The Technology and Applications of Protein Biochip Array Technology

Biochip Array TechnologyThe Technology and Applicationsof Protein Biochip ArrayTechnologyKatie WalkerBiochip Array Product SpecialistPanels of tests,allowing for thesimultaneous multianalytedetection ofa wide range ofanalytes, from asingle undividedsample•81mm 2 Solid StateDevice•Assays contained indiscrete reaction sites•Single patient sampleper biochip•Multiple assaysperformedsimultaneously

Development of Biochip Array• 1992 – 2003• Over 185 Scientists involved• $140,000,000.00 Investment• 45 new patents• Three main stages in Biochip development– Synthetic Chemistry– Nano Dispense– Detection technologyThe Biochip• The crux of the technology is the Biochip itself• Coated in a proprietary silanation process– Activation of the Biochip surface• Ensures uniformity and reproducibility– Modification of surface chemistry• Controlled anti-body binding in optimal orientation

Biochip Surface ChemistryNon -activated SurfaceActivated Surface

Biochip ManufacturingNano Dispense of Antibody solution• All manufactured by Randox at our HQ in theU.K.• 20 million chip per year capability• Fully automated clean-room facilityQuality ControlQuality Control• Quality control is at the heart of all that Randox does.• Quality is ensured at every level of product– Pre process• Quality components• Randox Antibodies– In process• Manufactured to ISO 13485• Ensures reproducibility– Post Process• Internal QC to ensure Functionality, integrity andstability

Immunoassay FormatsCompetitive ImmunoassayDETECTION• Tried and trusted EIA• Sandwich immunoassay– Cytokine and Growth Factor Array• Competitive immunoassay– Drugs of Abuse ArrayCONJUGATEEY YANALYTEANTIBODY• Horse Radish Peroxidase LabelSUBSTRATESignal Analysis• Chemiluminescence• L.O.D. compared– Fluorescence (ELISA) 2,000,000 zeptomoles– Radio Immunoassay 1,000,000 zeptomoles– Chemiluminescence 25,000 zeptomoles» Tietz ‘Clinical Chemistry’• Imaged with super cooled CCD camera• Quantified by custom software

Available ArraysDrugs of Abuse Array 1• Drugs of Abuse Array 1• Cytokine and GrowthFactor Array 1• Free Thyroid Array• Total Thyroid Array• Fertility Array• Tumour MonitoringArray• Tumour PSA Array• Adhesion MoleculeArray• Cardiac Array• Growth Promoter Array• Anti-Microbial Array 1• Cardiovascular DNAArray• Colorectal Cancer DNAArray• Anti-Microbial Array 2*• Anti-Microbial Array 3*• SyntheticSteroidsArray*• Drugs of Abuse Array2*• Amphetamine• Methamphetamine• Barbiturates• Benzodiazepine I• Benzodiazepine II• Cocaine Metabolite• Methadone• Opiates• Phencyclidine• Cannabinoids• Creatinine (urineapplication only)Drugs of Abuse Array IIDrugs of Abuse Arrays• Buprenorphine• Fentanyl• Ketamine• LSD• MDMA• Hydromorphone• Methaqualone• Oxycodone I• Oxycodone II• Propoxyphene• Creatinine*• Specific matrix optimised kits for– Urine (FDA cleared)– Urine Semi-Quant– Whole Blood Quant– Oral Fluid Quant

Amphetamine ClassUrineBloodOral FluidUrineBarbituratesBloodOral FluidTargetd-Amphetamine /+ Methamphetamined-Amphetamine /+ Methamphetamined-Amphetamine /+ MethamphetamineTargetPhenobarbitalPhenobarbitalPhenobarbitalCut-off1000 ng/mL /1000 ng/mL25 ng/mL /50 ng/mL50 ng/mL /50 ng/mLCut-off200 ng/mL50 ng/mL5 ng/mLCross reactivity (urine)Amphetamine assayMethamphetamine assayCross reactivity (urine)Barbiturates assayd-Amphetamine+ MethamphetamineMDAMDMA100%

CannabinoidsMethadoneUrineBloodOral FluidUrineBloodOral FluidTarget11 nor − 9 THC-COOH11 nor − 9 THC-COOH− 9 THCTargetMethadoneMethadoneMethadoneCut-off50 ng/mL10 ng/mL4 ng/mLCut-off300 ng/mL25 ng/mL5 ng/mLCross reactivity (urine)Cannabinoids assay11 nor − 9 THC-COOH11 nor − 8 THC-COOH11 hydroxy − 9 THC11 hydroxy − 8 THCCannabinolCannabidiol100%49%3%

TargetCut-offCocaine MetaboliteUrineBloodOral FluidBenzoylecgonine Benzoylecgonine Benzoylecgonine300 ng/mL50 ng/mL20 ng/mLCross reactivity (urine)Cocaine metabolite assayBenzoylecgonine100%Cocaine6.4%Cocaethylene4.1%Ecgonine

METHAMPHETAMINE AMPHETAMINE BENZODIAZEPINESGC-MSGC-MSGC-MS+ - + - + -EVIDENCE + 1 0 EVIDENCE + 0 1 EVIDENCE + 7 0- 0 32 - 0 30 - 0 23% AGREEMENT 100.0 % AGREEMENT 96.8 % AGREEMENT 100.0METHADONE OPIATES PHENCYCLIDINEGC-MSGC-MSGC-MS+ - + - + -EVIDENCE + 4 0 EVIDENCE + 3 0 EVIDENCE + 1 0- 0 29 - 0 29 - 0 32% AGREEMENT 100.0 % AGREEMENT 100.0 % AGREEMENT 100.0BENZOYLECGONINEOVERALLGC-MSGC-MS+ - + -EVIDENCE + 10 0 EVIDENCE + 26 1- 0 22 - 0 198% AGREEMENT 100.0 % AGREEMENT 99.6Urine Evaluation• FDA Trials• To assess the overall performance of drugsof abuse screening on the evidenceimmunoassay technology• To compare the evidence performance to analternate immunoassay system• To compare the evidence performance toresults obtained by GC/MSUrine EvaluationAmphetamines• More than 1300 clinical urine samplescovering the entire range of possible testresults for all ten drugs of abuse• Comparison carried out by independentclinical toxicology laboratory in Texas• 10% of samples fell within ±25% of theassay cut-off concentrations10080604020096.8 96.782.1% AGREEMENT% DISAGREEMENT17.93.23.3evidence with CEDIA evidence with GC/MS CEDIA with GC/MS% Agreement Total number of samples Discrepant samplesevidence with CEDIA 96.8 1264 40evidence with GC/MS 96.7 305 10CEDIA with GC/MS 82.1 234 42

MethamphetamineCannabinoids10096.7 97.010097.993.98082.28081.86040% AGREEMENT% DISAGREEMENT6040% AGREEMENT% DISAGREEMENT2017.803.33.0evidence with CEDIA evidence with GC/MS CEDIA with GC/MS20018.26.12.1evidence with CEDIA evidence with GC/MS CEDIA with GC/MS% Agreement Total number of samples Discrepant samplesevidence with CEDIA 96.7 1249 40% Agreement Total number of samples Discrepant samplesevidence with GC/MS 97.0 305 9evidence with CEDIA 97.9 1335 28CEDIA with GC/MS 82.2 219 39evidence with GC/MS 93.9 231 14CEDIA with GC/MS 81.8 231 42OpiatesOverall Summary1008092.689.1% Agreement604060.040.0% AGREEMENT% DISAGREEMENTEvidence with CEDIA 96.9207.410.9Evidence with GC/MS 90.50evidence with CEDIA evidence with GC/MS CEDIA with GC/MSCEDIA with GC/MS 83.4% Agreement Total number of samples Discrepant samplesevidence with CEDIA 92.6 1334 99evidence with GC/MS 89.1 320 35Total number of tests 11,750CEDIA with GC/MS 60.0 320 128

User Defined Cut-offselection.Analysers and SoftwareFeatures• Semi-automated• Based on Biochip Array Technology– Multiple test sites per biochip• Fast Results– 54 biochips ie. 540 Tests in 1 hour• Small sample volumes– Urine – 3.5μl per test– Whole Blood - 6μl per test– Oral Fluid - 10μl per test

Features• Quality of results– All Randox products are developed to ISO 13485,diagnostic standards– Full trace-ability through manufacture– Antibody Specificity– Self sufficient antibody supply– Specific Methamphetamine and LorazepamassaysFeatures• Cut-off selection– Cut-offs can be selected and reselected withoutrecalibration– Different cut-offs can be selected for the sameanalyte during the same run, ie. Opiates sample A25ng/mL, sample B 10ng/mL.• Fully Quantitative Analysis– Identified as positive/negative– Concentration of compound also displayedFeatures• Software– Windows XP® based– Colour coding easy to follow– Multiple search formats– Easy export of results– Fully LIMS connectable– Extensive QC facilities• Levy Jennings and Multi-point rules.– Enhance trouble shooting

Features• Fully Automated– Walk away system• High throughput– 130 samples per hour x no.of tests per chip• Chain of custody Features– Sample access doors lock once sample is loaded• Password ProtectedFeatures• Retrospective Testing– Although only invoiced for tests reported, all assaysare carried out simultaneously– If certain assays are not reported they can be recalledat a later stage.

ReferencesMolloy RM, McConnell RI, Lamont JV, FitzGerald SP.Automation of biochip array technology for quality resultsClin Chem Lab Med; 43 (12):1303-1313FitzGerald SP, Lamont JV, McConnell RI, Benchikh EO;Development of a high throughput automated analyserusing biochip array technology; Clinical Chemistry; 51 (7)1165-1176www..com