglobulin subclass response to blood stages of Plasmodium falciparum: a protocol for observational studies

  • Michael Eisenhut; 

ABSTRACT

Background:

Antibodies to bloodstages protective against complications of P. falciparum infection were found to be of IgG1 and IgG3 subclass and of high affinity to the target epitopes. Those target epitopes cannot be characterized using recombinant antigens because of lack of appropriate glycosylation, phosphorylation, methylation and bisulfide bond formation, which determine the structure of conformational and non-linear epitopes within the tertiary and quarternary structures of native P. falciparum antigens.

Objective:

To develop a method for comprehensive detection of all P. falciparum schizont antigens, eliciting a protective immune response.

Methods:

To get access to native schizont antigens Purified Parasitophorous Vacuole Membrane Enclosed Merozoite Structures (PEMS) are initially generated and separated by two- dimensional gel electrophoresis and blotted onto nitrocellulose. Antigens eliciting a protective antibody response are visualized by incubation with sera of patients with clinical immunity followed by elution of low affinity antibodies with urea and detection of protective antibody responses by incubation with anti-IgG1 and anti-IgG3 antibodies conjugated to horse radish peroxidase followed by visualisation with a colour reaction. Blot signals are normalised by relating to intensity of blot staining from a reference antibody and housekeeping antigens and related to intensity of exposure by relation of antibody responses to global P. falciparum antibody responses. Antigens eliciting the protective responses are identified as immunorelevant from comparison of spot positions indicating high affinity IgG1 or IgG3 responses on the Western blot unique to or consistently more intensive in clinically immune compared to non-immune.The results obtained are validated by affinity chromatography.

Results:

Another group previously applied two-dimensional Western blotting with serum of patients to analysis of antibody responses to P. falciparum. P. falciparum mature stages (trophozoite and schizont stages)were enriched by Plasmion flotation. Immunoblots were directly digitalized using an Image scanner. The sera allowed detection of 42 antigenic spots on the 2 D immunoblot. The spots detected were excised and submitted to mass spectrometry for identification. Nineteen protein spots (45%) were successfully identified and correspond to 13 distinct proteins. Immuno affinity chromatography used antigens bound by immunoglobulins produced by mice with enhanced immunity to Plasmodium yoelii. Immuno-relevant antigens have hereby been isolated and identified by immobilising immunoglobulin from immune mice to a sephadex column and then passing a blood stage antigen mixture through the column followed by elution of specific bound antigens with sodium deoxycholate and identification of those antigens on Western blotting by specific antibodies.

Conclusions:

Two dimensional Wester blotting using native antigens has the potential for identification of antibody responses selective for specific defined isomeric forms of the same protein including isoforms (“protein species”) generated by post-transcriptional modifications like phosphorylation, glycosylation and methylation. The process involved in two- dimensional Western blotting enables highly sensitive detection and high resolution and preservation of antibody responses during blotting. Validation by immunoaffinity chromatography can compensate for antigen loss through the blotting process and has the potential for exact quantification of antigens eluted possible through mass spectrometry and therefore indirectly quantification of protective antibody responses Clinical Trial: Not applicable


 Citation

Please cite as:

Eisenhut M

Identification of native epitopes eliciting a protective high affinity immunoglobulin subclass response to blood stages of Plasmodium falciparum: a protocol for observational studies

JMIR Preprints. 30/07/2019:15690

DOI: 10.2196/preprints.15690

URL: https://preprints.jmir.org/preprint/15690


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