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Evaluation of immuno-rolling circle amplification for multiplexed detection and profiling of antigen-specific antibody isotypes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Antibody characterization has become essential for diagnosis and development of therapeutic solutions in autoimmune, cardiovascular and infectious diseases. The specificity, affinity and isotype are crucial information for antibody studies and can be obtained from screening plasma samples or populations of B cells. Current technologies are mainly focusing on the discovery of abundant immunoglobulins, namely IgG, and are based on bulk measurements. In this study, we present a digital screening platform utilizing rolling circle amplification (RCA) for the detection of antigen-specific antibody isotypes in solution or secreted from single cells. To validate this approach, the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) was used as model disease and anti-ADAMTS13 antibodies were the target molecules. Antibody-oligonucleotide conjugates (AOCs) were designed for the multiplexed detection of human antibody isotypes IgA, IgG and IgM. Then, ADAMTS13 fragments were coated on glass slides and subsequently, target antibodies identified by specific AOC binding and visualized via an RCA assay. First, we validated the method by characterizing the assay specificity and limit of detection (LoD). When seeding different isotypes, we confirmed the high specificity of the assay (> 90%) and detection of monoclonal anti-ADAMTS13 IgG down to 0.3 ng/mL. A dilution series of a plasma sample of iTTP patient confirmed the multiplexed detection of the three isotypes with higher sensitivity compared to ELISA. Finally, we performed single cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving, achieving a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.

National Category
Immunology Biochemistry Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-185977OAI: oai:DiVA.org:su-185977DiVA, id: diva2:1478016
Available from: 2020-10-21 Created: 2020-10-21 Last updated: 2025-02-20Bibliographically approved
In thesis
1. Advancing isothermal nucleic acid amplification tests: Towards democratization of diagnostics
Open this publication in new window or tab >>Advancing isothermal nucleic acid amplification tests: Towards democratization of diagnostics
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Modern healthcare is the result of scientific advancement across disciplines and has enabled us to understand the rationale behind many diseases and how to treat or cure them; but still a myriad of unanswered questions remains. Especially infectious diseases play an important role in healthcare as they pose a constant threat for global health and well-being. This was painfully highlighted in this year's ongoing COVID-19 pandemic with more than 40 million people infected and over 1 million deaths. Pandemics like this have not only devastating effects on global health but also economy.

Therefore, scientific research in the field of infectious diseases is paramount to ensure outbreak control and surveillance of emerging threats. Current healthcare relies heavily on the diagnosis of infectious diseases in centralized healthcare centers thereby overlooking the access of molecular diagnostics for other areas such as airports, home-testing and especially the developing world with its limited resources. Towards this, various isothermal nucleic acid amplification technologies have been developed that hold the promise to bring state-of-the-art molecular diagnostics into these areas as they are versatile, sensitive and specific, and cost-effective. One such technique is rolling circle amplification which was used in this thesis.

This research work provides an overview of the developments in biochemistry, related disciplines and their combination to design methods for diagnostic platforms tackling infectious diseases. The studies conducted in this work can be considered as individual modules for addressing challenges, like typing of pathogens and disease-related antibodies, and inexpensive bulk as well as digital quantification and simplified assay schemes. These approaches and their combinations aim to bring rolling circle amplification-based assay schemes into the molecular diagnostic field and towards decentralized healthcare.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 68
Keywords
molecular diagnostics, infectious diseases, point-of-care, digital quantification, fluorescence detection, rolling circle amplification, padlock probes, microfluidic enrichment
National Category
Biochemistry Molecular Biology Infectious Medicine Microbiology in the medical area
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-185978 (URN)978-91-7911-317-9 (ISBN)978-91-7911-318-6 (ISBN)
Public defence
2020-12-10, Pascal, Science for Life Laboratory, Tomtebodavägen 23A or digitally via Zoom: https://stockholmuniversity.zoom.us/j/64931329555, Solna, 16:00 (English)
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Supervisors
Available from: 2020-11-17 Created: 2020-10-27 Last updated: 2025-02-20Bibliographically approved

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Neumann, FelixMattson Langseth, ChristofferMadaboosi, NarayananNilsson, Mats

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