Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

[Enzyme immunoassay of usnic acid in lichens].

An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families.

Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell.

Efaroxan induces membrane depolarization by interaction with the pore forming subunit of the ATP-sensitive potassium channel, Kir6.2. However, this effect is not responsible for its full secretory activity. In this study we have used an anti-idiotypic approach to generate antibodies that recognize additional proteins that may be regulated by efaroxan in pancreatic beta-cells. Using these antisera in an expression cloning strategy we have identified a monomeric GTP-binding protein, Rhes, as a potential target for regulation by imidazoline ligands. Rhes is shown to be expressed in beta-cells and its expression is regulated by efaroxan under conditions when a structurally related molecule, KU14R, is ineffective. The results reveal that beta-cells express Rhes and suggest that changes in the expression of this molecule may regulate the sensitivity of beta-cells to imidazoline secretagogues.

Immunochemical analysis for dioxins--progress and prospects.

Recent developments in antibody design and sample preparation have considerably enhanced the use of enzyme immunoassay (EIA) as an alternative to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of the trace organic pollutants polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in source and environmental samples. The EIA-specific sample preparation technique for solid matrices using a compatible extracting solvent coupled with an assay using a more sensitive antibody permits screening of source and environmental samples to be undertaken with minimal sample preparation. EIA has been validated on an increasing range of environmental and source samples as a cost effective I-TEQ screening tool, exploiting the commonly recognised general strengths of immunoassays such as speed, simplicity, low cost and parallel processing of many samples, greatly reducing the number of samples requiring conventional GCMS analysis. Further research and developmental work is required in the areas of sample extraction and extract cleanup to ensure compatibility with the immunoassay from the outset, and to standardise as far as is possible an assay format relevant to the application of interest, covering sample preparation, sample cleanup, quality control, assay procedure and data analysis.

Production of polyclonal antibodies for cyclopaldic acid, a major phytotoxic metabolite produced by the plant pathogen Seiridium cupressi.

An antiserum against cyclopaldic acid (CA), a phytotoxic metabolite produced by Seiridium cupressi, the fungal pathogen of a canker disease of cypress, was prepared by immunization of rabbits with a CA-bovine serum albumin conjugate (CA-BSA). Antibodies recognizing CA were purified by affinity chromatography through an immunoadsorbent prepared by conjugating CA to lysine-Sepharose 4B. The specificity of antibodies was assayed against CA and some of its derivatives by competitive indirect enzyme-linked immunoassay. The homologous antigen CA-BSA and CA showed the highest binding activity with antibodies. The derivatives of CA modified in 1 of the 2 aldehyde groups reacted with antibodies to a lesser extent compared with CA-BSA and CA. Isocyclopaldic acid and the tetraacetylderivative of CA, which have more than one modified functional group, showed a further decrease in their reactivity. Other derivatives, which have more substantial structural modifications of the carbon skeleton, did not react. The possible use of these antibodies in studies concerning the involvement of CA in the canker disease of cypress is discussed.