Evaluation of a multiplex ELISA for autoantibody profiling in patients with autoimmune connective tissue diseases.

The performance of immunoassays for the detection of autoantibodies is of critical importance in the diagnosis and assessment of patients with autoimmune connective tissue diseases (ACTD). Our objective was to compare the features of two multiplexed assays-INNO-LIA ANA and Gennova-PictArray ENA ELISA-for measurement of multiple autoantibodies and their utility as a clinical tool in ACTD diagnosis. The antigens included SS-A/Ro (60 and 52), SSB/La, Sm, Sm/RNP, CENP-B, Jo-1, and Scl-70. Stored sera from 85 ACTD patients and 80 controls consisting of patients with vasculitis, rheumatoid arthritis and infectious diseases, as well as healthy subjects were analyzed jointly with clinical and laboratory data. Agreement between the two methods varied between 58 and 99% (Cohen's kappa: 0.21-0.71) mostly for SSA and SSB. The frequency of specific autoantibodies measured using the two methods was more variable for SSA, SSB, and RNP/Sm. There were a higher number of ambiguous results when using INNO-LIA. The optimized cut-off values of the Gennova-PictArray resulted in over 99% specificities in samples obtained from the control group. Sensitivity patterns were more accurate in Gennova-PictArray than in INNO-LIA, as suggested in previously reported studies. A third method could be applied to determine which of the two methods is more accurate.

Inexpensive optical system for microarray ELISA.

The use of antibody-based diagnostic testing has increased significantly over the past decade, giving rise to a wide range of diagnostic devices. At one end of the cost-range are rapid inexpensive point-of-care tests based on immunochromatographic strips which provide a qualitative positive or negative test outcome. On the other hand, quantitative tests generally require the use of dedicated and expensive laboratory instruments. There remains a need for diagnostic instruments and tests that can provide quantitative assessment of disease markers at low cost. This paper describes the development of a novel low cost optical device for reading colorimetric and fluorescent immunodiagnostic test results. This portable instrument uses a webcam to capture test results from a specially designed 16-well slide containing a miniaturized array of test spots. Arrays are illuminated with either LEDs or lasers, while transmitted or emitted light is captured through a long-pass filter, allowing two different types of optical measurement to be performed within the same device. This device was used to read results from an array of antibodies conjugated with either an enzymatic or fluorescent tag resulting in a colored or fluorescent readout.

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne.

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.

An update on using protein microarrays in drug discovery.

Protein microarrays are evolving as useful tools for biopharmaceutical research. The differences in characteristics of individual proteins has made development challenging compared with DNA arrays. Nonetheless, significant advances have nontheless been made in developing protein microarray technology. Retention of function has been demonstrated for proteins belonging to various structural and functional classes after arraying. Focused arrays with small groups of proteins have been developed for a variety of applications, from biomarker validation to small molecule screening. Issues of protein stability as well as assay specificity and sensitivity, are being worked out for panels of arrayed proteins. The development of robust manufacturing methods has resulted in an increase in the number of commercially available protein array products. Quality control guidelines, which will also aid in accelerating development of the technology, are being established.

Simultaneous analysis of eight human Th1/Th2 cytokines using microarrays.

The adaptive immune system induces T cells to change from a naive phenotype to a Th1/Th2 phenotype each of which produce characteristic types of cytokines. Knowledge of whether a specific immune response is Th1 or Th2 is a useful indicator for diseases with basis in immune function disorder. An assay that can rapidly analyze multiple cytokines indicative of these two cell types from small sample quantities can be an extremely useful research and diagnostic tool. Silanized glass slides were printed with multiple arrays of capture antibodies to detect eight different cytokines involved in the Th1/Th2 response along with control proteins for assessing assay performance. Arrays were developed by sequential addition of known antigen amounts, detector antibodies and a fluorescent detection system followed by imaging and quantification. These arrays were used to determine the specificity, sensitivity and reproducibility of the assay and the performance compared with conventional ELISA. This multiplexed assay is able to measure human Th1/Th2 cytokines in sample volumes lower than 20 microl. The assay sensitivity for the eight cytokines range from 0.3 microg/l for IL-4 to 6.4 microg/l for IL-5 which are either comparable to or higher than those reported for conventional ELISA or bead-based multiplex ELISA methods. This assay can be automated to measure expression levels of multiple Th1/Th2 cytokines simultaneously from tens to hundreds of biological samples. This assay platform is more sensitive and has a larger dynamic range as compared to a conventional ELISA in addition to significantly reducing the time and cost of assay. This platform provides a versatile system to rapidly quantify a wide variety of proteins in a multiplex format.