Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target aff...

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Bibliographic Details
Published in:	International journal of molecular sciences Vol. 22; no. 23; p. 12845
Main Authors:	Sapozhnikova, Ksenia A, Misyurin, Vsevolod A, Ryazantsev, Dmitry Y, Kokin, Egor A, Finashutina, Yulia P, Alexeeva, Anastasiya V, Ivanov, Igor A, Kocharovskaya, Milita V, Tikhonova, Nataliya A, Popova, Galina P, Alferova, Vera A, Ustinov, Alexey V, Korshun, Vladimir A, Brylev, Vladimir A
Format:	Journal Article
Language:	English
Published:	Switzerland MDPI AG 27-11-2021 MDPI
Subjects:	antibodies Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - immunology Antigens Antigens, Neoplasm - analysis Antigens, Neoplasm - immunology Bone marrow Bone Marrow - immunology Bone Marrow - metabolism Bone Marrow - pathology Cell Line, Tumor Conjugates Conjugation Drug delivery Dyes ethoxyethylidene protecting group Flow cytometry Flow stability Fluorescein fluorescence imaging Fluorescent Antibody Technique - methods fluorescent dyes Fluorescent Dyes - chemistry Fluorescent indicators Humans IgG antibody Imaging techniques Immunoconjugates Immunoconjugates - chemistry Immunoconjugates - immunology Immunoconjugates - metabolism Immunoglobulin G Immunotherapy Labeling Leukemia Leukemia, Myeloid, Acute - diagnosis Leukemia, Myeloid, Acute - immunology Leukemia, Myeloid, Acute - metabolism Melanoma Monoclonal antibodies Multiple myeloma Oxidation oxime ligation Payloads Polysaccharides PRAME Proteins Reagents Stoichiometry PRAME antibodies fluorescent dyes oxime ligation fluorescence imaging ethoxyethylidene protecting group
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Summary:	Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1422-0067 1661-6596 1422-0067
DOI:	10.3390/ijms222312845