A Novel Peptide ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

BACKGROUND: Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met. METHODOLOGY/PRINCIPAL FINDINGS: Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA. CONCLUSION/SIGNIFICANCE: The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas

Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders using Supercritical or Near-Critical Fluids

Supercritical or near-critical fluid processes for generating microparticles have enjoyed considerable attention in the past decade or so, with good success for substances soluble in supercritical fluids or organic solvents. In this review, we survey their application to the production of protein particles. A recently developed process known as CO2-assisted nebulization with a Bubble Dryer® (CAN-BD) has been demonstrated to have broad applicability to small-molecule as well as macromolecule substances (including therapeutic proteins). The principles of CAN-BD are discussed as well as the stabilization, micronization and drying of a wide variety of materials. More detailed case studies are presented for three proteins, two of which are of therapeutic interest: anti-CD4 antibody (rheumatoid arthritis), α1-antitrypsin (cystic fibrosis and emphysema), and trypsinogen (a model enzyme). Dry powders were formed in which stability and activity are maintained and which are fine enough to be inhaled and reach the deep lung. Enhancement of apparent activity after CAN-BD processing was also observed in some formulation and processing conditions

\u201cChondroitin-4-sulphate exhibits inhibitory effect during Cu2+-mediated LDL oxidation\u201d.

AbstractChondroitin 4-sulphate (C4S), a basic component of the extracellular matrix of the artery wall, inhibited copper-induced low density lipoprotein (LDL) oxidation by prolonging the lag time and reducing the rate of propagation. LDL oxidation was assessed by monitoring conjugated dienes and low level chemiluminescence. A possible initial key reaction in LDL oxidation, the reduction of copper(II) to copper(I) by LDL, was decreased in the presence of C4S. Moreover, C4S protected tryptophan residues of Apo-B-100 in the early stage of LDL oxidation and during the subsequent propagation phase. The presence of the sulphate group in position 4 of N-acetylgalactosaminyl residues of C4S is crucial for protective activity. In fact, the structurally related chondroitin 6-sulphate, also present in tissues, had no effect on LDL oxidation. These data suggest that C4S may contribute to protect LDL against oxidation in arterial intima.© 1997 Federation of European Biochemical Societies