SYBR Green I: fluorescence properties and interaction with DNA.

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.

Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation.

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (~1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity.

Metal-enhanced PicoGreen fluorescence: application for double-stranded DNA quantification.

PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 microM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification.

The Dami cell possesses the platelet collagen receptor VLA-2, but does not mobilize calcium.

Flow cytometric and Western blot analysis showed that Dami cells possess the major platelet collagen adhesion receptor, the integrin VLA-2, and that VLA-2 was expressed in higher levels in a time-dependent manner in DMSO-induced Dami cells. Both control and DMSO-induced Dami cells were able to adhere to collagen as measured in a microtiter-based adhesion assay. It appeared that collagen adhesion was solely mediated by VLA-2, since inclusion of a monoclonal antibody directed against the alpha-2 subunit of VLA-2 in the adhesion assay was able to totally inhibit adhesion. Although Dami cells possess a variety of platelet markers, and are able to mobilize intracellular calcium in response to ADP, U-46619, and thrombin, they were unable to respond to collagen challenge. We concluded that Dami cells may lack some key transducing element present in platelets that prevents them from being activated by collagen.

Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA).

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.

Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin.

Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin. Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution. All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains. These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue. The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA. Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule. The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay. Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin. Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes.

Rapid detection of Clostridium perfringens type A enterotoxin by enzyme-linked immunosorbent assay.

Clostridium perfringens type A enterotoxin was specifically detected and readily quantified by indirect and four-layer sandwich enzyme-linked immunosorbent assays (ELISAs). With the indirect ELISA, enterotoxin was detected in quantities of as low as 2.5 ng (25 ng/ml). When the more sensitive sandwich ELISA procedures was used, 100 pg (1 ng/ml) of enterotoxin was detected. The sandwich ELISA procedure specifically detected enterotoxin in human fecal extracts. Additionally, the sandwich ELISA specifically differentiated enterotoxin-positive strains from enterotoxin-negative strains of C. perfringens. Both the indirect and sandwich ELISA procedures described for C. perfringens enterotoxin in this report are rapid, specific, sensitive, and easily adaptable for large-scale use by clinical or research laboratories.