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.

Edited by Renee Tsolis Keywords:Innate immunity Muramyl peptide Glucosaminyl-muramyl dipeptide Y-box protein 1 a b s t r a c tThe bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-jB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-jB2 cleavage, transport of activated NF-jB2 p52 to the nucleus, and upregulation of NF-jB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

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.

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.z 1998 Federation of European Biochemical Societies.