Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Potentiating TRPA1 by Sea Anemone Peptide Ms 9a-1 Reduces Pain and Inflammation in a Model of Osteoarthritis.

Progressive articular surface degradation during arthritis causes ongoing pain and hyperalgesia that lead to the development of functional disability. TRPA1 channel significantly contributes to the activation of sensory neurons that initiate neurogenic inflammation and mediates pain signal transduction to the central nervous system. Peptide Ms 9a-1 from the sea anemone Metridium senile is a positive allosteric modulator of TRPA1 and shows significant anti-inflammatory and analgesic activity in different models of pain. We used a model of monosodium iodoacetate (MIA)-induced osteoarthritis to evaluate the anti-inflammatory properties of Ms 9a-1 in comparison with APHC3 (a polypeptide modulator of TRPV1 channel) and non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and ibuprofen. Administration of Ms 9a-1 (0.1 mg/kg, subcutaneously) significantly reversed joint swelling, disability, thermal and mechanical hypersensitivity, and grip strength impairment. The effect of Ms 9a-1 was equal to or better than that of reference drugs. Post-treatment histological analysis revealed that long-term administration of Ms9a-1 could reduce inflammatory changes in joints and prevent the progression of cartilage and bone destruction at the same level as meloxicam. Peptide Ms 9a-1 showed significant analgesic and anti-inflammatory effects in the model of MIA-induced OA, and therefore positive allosteric modulators could be considered for the alleviation of OA symptoms.

Influence of Molecular Weight on Thermal and Mechanical Properties of Carbon-Fiber-Reinforced Plastics Based on Thermoplastic Partially Crystalline Polyimide.

For the first time, a study of the influence of the molecular weight of the thermoplastic partially crystalline polyimide R-BAPB on the thermophysical and mechanical properties of carbon plastics was presented. The molecular weight of polyimide was determined using the method of light scattering and the study of the intrinsic viscosity of polyamic acid solutions. To obtain CFRPs, the uniform distribution of polyimide powder on continuous carbon fibers via electrostatic spraying and further hot calendering and pressing were applied. The study of the structure of the obtained carbon plastics via scanning electron microscopy has shown that the growth of the molecular weight of polyimide prevents the impregnation of carbon fiber with the introduced polyimide. Moreover, an increase in the molecular weight of polyimide leads to a rise in glass transition and thermal decomposition temperatures up to 590 °C, while the degree of crystallinity of CFRP falls. Nonetheless, raising the molecular weight from 22,000 to 70,000 g/mol of a binder polymer improves the interlayer fracture toughness G1C by more than five times.

Synthesis, Characterization, Conformation in Solution, and Thermoresponsiveness of Polymer Brushes of methoxy[oligo (propylene glycol)-block-oligo(ethylene glycol)]methacrylate and N-[3-(dimethylamino)propyl]methacrylamide Obtained via RAFT Polymerization.

The thermo- and pH-responsive polymer brushes based on methoxy[oligo(propyleneglycol)8-block-oligo(ethyleneglycol)8]methacrylate with different concentrations of N-[3-(dimethylamino)propyl]methacrylamide (from 0% to 20%) were synthesized via RAFT polymerization. The "grafting-through" approach was used to prepare the low-molar-mass dispersion samples (Mw/Mn ≈ 1.3). Molar masses and hydrodynamic characteristics were obtained using static and dynamic light scattering and viscometry. The solvents used were acetonitrile, DMFA, and water. The molar masses of the prepared samples ranged from 40,000 to 60,000 g·mol-1. The macromolecules of these polymer brushes were modeled using a prolate revolution ellipsoid or a cylinder with spherical ends. In water, micelle-like aggregates were formed. Critical micelle concentrations decreased with the content of N-[3-(dimethylamino)propyl]methacrylamide. Molecular brushes demonstrated thermo- and pH-responsiveness in water-salt solutions. It was shown that at a given molecular mass and at close pH values, the increase in the number of N-[3-(dimethylamino)propyl]methacrylamide units led to an increase in phase separation temperatures.

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody-Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control.

A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.

The Solution Properties of Polymethacrylate Molecular Brushes with Oligo(ethylene glycol) and Oligo(propylene glycol) Side Chains.

The properties of polymer brushes based on three macromonomers were investigated in aqueous and organic solutions. Methacrylic monomers with different compositions of the oligo(oxyalkylene) substituents and arrangements of the oligo(ethylene glycol) and oligo(propylene glycol) blocks were used for the synthesis of polymers. There were methoxy [oligo(ethylene glycol)10.3-block-oligo(propylene glycol)4.7] methacrylate, methoxy [oligo(propylene glycol)8.3-block-oligo(ethylene glycol)6.6] methacrylate, and methoxy oligo(propylene glycol)4.2 methacrylate. Molecular brushes were investigated by the methods of molecular hydrodynamics and optics in dilute solutions in acetonitrile, chloroform, and water. The peculiarities of behavior of poly[oligo(oxyalkylene) methacrylates] in aqueous solutions and water-toluene systems have been found; in particular, the solubility of the polymers in water and organic solvents, the polymers equilibrium distribution between the phases, and the surface activity in the water-toluene system have been established. The thermo-responsibility in aqueous solutions and values of a critical concentration of micelle formation were shown. Depending on the arrangement of blocks in the side chains of molecular brushes, they are characterized by different intramolecular density.

Properties in Langmuir Monolayers and Langmuir-Blodgett Films of a Block Copolymer Based on N-Isopropylacrylamide and 2,2,3,3-Tetrafluropropyl Methacrylate.

The amphiphilic block copolymer poly(N-isopropylacrylamide)-Ge(C6F5)2-poly(2,2,3,3-tetrafluoropropyl methacrylate) was prepared by the reaction of chain transfer to bis-(pentafluorophenyl)germane during the polymerization of N-isopropylacrylamide and the subsequent postpolymerization of isolated functional polymers in 2,2,3,3-tetrafluoropropyl methacrylate. The conversion of the block copolymer was 68% and the molecular weight of the sample was 490,000 g/mol. The colloidal chemical properties of Langmuir monolayers and Langmuir-Blodgett films of synthesized block copolymer have been studied. For comparison, a functional polymer, namely, poly-N-isopropylacrylamide with terminal -Ge(C6F5)2H group, was synthesized and studied. The concentrations of spreading solutions were selected and the effect of subphase acidity on the formation of monolayers of macromolecules of the block copolymer was studied. It was found that regardless of the acidity of the subphase, high pressure of fracture of films are characteristic of monolayers of collapse pressures πmax = (48-61) mN/m. The morphology of the Langmuir-Blodgett films of functional polymer exhibit isolated elongated micelles with high densities in the form of "octopus" on the periphery of which there are terminal hydrophobic groups. For the Langmuir-Blodgett film of block copolymer, a comb-like structure is observed with characteristic protrusions.

Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay.

Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria.

The Influence of Synthesis Method on Characteristics of Buffer and Organic Solutions of Thermo- and pH-Responsive Poly(N-[3-(diethylamino)propyl]methacrylamide)s.

Thermo- and pH-responsive poly(N-[3-(diethylamino)propyl]methacrylamide)s were synthesized by free radical polymerization and RAFT polymerization. The molar masses of the samples were 33,000-35,000 g∙mol-1. Investigations of the dilute solutions showed that the prepared samples were flexible chain polymers. The behavior of the synthesized polymers in the buffer solutions was analyzed by turbidity and light scattering at a pH range of 7-13 and a concentration range of 0.0002-0.008 g·cm-3. When the concentrated solutions were at a low temperature, there were macromolecules and aggregates, which were formed due to the interaction of hydrophobic units. For the investigated samples, the lower critical solution temperatures were equal. The phase separation temperatures decreased as pH increased. The influence of polydispersity index on the characteristics of the samples in the solutions was analyzed. The radii of molecules of poly(N-[3-(diethylamino)propyl]methacrylamide) obtained by RAFT polymerization at this temperature at the onset and end of the phase separation interval were lower than ones for samples synthesized by conventional free radical polymerization.

Novel Amphiphilic Polyfluorene-Graft-(Polymethacrylic Acid) Brushes: Synthesis, Conformation, and Self-Assembly.

Novel polyfluorene polymer brushes with polymethacrylic acid side chains were obtained by atom transfer radical polymerization (ATRP) and activator generated by electron transfer (AGET) ATRP of tert-butyl methacrylate on polyfluorene multifunctional macroinitiator, followed by protonolysis of the tert-butyl groups of the side chains. Kinetics of polymerization and molecular weights were fully characterized. These polymer brushes luminesce in the blue region of the spectrum with high quantum yields (0.64-0.77). It was shown that the luminescence intensity of polymer brushes is higher than the luminescence intensity of the macroinitiator (0.61). Moreover, due to their amphiphilic nature, they can form unimolecular micelles when an alcohol solution of the polymer brush is injected into water. These properties can potentially be used in drug delivery and bioimaging.

Conformation, Self-Organization and Thermoresponsibility of Polymethacrylate Molecular Brushes with Oligo(ethylene glycol)-block-oligo(propylene glycol) Side Chains.

Polymethacrylic molecular brushes with oligo(ethylene glycol)-block-oligo(propylene glycol) side chains were investigated by static and dynamic light scattering and viscometry. The solvents used were acetonitrile, tetrahydrofuran, chloroform, and water. The grafted copolymers were molecularly dispersed and dissolved in tetrahydrofuran and acetonitrile. In these solvents, the molar masses of copolymers were determined. In thermodynamically good solvents, namely tetrahydrofuran and acetonitrile, investigated copolymers have a high intramolecular density and the shape of their molecules resembles a star-shaped macromolecule. In chloroform and water, the micelle-like aggregates were formed. Critical micelle concentrations decreased with the lengthening of the hydrophobic block. Molecular brushes demonstrated thermosensitive behavior in aqueous solutions. The phase separation temperatures reduced with an increase in the content of the oligo(propylene glycol) block.

Epitope-Specific Response of Human Milk Immunoglobulins in COVID-19 Recovered Women.

The breastfeeding of infants by mothers who are infected with SARS-CoV-2 has become a dramatic healthcare problem. The WHO recommends that infected women should not abandon breastfeeding; however, there is still the risk of contact transmission. Convalescent donor milk may provide a defense against the aforementioned issue and can eliminate the consequences of artificial feeding. Therefore, it is vital to characterize the epitope-specific immunological landscape of human milk from women who recovered from COVID-19. We carried out a comprehensive ELISA-based analysis of blood serum and human milk from maternity patients who had recovered from COVID-19 at different trimesters of pregnancy. It was found that patients predominantly contained SARS-CoV-2 N-protein-specific immunoglobulins and had manifested the antibodies for all the antigens tested in a protein-specific and time-dependent manner. Women who recovered from COVID-19 at trimester I-II showed a noticeable decrease in the number of milk samples with sIgA specific to the N-protein, linear NTD, and RBD-SD1 epitopes, and showed an increase in samples with RBD conformation-dependent sIgA. S-antigens were found to solely induce a sIgA1 response, whereas N-protein sIgA1 and sIgA2 subclasses were involved in 100% and 33% of cases. Overall, the antibody immunological landscape of convalescent donor milk suggests that it may be a potential defense agent against COVID-19 for infants, conferring them with a passive immunity.

Self-Assembly of Molecular Brushes with Polyimide Backbone and Amphiphilic Block Copolymer Side Chains in Selective Solvents.

Three-component molecular brushes with a polyimide backbone and amphiphilic block copolymer side chains with different contents of the "inner" hydrophilic (poly(methacrylic acid)) and "outer" hydrophobic (poly(methyl methacrylate)) blocks were synthesized and characterized by molecular hydrodynamics and optics methods in solutions of chloroform, dimethylformamide, tetrahydrofuran and ethanol. The peculiarity of the studied polymers is the amphiphilic structure of the grafted chains. The molar masses of the molecular brushes were determined by static and dynamic light scattering in chloroform in which polymers form molecularly disperse solutions. Spontaneous self-assembly of macromolecules was detected in dimethylformamide, tetrahydrofuran and ethanol. The aggregates size depended on the thermodynamic quality of the solvent as well as on the macromolecular architectural parameters. In dimethylformamide and tetrahydrofuran, the distribution of hydrodynamic radii of aggregates was bimodal, while in ethanol, it was unimodal. Moreover, in ethanol, an increase in the poly(methyl methacrylate) content caused a decrease in the hydrodynamic radius of aggregates. A significant difference in the nature of the blocks included in the brushes determines the selectivity of the used solvents, since their thermodynamic quality with respect to the blocks is different. The macromolecules of the studied graft copolymers tend to self-organization in selective solvents with formation of a core-shell structure with an insoluble solvophobic core surrounded by the solvophilic shell of side chains.

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR.

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.

Comparative diagnostics of allergy using quantitative immuno-PCR and ELISA.

AIM: Estimation of specific IgE is essential for the prevention of allergy progression. Quantitative immuno-PCR (qiPCR) can increase the sensitivity of IgE detection. We aimed to develop qiPCR and compare it to the conventional ELISA in identification of IgE to Alt a 1 and Fel d 1 allergens. RESULTS: Single stranded 60-mer DNA conjugated to streptavidin was used to detect antigen-IgE-biotin complex by qiPCR. In semi-logarithmic scale qiPCR data were linear in a full range of serum dilutions resulting in three- to ten-times higher sensitivity of qiPCR in comparison with ELISA in IgE estimation in low titer sera. CONCLUSION: Higher sensitivity of qiPCR in identification of low titer IgE is a result of a higher linearity of qiPCR data.

xMAP-based analysis of three most prevalent staphylococcal toxins in Staphylococcus aureus cultures.

Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods-biological tests on animals or cell cultures as well as ELISA-is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins-enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)-in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.

Development of xMAP assay for detection of six protein toxins.

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.

Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins.

Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.

GMDP augments antitumor action of the CP/TNFalpha combination in vivo.

We have shown that glucosaminyl muramyl dipeptide (GMDP) has been augmented the antitumor action of chemotherapy drug cisplatin and tumor necrosis factor-alpha (TNFalpha) on the Ehrlich ascites carcinoma and melanoma B-16 mouse tumor models. The doses of cisplatin, TNFalpha and GMDP and also the conditions of the drugs combination injection provided 100% survival of mice with Ehrlich ascites carcinoma were found. Furthermore, it was shown first that GMDP has been decreased toxicity of the cisplatin/TNFalpha combination and normalized the changes in the experimental mice hematological parameters which were produced by the CP/TNFalpha combination.