Modelling of the immunoassay element of an optical immunosensor.

Several factors affect the viability of biosensor design. A computer-based model is being developed to enable the sources and effect of noise and variability within the sensor to be analysed. The work now presented details the modelling of the biochemical aspect of the biosensor model-the immunoassay. The equilibrium equations that describe the chemical reactions that occur when a sample containing the analyte is added to the immunosensor are cast as a sum of squares function that can be minimized using an optimization procedure. The optimization returns the concentrations of each species at equilibrium and the procedure is incorporated within a Monte Carlo simulation, which allows the variations in the resulting concentrations to be determined. Three classes of optimization technique are considered, classical regression techniques and two intelligent optimization techniques: simulated annealing and genetic algorithms. Several methods of imposing constraints are implemented and the issue of local minima is discussed. Classical regression procedures were found to be superior to the intelligent optimizations examined.

Failure of meperidine to anesthetize human median nerve. A blinded comparison with lidocaine and saline.

BACKGROUND AND OBJECTIVES: Although meperidine safely produces clinical spinal anesthesia, the responsible mechanism is unknown. This study was undertaken to test the possibility that this drug acts as a local anesthetic by investigating its ability to inhibit conduction in a human peripheral nerve. METHODS: In a blinded fashion, the abilities of 5-mL injections of meperidine (0.5% and 1.5%), lidocaine (0.25%), and saline to produce median nerve block were tested in eight volunteer subjects, and these four solutions were compared with standard local anesthetic solutions that had been tested in previous studies. The extent of local anesthesia was measured objectively by electrodiagnostic tests, namely, compound motor action potentials (CMAPs) and sensory nerve action potentials (SNAPs), as well as by qualitative tests of sensation. RESULTS: Lidocaine (0.25%) prolonged median SNAP latency from 3.1 ms to 3.3 ms (P < .015) and prolonged mean CMAP latency from 4.1 ms to 4.7 ms (P < .002). The SNAP amplitude trended downward after lidocaine (0.25%), but the decrease did not reach statistical significance (35 microV to 25 microV, P < .19). Neither meperidine solution (0.5% or 1.5%) nor saline inhibited SNAP or CMAP amplitudes or prolonged SNAP or CMAP latencies. Also, in contrast to previous findings with more potent local anesthetic solutions (eg, lidocaine 1%, mepivacaine 1%, and bupivacaine 0.33%), none of the four solutions tested in this study altered subjective sensations of hot, cold, or pinprick. Meperidine 1.5% produced systemic side effects, including vertigo, nausea, and flushing, in all subjects. CONCLUSIONS: Meperidine produced no signs of local anesthesia, even when given at a dose (75 mg) and concentration (1.5%) that consistently produced systemic side effects. Thus, the coequivalent ability of meperidine and lidocaine to produce spinal anesthesia contrasts with their discordant ability to produce local anesthesia. This disparity suggests that meperidine may produce spinal anesthesia through mechanisms other than inhibition of sodium channel function.

A Kalman filter algorithm and monitoring apparatus for at-line control of fractional protein precipitation.

Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. (c) 1997 John Wiley & Sons, Inc.

Isolation of decidual lymphocytes from chorionic villus samples: phenotypic analysis and growth in vitro.

PROBLEM: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CONCLUSION: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

Metal phosphate planar waveguides for biosensors.

Hard, impermeable, glassy, metal phosphate films have been fabricated inexpensively by the use of a spin-coating and low-temperature-curing technique. Films that are suitable for use as monomode waveguides in biosensors have been identified through an examination of the optical and chemical properties of films containing Fe, Al, Ga, In, Cr, or V. The refractive index is controlled over the range 1.49-1.78 by varying the film composition. The film thickness is controlled over the range 50-1200 nm by varying the spin speed and the deposition temperature. Films can be patterned by photolithography or by embossing. Input coupling through an embossed grating of 833-nm pitch is demonstrated.

Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination.

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film antimony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)M to 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.

Mechanism of lymphocyte activation: the binding of phytohemagglutinin to the lymphocyte surface.

The dynamics of phytohemagglutinin (PHA)-lymphocyte interaction was studied using 125I-labeled PHA (leucoagglutinin) and pig mesenteric lymph node lymphocytes that had been depleted of erythrocytes, dead cells, adherent cells and immunoglobulin-bearing cells. Evidence was obtained that PHA stimulated the majority of the lymphocytes to transform. Binding of PHA at 37 degrees C was fairly rapid (rate constant for association: 2.6 X 10(5) M-1 sec-1), saturable, reversible and specifically inhibited by N-acetylgalactosamine (Kdiss: 3 X 10(-4) M) and unlabeled PHA. A Scatchard plot was curvilinear and gave evidence for 3.6 X 10(5) binding sites per cell comprising 8.7% of high affinity sites (Kdiss: 3.7 X 10(-9) M) and 91.3% of lower affinity (Kdiss: 1.4 X 10(-7) M). About 20% of the sites were occupied under culture conditions giving maximal transformation. Alternative explanations for the curvilinear plot included negative cooperative interactions and/or increase in affinity through multivalent interaction. Negative cooperativity was supported by the demonstration that free PHA promoted the dissociation of bound PHA. Binding was not affected by metabolic inhibitors, and binding to purified lymphocyte plasma membrane resembled that to whole cells. These results suggested that PHA binding to whole lymphocytes was not grossly influenced by "capping", endocytosis and shedding.

The prediction of the conformation of membrane proteins from the sequence of amino acids.

The methods of Chou & Fasman [Biochemistry (1974) 13, 211-222, 222-245] and of Lim [J. Mol. Biol. (1974)88, 857-872, 873-894] for predicting secondary structure from amino acid sequence have been applied to five predominantly helical membrane-associated peptides. The predictions from the method of Lim (1974a,b) are consistent with the experimental observations, whereas those from Chou & Fasman (1974a,b), although not inconsistent with alpha-helix, favour a beta-structure for several very hydrophobic regions. The results may be rationalized in terms of the effect of the solvent on the conformation of a polypeptide.

The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes.

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.