SARS-CoV-2 Peptide Bioconjugates Designed for Antibody Diagnostics.

In the near future, the increase in the number of required tests for COVID-19 antibodies is expected to be many hundreds of millions. Obviously, this will be done using a variety of analytical methods and using different antigens, including peptides. In this work, we compare three method variations for detecting specific immunoglobulins directed against peptides of approximately 15-aa of the SARS-CoV-2 spike protein. These linear peptide epitopes were selected using antigenicity algorithms, and were synthesized with an additional terminal cysteine residue for their bioconjugation. In two of the methods, constructs were prepared where the peptide (F, function) is attached to a negatively charged hydrophilic spacer (S) linked to a dioleoylphosphatidyl ethanolamine residue (L, lipid) to create a function-spacer-lipid construct (FSL). These FSLs were easily and controllably incorporated into erythrocytes for serologic testing or in a lipid bilayer deposited on a polystyrene microplate for use in an enzyme immunoassays (EIA). The third method, also an EIA, used polyacrylamide conjugated peptides (peptide-PAA) prepared by controlled condensation of the cysteine residue of the peptide with the maleimide-derived PAA polymer which were immobilized on polystyrene microplates by physisorption of the polymer. In this work, we describe the synthesis of the PAA and FSL peptide bioconjugates, design of test systems, and comparison of the bioassays results, and discuss potential reasons for higher performance of the FSL conjugates, particularly in the erythrocyte-based serologic assay.

Immuno-PCR technology for detection of natural human antibodies against Lec disaccharide.

The development of an immuno-PCR assay for quantitation of low amounts of anti-glycan human antibodies is described. The sensitivity of the assay for determination of low-affinity anti-LeC IgM has been found to be 4 ng/ml (~100 pg per sample), thus being two orders of magnitude higher compared to the conventional ELISA with the same antigen.

Immobilization of polyacrylamide-based glycoconjugates on solid phase in immunosorbent assays.

Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbohydrate antibodies are considered, with special emphasis on the application niches of different immobilization techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results.

Biotinylated multivalent glycoconjugates for surface coating.

Systematic studying of biological processes driven by multipoint high-cooperative carbohydrate recognition requires application of multivalent carbohydrates as tools. In this regard polyacrylamides with various pendant carbohydrate residues and labels are probably the most well advanced class of carbohydrate multimerics. Here we describe a synthetic approach to polyacrylamide-based glycoconjugates with biotin tag, with special emphasis to development of carbohydrate biosensors and arrays.

High molecular weight neoglycoconjugates for solid phase assays.

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc). The first of them is based on the covalent immobilization of neoglycoconjugates on the NH(2)-modified polystyrene; the second one is based on the elevated adherence of high m.w. neoglycoconjugates to polystyrene. Covalent immobilization of polyacrylamide conjugates, Glyc-PAA, provided a possibility to solve the problem, but the nonspecific binding of antibodies in ELISA proved to be unacceptably high. At the same time, the increase of the Glyc-PAA m.w. from 30 kDa to 2,000 kDa allowed a 10-20 fold decrease of its consumption, when using physical adsorption, whereas the assay background remained at the low level. The amount of 2,000 kDa Glyc-PAA that is sufficient for the coating of a standard 96-well plate corresponds to the nanomole level of oligosaccharide, this providing a possibility to use saccharides that are available in a very limited amount when studying the carbohydrate-protein interaction with solid-phase techniques.

Assembly of p-selectin ligands on a polymeric template.

High-affinity receptor-ligand interactions frequently involve molecular interactions at two distinct sites. A derivatized polyacrylic-based polymer was synthesized to allow substitution with multiple ligands (e.g., L(1) and L(2)) on the backbone. Two-site P-selectin-ligand interactions were first studied with SiaLe(x) (L(1)) and tyrosine sulfate (L(2)) covalently incorporated onto the flexible polymer. In competition assays, a marked synergistic inhibitory effect was observed when the polymer presented both L(1) and L(2) as opposed to either ligand alone. In a second approach, the SiaLe(X) ligand was reduced in complexity so that L(1) was fixed as Le(x) or Le(a), and alternative L(2) groups (to mimic sialic acid) were investigated. Certain combinations of L(1) and L(2) were better antagonists of P-selectin than SiaLe(x) itself. These approaches offer the potential of facilitating the discovery of novel inhibitors of receptors or enzymes.