Enzyme-linked Sandwich Assay Research Articles

Electrochemical Enzyme-Linked Sandwich Assay with a Cellulase Label for Ultrasensitive Analysis of Synthetic DNA and Cell-Isolated RNA.

Electrochemical enzyme-linked sandwich assays on magnetic beads (MBs) refer to one of the most sensitive approaches for analysis of nonamplified nucleic acid samples, with redox enzymes being routinely used as labels. Here, we report a sensitive and inexpensive electrochemical nucleic acid sandwich assay on MBs that exploits a hydrolytic enzyme cellulase as a label, while MBs are used for preconcentration and bioseparation of analyzed samples. Binding of target DNA or RNA to capture DNA-modified MB triggers sandwich assembly and its labeling with cellulase. Application of the assembled sandwich to the electrodes covered with insulating nitrocellulose films induces film digestion by the cellulase label and pronounced changes in the electrical properties of the electrodes, the extent of the changes being proportional to the concentration of the analyzed nucleic acids. Down to 1 amol of Lactobacillus brevis specific synthetic DNA and rRNA isolated from L. brevis cells could be detected in 1 mL samples in the overall from 2 to 3 h assay. The assay is universal and can be adapted for point-of-care-testing and for in-field environmental and microbiomic analysis of unamplified samples of any DNA/RNA sequences.

High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins

Efforts to elucidate the role of lipoprotein [a] (Lp[a]) in atherogenesis have been hampered by the lack of an animal model with high plasma Lp[a] levels. We produced two lines of transgenic mice expressing apolipoprotein [a] (apo[a]) in the liver and crossed them with mice expressing human apolipoprotein B-100 (apoB-100), generating two lines of Lp[a] mice. One had Lp[a] levels of approximately 700 mg/dl, well above the 30 mg/dl threshold associated with increased risk of atherosclerosis in humans; the other had levels of approximately 35 mg/dl. Most of the LDL in mice with high-level apo[a] expression was covalently bound to apo[a], but most of the LDL in the low-expressing line was free. Using an enzyme-linked sandwich assay with monoclonal antibody EO6, we found high levels of oxidized phospholipids in Lp[a] from high-expressing mice but not in LDL from low-expressing mice or in LDL from human apoB-100 transgenic mice (P <0.00001), even though all mice had similar plasma levels of human apoB-100. The increase in oxidized lipids specific to Lp[a] in high-level apo[a]-expressing mice suggests a mechanism by which increased circulating levels of Lp[a] could contribute to atherogenesis.

An enzyme-linked oligonucleotide assay.

The recent development of in vitro methods to select high-affinity ligands by combinatorial chemistry methodologies promises unique and theoretically unlimited supplies of novel therapeutic and diagnostic reagents. One such combinatorial chemistry process, systematic evolution of ligands by exponential enrichment (SELEX), allows rapid identification, from large random sequence pools, of the few oligonucleotide sequences that bind to a desired target molecule with high affinity and specificity. We describe an enzyme-linked sandwich assay that uses a SELEX-derived oligonucleotide. This assay demonstrates that these oligonucleotides can be effective and useful analytical reagents.

A colourimetric enzyme-linked sandwich assay for the detection of hunan platelets bound to a fibrinogen-coated surface

A colourimetric enzyme-linked sandwich assay has been developed to investigate the binding of human platelets to fibrinogen. The presence of platelets bound to fibrinogen-coated plastic can easily be detected and quantitated. Platelets treated with chymotrypsin to expose the fibrinogen receptor, are fixed with paraformaldehyde, and stored frozen. The detection sandwich consists of a mouse monoclonal antibody directed against the human platelet CD9 antigen, and a rabbit anti-mouse immunoglobulin conjugated to the enzyme alkaline phosphatase. The cleavage of the phosphatase substrate p-nitrophenyl phosphate can be monitored colourimetrically. The data presented provide evidence that this method is capable of detecting platelet-fibrinogen binding in a physiologically relevant manner. The binding is inhibited by EDTA or excess fibrinogen. The fibrinogen α and γ chain peptides, RGDS and LGGAKQAGDV, and the snake venom echistatin are also inhibitory with IC 50 values of 135 μM, 1.8 mM and 100 nM respectively.