Skip to main content
main-content

01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Anthony Ajua, Thomas Engleitner, Meral Esen, Michael Theisen, Saadou Issifou, Benjamin Mordmüller
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-367) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

BM conceived the study and directed the experimental work. AA performed the experimental work shown in this paper. TE, BM and AA analysed the data. AA, TE and BM wrote the paper. ME collected samples and performed ELISA experiments. MT invented the GMZ2 vaccine and donated antigens for use in ELISA. SI contributed to the study design and reviewed the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, although they provide important information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for quantitative detection of anti-plasmodial antibodies in human serum is presented.

Methods

Fixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria vaccine trials in semi-immune adults and pre-school children residing in a malaria endemic area.

Results

Fixation, permeabilization, and staining of infected RBCs were adapted for best operation in flow cytometry. As asexual blood-stage vaccine candidates are designed to induce antibody patterns similar to those in semi-immune adults, serial dilutions of sera from heavily exposed individuals were compared to naïve controls to determine optimal antibody dilutions. To eliminate investigator effects introduced by manual gating, a non-biased algorithm (OSA) for data-driven gating was developed. OSA-derived results correlated well with those obtained by manual gating (r between 0.79 and 0.99) and outperformed other model-driven gating methods. Bland-Altman plots confirmed the agreement of manual gating and OSA-derived results. A 1.33-fold increase (p=0.003) in the number of positive cells after vaccination in a subgroup of pre-school children vaccinated with 100 μg GMZ2 was present and in vaccinated adults from the same region we measured a baseline-corrected 1.23-fold, vaccine-induced increase in mean fluorescence intensity of positive cells (p=0.03).

Conclusions

The current workflow advances detection and quantification of anti-plasmodial antibodies through improvement of a bias-prone, low-throughput to an unbiased, semi-automated, scalable method. In conclusion, this work presents a novel method for immunofluorescence assays in malaria research.
Zusatzmaterial
Authors’ original file for figure 1
12936_2012_2595_MOESM1_ESM.pdf
Authors’ original file for figure 2
12936_2012_2595_MOESM2_ESM.pdf
Authors’ original file for figure 3
12936_2012_2595_MOESM3_ESM.pdf
Authors’ original file for figure 4
12936_2012_2595_MOESM4_ESM.pdf
Authors’ original file for figure 5
12936_2012_2595_MOESM5_ESM.pdf
Authors’ original file for figure 6
12936_2012_2595_MOESM6_ESM.pdf
Authors’ original file for figure 7
12936_2012_2595_MOESM7_ESM.pdf
Literatur
Über diesen Artikel

Weitere Artikel der Ausgabe 1/2012

Malaria Journal 1/2012 Zur Ausgabe

Neu im Fachgebiet Innere Medizin

Meistgelesene Bücher aus der Inneren Medizin

2017 | Buch

Rheumatologie aus der Praxis

Entzündliche Gelenkerkrankungen – mit Fallbeispielen

Dieses Fachbuch macht mit den wichtigsten chronisch entzündlichen Gelenk- und Wirbelsäulenerkrankungen vertraut. Anhand von über 40 instruktiven Fallbeispielen werden anschaulich diagnostisches Vorgehen, therapeutisches Ansprechen und der Verlauf …

Herausgeber:
Rudolf Puchner

2016 | Buch

Ambulant erworbene Pneumonie

Was, wann, warum – Dieses Buch bietet differenzierte Diagnostik und Therapie der ambulant erworbenen Pneumonie zur sofortigen sicheren Anwendung. Entsprechend der neuesten Studien und Leitlinien aller wichtigen Fachgesellschaften.

Herausgeber:
Santiago Ewig

Mail Icon II Newsletter

Bestellen Sie unseren kostenlosen Newsletter Update Innere Medizin und bleiben Sie gut informiert – ganz bequem per eMail.

© Springer Medizin 

Bildnachweise