Anna O. Shepelyakovskaya

Magnetic Immunoassay for Detection of Staphylococcal Toxins in Complex Media

Method of highly sensitive registration of magnetic nanoparticles by their nonlinear magnetization is used in a novel sandwich-type immunoassay for detection of staphylococcal toxins in complex media of virtually any volume, with increasing sensitivity at higher sample volume. The signal is read out from the entire volume of a nontransparent 3D fiber structure employed as a solid phase, which provides large reaction surface, quick reagent mixing, as well as antigen immunofiltration directly in the course of the assay. The method has demonstrated near-linear dose-response curves within a wide range of ~3 decades, while detection of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST) in neat milk without sample preparation. The limits of detection (LOD) as low as 4 and 10 pg/mL for TSST and SEA, respectively, were obtained in 2-h format using 30-mL samples. The second, 25-min format, showed the LOD of 0.1 and 0.3 ng/mL for the same toxins in a 150 μL sample. The developed immunoassay can be applied in food safety control, in vitro diagnostics, and veterinary for a variety of research from express tests in the field to highly sensitive laboratory tests.

Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function

Cytokinins regulate chloroplast differentiation and functioning, but their targets in plastids are not known. In this connection, the plastid localization of the 70 kDa cytokinin-binding protein (CBP70) was studied immunocytochemically in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus) using monoclonal antibodies (mAbs) against CBP70 recognizing this protein not only in nuclei and cytoplasm, but also in plastids. CBP70 was detected in the amyloplasts of the root cap and etioplasts of the mesocotyl, stem apex, and leaves encircling the stem axis in the node. Immunogold electron microscopy demonstrated CBP70 localization in amyloplasts outside starch grains and revealed a dependence of CBP70 content in etioplasts on the degree of their inner membrane differentiation: the low CBP70 amount in etioplasts at the early stages of membrane development, the high content in etioplasts with actively developing membranes, and a considerable decrease in plastids with the formed prolamellar body. This suggests that CBP70 is involved in etioplast structure development. CBP70 was also observed in chloroplasts of the bundle sheath of green maize leaves. CBP70 purified from etioplasts mediated trans-zeatin-dependent activation of transcription elongation in vitro in the transcription systems of maize etioplasts and barley chloroplasts, suggesting that CBP70 is a plastid transcription elongation factor or a modulator of plastid elongation factor activity. CBP70 involvement in the cytokinin-dependent regulation of plastid transcription elongation could be essential for the cytokinin control of the biogenesis of this organelle.

Muramyl peptides activate innate immunity conjointly via YB1 and NOD2

Bacterial cell wall muramyl dipeptide (MDP) and glucosaminyl-MDP (GMDP) are potent activators of innate immunity. Two receptor targets, NOD2 and YB1, have been reported; we investigated potential overlap of NOD2 and YB1 pathways. Separate knockdown of NOD2 and YB1 demonstrates that both contribute to GMDP induction of NF-κB expression, a marker of innate immunity, although excess YB1 led to induction in the absence of NOD2. YB1 and NOD2 co-migrated on sucrose gradient centrifugation, and GMDP addition led to the formation of higher molecular mass complexes containing both YB1 and NOD2. Co-immunoprecipitation demonstrated a direct interaction between YB1 and NOD2, a major recombinant fragment of NOD2 (NACHT–LRR) bound to YB1, and complex formation was stimulated by GMDP. We also report subcellular colocalization of NOD2 and YB1. Although YB1 may have other binding partners in addition to NOD2, maximal innate immunity activation by muramyl peptides is mediated via an interaction between YB1 and NOD2.

Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic

The method for searching for ligands exerting an adjuvant effect is described. The method involves isolation of polysomes using an immobilized peptide mimetic of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) — RN-peptide. After the affinity chromatography and washing, RN-peptide complexes with the target sequences were dissociated with guanidine hydrochloride. The obtained mRNA was used for cDNA synthesis and subsequent cloning in an expression vector. Further studies showed the effectiveness of this method. Clones interacting with the peptide were selected using biotinylated RN-peptide. It was found that all clones encode a sequence identical to the protein YB-1. Recombinant antibodies against protein YB-1 were selected from a phage display human scFv library. Using these antibodies, we determined the binding constant of RN-peptide to protein YB-1. Competitive analysis showed that RN-peptide and GMDP compete for the same portion of YB-1 at molar ratio 1: 12.

Staphylococcal enterotoxin a detection with phage displayed antibodies

A technique for detection of staphylococcal enterotoxin A with sandwich enzyme-linked immunoassay (ELISA) was developed. Mouse monoclonal anti-SEA antibodies were used as capture antibodies and phage displayed anti-SEA scFv were used as detection antibodies. The limit of detection was 6–12.5 ng/mL for different pairs of antibodies. Some conditions of phage-displayed antibodies for storage in dissolved and lyophilized state were examined. It was shown the use of trehalose and arginine preserved the functional activity of phage-displayed antibodies.

Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens

A method for generation of highly specific miniantibodies within the phage particle has been developed, and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G, and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.

Detection of staphylococcal enterotoxin a by phage display mediated immuno-PCR method

A method of staphylococcal enterotoxin A (SEA) detection in pasteurized milk using recombinant miniantibodies exposed on phage particles was developed. Miniantibodies bind to the antigen, while phage DNA serves as a matrix in polymerase chain reaction (PCR). The SEA detection limit in milk by a phage display mediated immuno-PCR method is 100 pg/mL that is much lower than the level of toxic and allergic dose.