Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones.

Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC(50) (test midpoint) was 134 ± 30 µg L(-1) (n = 54) for N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1-HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-L-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.

Interaction behaviour of a PDMS-calixarene system and polar analytes characterised by microcalorimetry and spectroscopic methods.

Spectroscopic techniques and microcalorimetry were applied to investigate a polymer-(polydimethylsiloxane; PDMS) calixarene system during interaction with propylamine and n-propanol as analyte molecules. This was done to understand the sensitivity and selectivity of this system. By these means the interesting binding site of the calixarene selector was identified and dependencies on specific properties of the polymer and the functional groups were determined. Reflectometric interference spectroscopy (RIfS) was used to characterize the kinetics whereas isothermal titration calorimetry (ITC) yielded thermodynamic data. Infrared (IR) and (1)H NMR spectroscopy allowed identification of the sensing process as an interaction between the selective group of the PDMS-calixarene system and the amino group of propylamine, and measurement of the effects on hydrogen bonds. The combination of the different spectroscopic methods and the microcalorimetric measurements broadened the understanding of this system, regarded as a model system. Thus, future tailoring of functional groups designed for improved and more selective analyte detection is possible.

Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR.

Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH(2) as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 muM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. (1)H,(15)N heteronuclear NMR localised the interaction site across the beta sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.