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 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.     

DNA‐Templated Introduction of an Aldehyde Handle in Proteins

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site‐specific conjugates. This can rarely be achieved by simple residue‐specific random labeling, but generally requires genetic engineering. Using site‐selective DNA‐templated reductive amination, we created DNA–protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal‐binding functionality facilitates site‐selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal‐binding site. We demonstrate DNA‐templated reductive amination for His₆‐tagged proteins and metal‐binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.     

Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.     

2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching,Secondary Structure Alteration and Binding Sites Localization

The first step in determining whether a fluorescent dye can be used for antibody labeling consists in collecting data on its physical interaction with the latter. In the present study, the interaction between the 2-(2-hydroxy-5-nitrobenzylidene)-1,3-indanedione (HNBID) dye and the IgG1 monoclonal mouse antibody anti-human heart fatty acid binding protein (anti-hFABP) has been investigated by fluorescence and circular dichroism spectroscopies and complementary structural results were obtained by molecular modeling. We have determined the parameters characterizing this interaction, namely the quenching and binding constants, classes of binding sites, and excited state lifetimes, and we have predicted the localization of HNBID within the Fc region of anti-hFABP. The key glycosidic and amino acid residues in anti-hFABP interacting with HNBID have also been identified. A similar systematic study was undertaken for the well-known fluorescein isothiocyanate fluorophore, for comparison purposes. Our results recommend HNBID as a valuable alternative to fluorescein isothiocyanate for use as a fluorescent probe for IgG1 antibodies.     

Interaction of dis‐azo dyes with quaternized poly(dimethylaminoethyl methacrylate) as a function of the dye structure and polycation charge density

Interactions between some dis‐azo dyes, different by either the position of their sulfonic groups or their number (Ponceau SS, Crocein Scarlet MOO, Congo Red, and Direct Blue 1), and some strong polycations (PCs), with cationic centers in their side chains and dye removal from artificial wastewaters were systematically investigated in this study. PCs with variable charge densities (CDs) were prepared from poly(dimethylaminoethyl methacrylate) by controlled quaternization with benzyl chloride. Even when the main process in the dye removal was charge neutralization (coagulation) for all of the dyes, significant effects of the CD and dis‐azo dye structure on the metachromatic behavior of the dyes in dilute aqueous solutions and on the dye removal efficiency were observed. The stability of the PC/azo dye complex and, connected with this, the flocculation window were higher when the PC with the highest CD was used. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

The Exception That Proves the Rule: How Sodium Chelation Can Alter the Charge-Cell Binding Correlation of Fluorescein-Based Multimodal Imaging Agents

In the present study we describe and explain an aberrant behavior in terms of receptor binding profile of a fluorescein-based multimodal imaging agent for gastrin releasing peptide receptor (GRPR) visualization by elucidating a chelating mechanism on sodium ions of its fluorescent dye moiety. This hypothesis is supported by both biological results and spectroscopic analyses of different fluorescein-carrying conjugates and an equally charged set of analogous tartrazine-based GRPR-binding imaging agents. Fluorescein interacts with sodium which reduces the overall negative charge of the dye molecule by one. This reduction in apparent total net charge explains the exceptional behavior found for the fluorescein-based multimodal bioconjugate in the context of the charge-cell binding correlation hypothesis.     

The role of membrane properties in Mistic folding and dimerisation

Membranes not only provide cellular compartmentalization but influence protein behavior and folding by virtue of the multitude of different lipid types. We have studied the impact of lipid composition on the folding of the membrane-associated protein Mistic from B. subtilis. We use dimerisation via the single Cys3 residue as monitor for the degree of correct folding, since mis- or unfolding will expose the otherwise buried Cys3. We find great variability in how lipids affect protein production and dimerization, ranging from high production and low dimerization via increased production and higher dimerization to low production and low dimerization. Phosphocholine (PC) vesicles, in particular di-oleoyl-PC, lead to the highest production levels. Shorter chain lengths lead to reduced production but higher levels of dimerization. Different lipids may promote correct folding of Mistic to different extents, mediated by proper hydrophobic matching (attained for long-chain but not short-chain PC vesicles) and the existence of a fluid phase (the gel phase reduces production as well as dimerization, probably by immobilizing Mistic on the surface). The very fact that different lipids have an effect indicates that Mistic behaves like a bona fide membrane protein with a clear preference for membranes of a certain thickness and flexibility.     

Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.     

Relationship between the dye/additive interaction and inkjet ink droplet formation

To design adequate ink composition for textile printing, the relationship between the dye/additive interaction and ink performance is investigated. In the present study, the three acid dyes C. I. Acid Red 88, 13, and 27, a water-soluble polymer poly(vinylpyrrolidone) (PVP) and three surfactants, sodium dodecyl sulfate (SDS), octaethylene glycol monododecyl ether (OGDE), and Surfynol 465 (S465) were used and the dye/additive interaction was investigated by means of visible absorption measurements. The visible absorption spectra of aqueous dye solutions changed with the addition of the nonionic surfactants, but further addition of PVP had little effect on the spectra, indicating that the strong binding of the dye molecules with the nonionic surfactant micelles is maintained even in the presence of PVP. In contrast, in the case of SDS, the spectra changed with the addition of the surfactant as well as with further addition of PVP. This indicates that the behavior of the acid dyes in the three-species system depends on the dye structure, the surfactant structure, and the molecular weight of PVP. Furthermore, to estimate the ink performance, the physical properties of the ink, such as viscosity, surface tension, and ink droplet formation were determined. Ink solutions with favorable physicochemical properties and low molecular weight PVP showed good ink droplet formation. In the optimized ink composition (PVP-1/S465: 1.4/0.004 mol dm⁻³) most of the dye molecules are strongly bound to the PVP chain, but the binding is not significantly affected by the addition of S465.     

An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition

Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (～ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.     

Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability

Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy. In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL-barnase. The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C-terminus of the light chain variable domain (VL) of the anti-human ferritin monoclonal antibody F11. While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant ( approximately 70%) fraction of soluble protein, with only a minor insoluble fraction ( approximately 30%) packed into inclusion bodies. The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone-like role that barnase exerted with regard to the N-terminal VL domain. Cytoplasmic VL-barnase was analyzed for structural and functional properties. The dimeric state of the chimeric protein was demonstrated by size-exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain. Ferritin-binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion. Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein. However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH-induced changes in both intrinsic fluorescence and binding of ANS. Together, the results indicate that VL-barnase retained the antigen-binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single-chain chimeric protein resulted in an altered tertiary fold and pH stability.     

Exploiting Radiation Induction of Antigens in Cancer: Targeted Drug Delivery

Therapeutic antibodies used to treat cancer are effective in patients with advanced-stage disease. For example, antibodies that activate T-lymphocytes improve survival in many cancer subtypes. In addition, antibody–drug conjugates effectively target cytotoxic agents that are specific to cancer. This review discusses radiation-inducible antigens, which are stress-regulated proteins that are over-expressed in cancer. These inducible cell surface proteins become accessible to antibody binding during the cellular response to genotoxic stress. The lead antigens are induced in all histologic subtypes and nearly all advanced-stage cancers, but show little to no expression in normal tissues. Inducible antigens are exploited by using therapeutic antibodies that bind specifically to these stress-regulated proteins. Antibodies that bind to the inducible antigens GRP78 and TIP1 enhance the efficacy of radiotherapy in preclinical cancer models. The conjugation of cytotoxic drugs to the antibodies further improves cancer response. This review focuses on the use of radiotherapy to control the cancer-specific binding of therapeutic antibodies and antibody–drug conjugates.     

Amino acid chemoreception: Effects of ph on receptors and stimuli

A current model of amino acid chemoreception has generated the idea that pH affects the efficiency of stimulus-receptor binding by altering the charge distribution on stimulus molecules. The model suggests that amino acids are maximally stimulatory near their isoelectric points. We point out that, within a broad range of pH values, changes in stimulant amino acids cannot account for altered chemoresponsiveness. We suggest instead that pH-induced changes in chemoreception are a result of changes in charge distribution on the protein receptor.     

噻唑橙类菁染料与生物大分子结合的光谱特性的研究

研究了两个噻唑橙类染料TO-1和TO-2与核酸、蛋白质结合的光谱特性，发现染料喹啉环中氮上取代基对染料标记生物大分子的荧光特性有着大的影响，羟基的引入，使染料TO-2与核酸结合后的荧光性能下降，但同时也提高了它与蛋白质结合的光物理性能，研究表明：在喹啉环氮上引入适当的亲水性取代基，可以使噻唑橙类菁染料的应用范围和拓展到蛋白质标记领域，并初步研究了蛋白质浓度的变化对染料TO-2的荧光光谱的影响以及TO-2在不同PH值的蛋白质溶液中的荧光性质。     

Grafting of polypropylene fibers. II. Electrokinetic properties of grafted polypropylene fibers

Electrokinetic properties of methacrylic acid- and acrylonitrile-grafted polypropylene fibers measured in the presence of cationic dyes are reported. The zeta potential of polypropylene fibers decreases, and the surface charge density along with surface conductivity increases as the concentration of the dyes in the streaming solution increases. The zeta potential at pH 7 decreases as the amount of graft increases in case of both acrylonitrile- and methacrylic acid grafted fibers. Both surface charge density and surface conductivity increase with the increase in dye concentration for both acrylonitrile- and methacrylic acid-grafted fibers. The results are explained on the basis of the cationic dye adsorption on the grafted fiber in the case of methacrylic acid graft. In the case of acrylonitrile-grafted fibers, this could be due to the strong attraction of cationic dye to the nitrile group of the grafted fibers.     

A Concise,Modular Antibody–Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry

The synthesis of antibody–oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves “plug-and-play” antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5′-amine-modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl– oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide–antibody and oligonucleotide–streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein–oligonucleotide conjugates.     

Brain Disposition of Antibody-Based Therapeutics: Dogma,Approaches and Perspectives

Due to their high specificity, monoclonal antibodies have been widely investigated for their application in drug delivery to the central nervous system (CNS) for the treatment of neurological diseases such as stroke, Alzheimer’s, and Parkinson’s disease. Research in the past few decades has revealed that one of the biggest challenges in the development of antibodies for drug delivery to the CNS is the presence of blood–brain barrier (BBB), which acts to restrict drug delivery and contributes to the limited uptake (0.1–0.2% of injected dose) of circulating antibodies into the brain. This article reviews the various methods currently used for antibody delivery to the CNS at the preclinical stage of development and the underlying mechanisms of BBB penetration. It also describes efforts to improve or modulate the physicochemical and biochemical properties of antibodies (e.g., charge, Fc receptor binding affinity, and target affinity), to adapt their pharmacokinetics (PK), and to influence their distribution and disposition into the brain. Finally, a distinction is made between approaches that seek to modify BBB permeability and those that use a physiological approach or antibody engineering to increase uptake in the CNS. Although there are currently inherent difficulties in developing safe and efficacious antibodies that will cross the BBB, the future prospects of brain-targeted delivery of antibody-based agents are believed to be excellent.     

Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group

Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.     

The influence of metal ions on the aggregation and hydrophobicity of dyes in solutions

The effect of the addition of metal ions to dye solutions in promoting dye aggregation and in changing dye association in solution has been studied. Experimental findings indicate that the promoting effects of calcium and magnesium ions are greater than that of sodium ions. It is expected that any change in dye hydrophobicity would have a direct influence on the extent of dye aggregation. Under acid conditions, a higher concentration of metal ions would result in an increase in dye aggregation with a reduction in dye hydrophobicity. On the other hand, while the dye aggregation is increased under alkaline conditions under the influence of metal ions, hydrophobicity was shown to increase. These changes in dye hydrophobicity could be used to explain the dyeing behaviour of reactive dyes in the dyeing of silk.