F. A. Brovko

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.

Simultaneous detection of seven staphylococcal enterotoxins: development of hydrogel biochips for analytical and practical application.

A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.

A NEW FAMILY OF CYTOKININ RECEPTORS FROM CEREALES

The highly specific recognition of a natural cytokinin, trans‐zeatin, by cytokinin‐binding protein (CBP) of 67 kDa from barley leaves was detected with an assay developed on the basis of cytokinin competition in ELISA with anti‐idiotype antibodies (raised against antibodies to zeatin) for complex formation with CBP. Monoclonal antibodies (mAbs) raised against 70 kDa CBP from etiolated maize seedlings cross‐reacted with barley 67 kDa CBP and prevented barley CBP and trans‐zeatin induced activation of transcription elongation directed by RNA polymerase I associated with barley chromatin. One mAb (Z‐6) had an agonistic effect. Maize CBP replaced barley CBP in activation of RNA synthesis with cytokinin in the barley transcription system. Hence, a new family of cytokinin receptors with common functions and immunodeterminants including maize and barley CBPs was found.

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.

The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library.

A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K(d) of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.

The library of human miniantibodies in the phage display format: designing and testing.

Antibodies are widely used in scientific studies, medicine, and veterinary owing to their ability for highaffinity and specific binding of compounds of various chemical nature. They are the main tool in different immunoassay techniques [1]. Additionally, antibodies are used more and more frequently as therapeutic agents in transplantology, oncology, and infectious immunology [2]. The wide therapeutic use of antibodies was preceded by the development of the technology of monoclonal antibodies, the development of procedures of obtaining monoclonal antibody-based chimerical and humanized analogues that retain the specificity of the original monoclonal antibody but exhibit a decreased immunogenicity [3].

Methods of express analysis of staphylococcal enterotoxin a in food products

Instrument-free methods for detection of staphylococcal enterotoxin A (SEA) in samples of drinkable dairy products and meat broth using immunochromatography (IC) and dot-assay were developed. A conjugate of monoclonal antibodies (MA) to SEA with colloid gold (CG) was used as the detecting component in IC; the MA-CG conjugate or biotinylated SEA antibodies and streptavidin-peroxidase conjugate (STR-HRP) were used for conducting the dot-assay. The results of the SEA analysis with the developed method and with the immunoenzymatic assay (IEA) were compared. The limit of detection in ng/mL was: 10 (IC), 20 (dot analysis, MA-CG), 10 (dot analysis, STR-HRP), and 4 (IEA). The analysis time in min was: 25 (IC), 60 (dot-assay, MA-CG), 70 (dot-assay, STR-HRP), and 150 (IEA). The simplicity and availability of the developed instrumentless detection methods allow conducting single and mass testing in low-resource laboratories as well as outside laboratories.

Detection of propeptides of AlpA and AlpB lytic endopeptidases from Lysobacter sp. XL1 by the sandwich enzyme immunoassay based on monoclonal antibodies

Extracellular lytic endopeptidases AlpA and AlpB of the Gram-negative bacterium Lysobacter sp. XL1 have a high degree of homology and are synthesized as preproproteins consisting of a signal peptide, a propeptide, and the mature protein. In the present work, two monoclonal antibodies against the AlpA propeptide (ProA) and eleven antibodies against the AlpB propeptide (ProB) have been obtained. The affinity constants for antibodies to ProA were 2.9 × 109 and 3.5 × 109 M−1, and those for antibodies to ProB were from 1.5 × 108 to 2.2 × 109 M−1. The antibodies showed no immune cross-reactivity with each other and with mature forms of the enzymes. On the basis of monoclonal antibodies, a sandwich enzyme immunoassay has been developed, which makes it possible to detect these propeptides in the dissolved native form. The linear range of the detection of ProA was 1.5–100.0 ng/mL with an error of measurement of 6%, and that of the determination of ProA was 0.2–6.25 ng/mL with an error of measurement of 6%. By using the assay, propeptides ProA and ProB were detected in cell lysates of Lysobacter sp. XL1 in an amount of 1.18 ± 0.03 and 0.096 ± 0.002 ng per 1 OD540 of bacterial culture, respectively. The immunochemical assay for the detection of different forms of AlpA and AlpB can be useful in solving the problems associated with their secretion into environment.

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.

Zeatin-binding proteins participating in cytokinin-dependent activation of transcription

A new zeatin-binding protein (ZBP) with molecular weight of 67± 2 kD was isolated from cytosol of first leaves of 10-day-old barley plants and revealed by non-denaturing PAGE as a single polypeptide. Its zeatin-binding capacity was established (i) by its ability to bind reversible [3H]trans-Zeatin, (ii) by its interaction with anti-idiotype antibodies (Aba-i) from antiserum raised against zeatin, and (iii) by its ability to displace antibodies to zeatin (Abz) from its complex with immobilized trans-zeatin in competitive ELISA. In concert with trans-zeatin the ZBP activated transcription elongation in the system containing chromatin-bound RNA polymerase I from barley leaves and in isolated nuclei. Our results proved that 67 ± 2 kD ZBP is a receptor of natural cytokinin in leaf cells which mediates cytokinin dependent activation of transcription elongation. ZBP- and mzns-zeatin-indueed activation of RNA synthesis in the system containing chromatin-bound RNA polymerase I depended on leaf age used for chromatin isolation and corresponded to age-dependence of leaf response to cytokinins. ZBP of 70 ± 2 kD was also isolated from the shoots of etiolated 5-day-old maize seedlings. This protein reversibly bound [3H]dihydrozeatin, was recognized by Aba-i isolated from antiserum raised against zeatin and activated RNA synthesis in vitro in the system containing chromatin-bound RNA polymerase from barley leaves in the presence of trans-zeatin. Hence, ZBP-mediated cytokinin-dependent activation of RNA synthesis is not species-specific. Thus, it was discovered the new family of ZBPs with the properties of cytokinin receptor which is involved in cytokinin-dependent activation of transcription elongation in plant cells.