A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals
<p>Abstract</p> <p>Background</p> <p>Antibodies play a central role in naturally acquired immunity against <it>Plasmodium falciparum</it>. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2012-11-01
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| Series: | Malaria Journal |
| Subjects: | |
| Online Access: | http://www.malariajournal.com/content/11/1/367 |
| _version_ | 1811283345400659968 |
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| author | Ajua Anthony Engleitner Thomas Esen Meral Theisen Michael Issifou Saadou Mordmüller Benjamin |
| author_facet | Ajua Anthony Engleitner Thomas Esen Meral Theisen Michael Issifou Saadou Mordmüller Benjamin |
| author_sort | Ajua Anthony |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Antibodies play a central role in naturally acquired immunity against <it>Plasmodium falciparum</it>. 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.</p> <p>Methods</p> <p>Fixed red blood cells (RBCs), infected with late stages of <it>P</it>. <it>falciparum</it> 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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p> |
| first_indexed | 2024-04-13T02:09:50Z |
| format | Article |
| id | doaj.art-e6df35e18d054f2e8d41c125c8e5925a |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-04-13T02:09:50Z |
| publishDate | 2012-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Malaria Journal |
| spelling | doaj.art-e6df35e18d054f2e8d41c125c8e5925a2022-12-22T03:07:21ZengBMCMalaria Journal1475-28752012-11-0111136710.1186/1475-2875-11-367A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individualsAjua AnthonyEngleitner ThomasEsen MeralTheisen MichaelIssifou SaadouMordmüller Benjamin<p>Abstract</p> <p>Background</p> <p>Antibodies play a central role in naturally acquired immunity against <it>Plasmodium falciparum</it>. 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.</p> <p>Methods</p> <p>Fixed red blood cells (RBCs), infected with late stages of <it>P</it>. <it>falciparum</it> 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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p>http://www.malariajournal.com/content/11/1/367MalariaFlow cytometry-based IFAAlgorithmic data analysisAnti-malarial antibodiesHuman serum |
| spellingShingle | Ajua Anthony Engleitner Thomas Esen Meral Theisen Michael Issifou Saadou Mordmüller Benjamin A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals Malaria Journal Malaria Flow cytometry-based IFA Algorithmic data analysis Anti-malarial antibodies Human serum |
| title | A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals |
| title_full | A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals |
| title_fullStr | A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals |
| title_full_unstemmed | A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals |
| title_short | A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals |
| title_sort | flow cytometry based workflow for detection and quantification of anti plasmodial antibodies in vaccinated and naturally exposed individuals |
| topic | Malaria Flow cytometry-based IFA Algorithmic data analysis Anti-malarial antibodies Human serum |
| url | http://www.malariajournal.com/content/11/1/367 |
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