Development of surface plasmon resonance-based immunoassay for aflatoxin B(1).

Aflatoxins are a group of highly toxic fungal secondary metabolites that occur in Aspergillus species and may contaminate foodstuffs and feeds. Two different anti-aflatoxin B(1) antibodies were examined to develop a surface plasmon resonance (SPR)-based immunoassay to aflatoxin B(1). A conjugate consisting of aflatoxin B(1)-bovine serum albumin (BSA) was immobilized on the dextran gel surface. Competition between immobilized aflatoxin B(1) conjugate and free aflatoxin B(1) in solution for binding to antibody injected over the surface formed the basis for the assay. Regeneration of the antibody from the immobilized conjugate surface is essential for the development of such an inhibitive immunoassay. Problems were encountered with the regeneration of the sensor surface, due to the high-affinity binding of the antibodies. Conventional regeneration solutions consisting of low concentrations of NaOH and HCl worked to a degree, but regeneration was at the expense of the integrity of the immobilized conjugate. A polyclonal anti-aflatoxin B(1) antibody was produced and was found to be regenerable using an organic solution consisting of 1 M ethanolamine with 20% (v/v) acetonitrile, pH 12.0. This combined high ionic strength and extreme pH, as well as chaotrophic properties and allowed the development of an inhibitive immunoassay. The assay had a linear range of 3.0-98.0 ng mL(-1) with good reproducibility.

Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody.

Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.

The effects of continuous superfusion of L-aspartate and L-glutamate on horizontal cells of the turtle retina.

We have studied the effects of prolonged superfusion with L-aspartate and L-glutamate on the membrane potential and photoresponses recorded in luminosity type horizontal cells in the turtle retina using an everted eyecup preparation. These acidic amino acids produce effects which are a function of the past history of the impaled cell. Initial prolonged superfusions with 30 mM of either drug has no pronounced effect on the membrane potential and photoresponses of horizontal cells. Subsequent superfusions with either agent eventually produce depolarizations with reductions in the amplitudes of the light evoked responses. These effects, however, are transient; the horizontal cell rehyperpolarizes and the light evoked response grows with time. In a retina which had been stored at 4 degrees centrigrade for 20 hours, an initial superfusion with L-aspartate solution produced an immediate depolarization of the horizontal cell and complete suppression of the light evoked response for as long as the amino acid was present. The data are consistent with the existence of powerful amino acid uptake mechanisms operating at a number of sites within the inner and outer retina but also raise questions about the role of acidic amino acids in the outer plexiform layer of the turtle retina.

Mixing of color signals by turtle cone photoreceptors.

The direct mixing of red and green cone signals in the outer plexiform layer of the turtle retina was studied by using intracellular recordings from red cone photoreceptors. Cone photoresponses were a function of the wavelength of the photons that stimulated them, even when small-diameter stimuli were used. The intensity response curves measured with red and green test flashes had different shapes. The kinetics of approximately equal amplitude red and green responses also differed. To quantify the short wavelength input onto red cones, differential chromatic adaptation was used. The relative sensitivity of the red cone to red and green test flashes was a function of the color and intensity of the background illumination; red backgrounds decreased relative red sensitivity, and green backgrounds increased relative red sensitivity. The spectral sensitivity of the additional short wavelength input onto red cones was determined by using differential chromatic adaptation, and was found to peak approximately 550 nm. We conclude that red cones receive an additional excitatory input from green cones (and possibly blue cones). A model of the cone mosaic suggests that approximately 50% of the red cone response (linear range) to a dim green test flash arises from neighboring green cones.

Temporal information processing in cones: effects of light adaptation on temporal summation and modulation.

We have studied the temporal information processing of turtle cones in steady states of light adaptation using intracellular recording techniques. We measured the linear range incremental sensitivity of cones as a function of the stimulus duration. Linear range incremental sensitivity is a function of the background intensity. It is also proportional to the duration of short duration stimuli but is independent of duration for long duration stimuli. The plot of log sensitivity versus log stimulus duration displays two straight line asymptotes; a slope of one for short durations and a slope of zero for long durations. These asymptotes intersect at a time, the critical duration, which decreases with increasing background intensity. Linear systems theory was used to predict these results in addition to the interdependence of critical duration, response kinetics, and sensitivity for any state of adaptation. We have also calculated cone sensitivity as a function of sinusoidal frequency for a variety of background intensities. Correlations between these results and psychophysical studies suggest that the limits on temporal summation established by the cones appear not to be substantially altered by the rest of the retina.

Chromatic and spatial information processing by red cones and L-type horizontal cells in the turtle retina.

Flash sensitivities to red (694 nm) and green (546 nm) light were measured intracellularly from red cones and L-type horizontal cells in the turtle retina. The horizontal cells' sensitivity to short wavelength light was significantly larger than expected from the model where horizontal cells receive input mainly from red cones. The red cone responses to chromatic stimuli were strongly dependent upon the flash colour and size. Increasing the spot size reduced the sensitivity to green relative to red and augmented the differences in kinetics between the response to red light and that evoked by green light. It was concluded that L-type horizontal cells received a short wavelength input in addition to the input from red cones. This additional green input coupled with the negative feedback pathway from horizontal cells onto red cones determined the differential spatial properties of the red cone responses to red and green test flashes.

Direct excitatory interactions between cones of different spectral types in the turtle retina.

Cone linear sensitivities to red and green stimuli were measured intracellularly in the dark- and light-adapted turtle retina. Test flashes of small diameter were used to minimize horizontal cell feedback. Light adaptation was achieved with either green or red background illumination. The ratio of cone sensitivities to red and green light depended on the color of the background light and differed from the ratio measured in the dark. Electron microscope studies of Golgi-stained turtle cones revealed direct synaptic connections between red and green cones mediated by cone telodendria. These data indicate that the red cone photoresponse is not univariant as has been previously supposed and suggest that mixing of signals from different spectral classes of cones can occur via direct excitatory connections between cones.