A novel sensitive immunoassay method based on the Invader technique.

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-alpha (hTNF-alpha). Detection limits obtained were 0.1 pg/ml hTNF-alpha when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-alpha enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.

4-Hydroxynonenal modifies IgA in rat intestine after lipopolysaccharide injection.

The lipid peroxide 4-hydroxynonenal (HNE) was measured in rat intestinal mucosa after lipopolysaccharide (LPS) injection (0.5 mg/kg, ip) by a highly sensitive time-resolved fluoroimmunoassay. HNE was increased, with a small peak at 20 min followed by a sustained elevation at 2-4 h, after injection of LPS. Immunohistochemistry demonstrated enhanced labeling with anti-IgA and anti-HNE antibodies in the plasma cells followed by diffusion of the labeled materials into the submucosal tissue after LPS injection. Immunoprecipitation with anti-IgA antibody and Western blotting with anti-HNE antibody showed that IgA is modified with HNE after LPS injection. The HNE (5 microM-5 mM) modification in vitro reduced the bactericidal activity of IgA and anti-Escherichia coli serum. The HNE modification in vitro also promoted polymerization of IgA as shown by nondenaturing gel electrophoresis. This is the first demonstration of the modification of IgA with HNE in an in vivo model of intestinal inflammation as well as in vitro effects of HNE on bactericidal activity and polymerization of IgA. These findings will help in understanding the involvement of oxidative stress in the IgA-mediated immune response exerted by plasma cells in early intestinal inflammation.

Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis of luminescent lanthanide chelate-labeled proteins with time-resolved detection.

Proteins labeled with a luminescent lanthanide chelate, {{2,2',2'',2'''-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2':6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu(3+)),were analyzed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis with a time-resolved fluorometric detector. The metal ion of the luminescent lanthanide chelate did not dissociate from the ligand during electrophoresis, and the luminescence was retained. On a slab gel, the band of DTBTA-Eu(3+)-labeled streptavidin was apparently less broad than that of AlexaFluor 488-labeled streptavidin. DTBTA-Eu(3+) in SDS-PAGE slab gel is stable, and the gel after electrophoresis can be dried and stored for at least one year without luminescence fading. In capillary gel electrophoresis (CGE), five labeled proteins with different molecular weight were separated, and a good correlation was observed between the molecular weight and the migration time. Although the detection limits of these proteins determined in buffer solutions of the capillary electrophoresis were not better than those reported for CGE with laser-induced-detection, the detection limits of the same proteins in the present CGE system were not significantly deteriorated in serum solutions compared to those in buffer solutions, and the advantage of using time-resolved detection has been shown.

Application of a lanthanide fluorescent chelate label for detection of single-nucleotide mutations with peptide nucleic acid probes.

We developed the approach to detect single-nucleotide mutation with peptide nucleic acid (PNA) probes and time-resolved fluorometry using a fluorescence lanthanide chelate label, {2,2',2'',2'''-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2': 6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu3+). Compared with DNA probes, PNA probes showed lower mismatch signals and gave higher signal/noise (S/N) ratios. Using the system, we examined the single-nucleotide mutations of codon 12 in the c-Ha-ras gene of PCR amplicons of genome DNAs isolated from human umbilical vein endothelial cells (HUVECs) and T24 cells.

New luminescent europium(III) chelates for DNA labeling.

The new europium(III) chelate [2,2',2'',2'''-[[4'-(aminobiphenyl-4-yl)-2,2':6',2''-terpyridine- 6,6''-diyl]bis(methylenenitrilo)]tetrakis(acetato)] europium(III) (ATBTA-Eu3+) and its 4,6-dichloro-1,3,5-triazinyl and succinimidyl derivatives (DTBTA and NHS-ATBTA, respectively) were synthesized and characterized. Both labeling complexes DTBTA-Eu3+ and NHS-ATBTA-Eu3+ are luminescent. Especially DTBTA-Eu3+ is strongly luminescent, with a luminescence quantum yield of 9.1%, molar extinction coefficient of 3.1 x 10(4) cm(-1) M(-1) (335 nm), and luminescence lifetime of 1.02 ms. The excitation and emission maximum wavelengths of DTBTA-Eu3+ are 335 and 616 nm, respectively. The complex is very stable in aqueous buffers, with a conditional formation constant log K(DTBTA-Eu) of 25.0 at pH 8, and can be conjugated to DNA and proteins. The chelates are also highly resistant to thermal decomposition, photodegradation, and ozone oxidation. These properties prove that DTBTA-Eu3+ is suitable as a luminescence label in DNA assays.