Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods.

BACKGROUND: Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of the E4 allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous. METHODS: Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels. RESULTS: Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for the E4 allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity. CONCLUSIONS: This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual's APOE4 status directly from plasma. The E4 genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer's disease. The biochip's potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-based APOE genotyping and may deliver additional protein-specific information on a variety of diseases in the future.

Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay.

A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis™ SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 µg kg(-1). The detection capability was below 0.5 µg kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 µg kg(-1) for SEM. IC(50) values ranged from 0.14 µg kg(-1) (AMOZ) to 2.19 µg kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology.

Stable competitive enzyme-linked immunosorbent assay kit for rapid measurement of 11 active beta-lactams in milk, tissue, urine, and serum.

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.

Application of Evidence Investigator for the simultaneous measurement of soluble adhesion molecules: L-, P-, E- selectins, VCAM-1, ICAM-1 in a biochip platform.

The semiautomated Evidence Investigator has been applied to the simultaneous specific measurement of soluble adhesion molecules: L-, P-, E- selectins, VCAM-1, ICAM-1 using a reduced volume of sample. The biochip is the solid support and vessel where the sandwich immunoassay takes place. Signal detection, imaging, data processing, and storage are fully automated. Calibration curves are generated simultaneously for each analyte, with automatic validation against supplied calibration data. These curves are used for the calculation of the concentrations in multi-analyte controls and human serum samples. Data from the evaluation parameters assessed indicate suitability of the Evidence Investigator system for the application.

Development of a high-throughput automated analyzer using biochip array technology.

BACKGROUND: Use of protein array technology over conventional assay methods has advantages that include simultaneous detection of multiple analytes, reduction in sample and reagent volumes, and high output of test results. The susceptibility of ligands to denaturation, however, has impeded production of a stable, reproducible biochip platform, limiting most array assays to manual or, at most, semiautomated processing techniques. Such limitations may be overcome by novel biochip fabrication procedures. METHODS: After selection of a suitable biochip substrate, biochip surfaces were chemically modified and assessed to enable optimization of biochip fabrication procedures for different test panels. The assay procedure was then automated on a dedicated instrument, and assay performance was determined for a panel of cytokine markers. Assay results were then compared with a commercial method for measurement of cytokine markers. RESULTS: Secondary ion mass spectrometry and x-ray photoelectron spectroscopy demonstrated appropriate and reproducible modification of the biochip surface. Contact-angle studies also confirmed generation of hydrophobic surfaces that enabled containment of droplets for fabrication of discrete test regions. Automation of the biochip assays on a dedicated instrument produced excellent cytokine marker performance with intra- and interassay imprecision <10% for most analytes. Comparison studies showed good agreement with other methods (r = 0.95-0.99) for cytokines. CONCLUSION: Performance data from this automated biochip array analyzer provide evidence that it is now possible to produce stable and reproducible biochips for output of more than 2000 test results per hour.