Modeling the antioxidant properties of the eye reduces the false-positive rate of a nonanimal eye irritation test (OptiSafe).

We recently identified a group of chemicals that are misclassified by most, if not all, in vitro alternative ocular irritation tests, suggesting that nonanimal tests may not fully model the ocular environment in which these chemicals interact. To address this, we evaluated the composition of tears, the first defense against foreign substances, and identified the presence of antioxidants that could detoxify reactive chemicals that otherwise may be falsely identified as irritants in alternative irritation tests. In this study, we evaluated the effects of tear antioxidants on the ocular irritation scoring of commonly overclassified chemicals (false positives) using the OptiSafe™ ocular irritation test. Six tear-related antioxidants were individually added to the OptiSafe formulation, and the effects on test outcome were determined. Ascorbic acid, the most abundant water-soluble antioxidant in tears, specifically reduced the OptiSafe false-positive rate. Titration curves showed that this reduction occurs at in vivo concentrations and is specific to chemicals identified either as producing reactive oxygen species or as crosslinkers. Importantly, the addition of tear antioxidants did not impact the detection of true negatives, true positives, or other false positives unassociated with reactive oxygen species or crosslinking. These results suggest that the addition of tear antioxidants to in vitro alternative test systems may substantially reduce the false-positive rate and improve ocular irritant detection.

Performance characteristics of colorimetric protein microarrays compared to ELISA.

Miniaturization and multiplexing are two areas that need improvement in immunoassays. However, it is unclear how associated changes in size, substrate, binding capacity, protein concentration, and other parameters affect sensitivity and quantitative range. In the study here we compared a new protein microarray substrate and system (Zeta-Grip, Miragene Inc., Anaheim, CA) with enzyme-linked immunosorbent assay (ELISA) for the application of immunoassays. We found that the protein microarray system performed better than ELISA in both sensitivity and quantitative range for use in immunoassay.

Development of a sensitive, colorometric microarray assay for allergen-responsive human IgE.

With the existence of several thousand unique human allergens, a multiplex format, such as protein microarrays, is an attractive option for allergy screening. To determine the feasibility and sensitivity of using an enzyme-based, colorimetric protein microarray assay, three common allergens (mold, dust-mite, grass) were arrayed and added sera assayed for responsive human IgE. Normal, low positive, and negative control samples were assayed to determine optimal reaction parameters. Sensitivity of the assay (in international units, IU) was determined by constructing a standard curve using World Health Organization (WHO) standards. The system described here can reliably detect allergen-specific IgE below 0.35 IU, the current WHO standard cutoff. By taking advantage of the sensitivity of enzyme-linked immunosorbent assays (ELISAs) and the multiplex format of microarrays, we have achieved a high throughput system, capable of screening patients for allergen-susceptibility with optimal sensitivity.

Development of a protein microarray library and a system for rheumatoid-arthritis disease marker screening.

Early detection and intervention can delay the onset of symptoms of RA (rheumatoid arthritis), but current diagnostic tests suffer from low sensitivity and specificity, making such early disease detection difficult. Analysis of body fluids for the identification of multiple molecular markers and the subsequent determination of modifications in their associated protein structures emerges as a possibility for early detection. The structural modifications of these markers are thought to play a pivotal role in defining the pathological state of diseased joints. Therefore a proteome library and system for RA disease screening has been developed and investigated in the present study. The large (39 S) mammalian ribosomal subunit has been identified as a potential RA marker. This protein is known as L 35, and antibodies to this protein have been found to be expressed in patients suffering from RA.

Development of an individual-specific autoantibody (ISA) protein microarray.

We have developed an ISA (individual-specific autoantibody) identification method to identify biological samples based on an individual's unique class of autoantibodies. This method involved the presentation of human proteins derived from crude lysates after SDS/PAGE separation and transferance to a solid support. In the present study, ISA strips are produced and developed on a new protein microarray. In making the ISA strips, it was found that variation in protein migration during electrophoresis and strip-manufacturing quality control limit the reliability of the assay. Therefore it was decided to semi-purify and separate proteins by column chromatography in large batches and to develop an ISA-specific protein microarray. It was found that this ISA protein microarray approximates a similar serum titre as the ISA strip and is predicted to circumvent the batch-to-batch production issues related to SDS/PAGE.