An Open Sandwich Immunochromatography for Non-competitive Detection of Small Antigens.

Immunochromatography assay is an easy and rapid on-site detection method. However, conventional sandwich immunochromatographies using two antibodies can only detect target molecules above a threshold size. Small molecules below 1000 in molecular weight are usually detected using competitive immunoassay. However, competitive immunoassay is not suitable for visual detection of low concentration samples. Based on the principles of open sandwich immunoassay, which detects small molecules via interchain interaction of separated variable region fragments (VH and VL) from a single antibody, we developed non-competitive open sandwich immunochromatography. Bone Gla protein (BGP)-C7, a peptide containing the seven C-terminal amino acids of human osteocalcin, was selected as the target. By using VL fragments fixed on a nitrocellulose membrane, and colored cellulose bead-labeled VH fragments, we specifically detected 10 ng/mL of BGP-C7. This is the first report of open sandwich immunochromatography, which is an easy and rapid method for on-site, signal-on detection of small molecules.

Significance of S100A4 as a prognostic marker of lung squamous cell carcinoma.

BACKGROUND: The prognostic significance of S100A as potential biomarker for breast cancer was reported; however, this finding has recently been challenged. Here, the aim was to assess whether S100A4 could also be a prognostic biomarker of lung cancer. MATERIALS AND METHODS: A specific high-titer anti-S100A4 monoclonal antibody was developed. The utility and specificity of this antibody was validated by immunostaining experiments. The antibody was tested against a newly developed high-density tissue microarray including 400 lung cancer tissues to examine the clinico-pathological and prognostic significance of S100A4 in lung cancer. RESULTS: The staining of S100A4 was significantly associated with patients' poor prognosis in lung squamous cell carcinoma but not lung adenocarcinoma. CONCLUSION: S100A4 seems to be a prognostic biomarker of lung squamous cell carcinoma (5-year survival rate of 38.5% versus 7.4%, p<0.01), but not of adenocarcinoma.

Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) mediated PCR clamping.

Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57 degrees C and 45 degrees C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.