An ImmunoChip prototype for simultaneous detection of antiepileptic drugs using an enhanced one-step homogeneous immunoassay.

The development and characterization of a one-step homogeneous immunoassay-based multiwell ImmunoChip is reported for the simultaneous detection and quantitation of antiepileptic drugs (AEDs). The assay platform uses a cloned enzyme donor immunoassay (CEDIA) and a Beta-Glo assay system for generation of bioluminescent signal. Results of the one-step CEDIA for three AEDs (carbamazepine, phenytoin, and valproic acid), in the presence of serum, correlate well with the values determined by fluorescence polarization immunoassay. CEDIA intra- and interassay coefficients of variation are less than 10%. A microfabrication process, xurography, was used to produce the multiwell ImmunoChip. Assay reagents were dispensed and lyophilized in a three-layer pattern. The multiwell ImmunoChip prototype was used to detect and quantify AEDs in serum samples containing all three drugs. Luminescent signals generated from each well were recorded with a charge-coupled device (CCD) camera. The assays performed on an ImmunoChip were fast (5 min), requiring only small volumes of both the reagents (<1 microl/well) and the serum sample. The ImmunoChip assay platform described in this article may be well suited for therapeutic monitoring of drugs and metabolites at the point-of-care setting.

Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors.

Application of immunoassay to biosensors for use in the point-of-care setting ideally requires immunoassay without separation steps and with small volumes of both sample and reagents. The suitability of cloned enzyme donor immunoassay (CEDIA), one of a few homogeneous immunoassays available, was investigated for application to biosensors. This method is based on the bacterial enzyme beta-galactosidase, which has been genetically engineered by others into two inactive fragments, enzyme donor (ED) and enzyme acceptor (EA). Association of the ED and EA fragments in the assay results in formation of active enzyme, which acts on substrate to generate a detectable signal. Sensitivity of commercially available CEDIA kits were compared, with respect to the sample and reagent volumes, using three different signal generation processes. The CEDIA kit for valproic acid and three substrates, a colorimetric (chlorophenol red-beta-D-galactopyranoside), a chemiluminescent (Lumi-Gal 530), and a bioluminescent (Beta-Glo Assay System), were employed in the study. Our results indicate that the high sensitivity of the bioluminogenic substrate, D-luciferin-O-beta-galactopyranoside, with short assay time and small volumes of sample and reagents required for the assay, simple handling, and relatively low expense, make this substrate, together with CEDIA, suitable for application to biosensors intended for drug and metabolite monitoring in the point-of-care setting.

The inhibitory activity of serum to prevent bacterial adhesion is mainly due to apo-transferrin.

A marked, up to 5-fold, reduction in bacterial adhesion to Tecoflex polyurethane (PU) surfaces was observed in the presence of bovine/human serum or plasma at 0.5% or higher concentrations in the medium. Further investigation of the phenomenon resulted in identification, isolation, and characterization of the serum component with the ability to significantly reduce bacterial adhesion. Upon fractionation of bovine serum by an anion exchange chromatography, protein pools were made and analyzed by immunoelectrophoresis and by polyacrylamide gel electrophoresis in the presence of SDS and were examined for their effect on the adhesion of Staphylococcus epidermidis to PU surfaces. The pool exhibiting a significant inhibitory effect was subjected to further biochemical tests, which resulted in the identification of transferrin (Tf) as its predominant protein. Bacterial adhesion studies in the presence of purified Tf revealed that holo-Tf (iron-containing form) had no influence on bacterial adhesion at any concentration. Only apo-Tf (iron-lacking form) exerted the inhibitory effect, in a dose responsive manner at concentrations of 10 microg/mL or higher. Bacteria remained viable when suspended at the low apo-Tf concentrations, sufficient to prevent bacterial adhesion.

The effect of apo-transferrin on bacterial adhesion to biomaterials.

Apo-transferrin (apo-Tf), the iron deficient form of Tf, has been identified previously as a potent inhibitor of Staphylococcus epidermidis adhesion to polyurethane surfaces. In this study, the ability of apo-Tf to suppress the adhesion of two other strains of bacteria, namely a Gram-positive Staphylococcus aureus and a Gram-negative Pseudomonas aeruginosa to several biomaterials, including polystyrene, polymethylmethacrylate, and silicone, is documented. The presence of apo-Tf in the medium at 20 microg/ml lowered bacterial adhesion to all tested biomaterials more than fourfold. Moreover, apo-Tf exerted its inhibitory activity even when protein coated surfaces were used as substrates for bacterial adhesion. To emphasize the importance of apo-Tf in the prevention of bacterial adhesion, human serum was depleted of Tf, employing affinity chromatography, and was shown to lose its inhibitory activity toward bacterial adhesion. Upon addition of apo-Tf to Tf-depleted serum, the activity was reestablished, resulting in a marked reduction in the number of bacteria adhered to the surfaces. Following the enzymatic deglycosylation, apo-Tf retained its ability to prevent bacterial adhesion. These results indicate that the carbohydrate moiety does not seem to play a role in this activity. The presented data provide the evidence that the inhibitory activity of apo-Tf is not bacterial strain specific and that the presence of apo-Tf in the medium results in a significant reduction of bacterial adhesion to a variety of neat and/or protein coated surfaces.