Errors Requesting appropriate tests Writing prescription Reading prescription Sample collection Sampling times environmental factors Drug interferences Patient identification Sample transfer Technician errors Instrumental errors Method limitations Data entry mistakes Interpretation of results How can we trust

1-Chemiluminescence is the best known method of immunoassay and 2-Automation systems are the best solutions to reduce errors and Combination of these two allow us to get accurate, precise and reliable results from laboratories

Which parameters are important in evaluating a method Signal / noise User safety Sensitivity Specificity Incubation time Ease of carrying out

Hormone measurement The Problem –very small amounts of hormone in a very complex mixture Pre-immunoassay –complex and insensitive methods (chemical methods, whole animal or tissue bioassay) –insensitive –imprecise –inaccurate Immunoassay –first described in 1960 –very rapid expansion since early 1970s –advantages (simplicity, speed, precision, accuracy, sensitivity)

In fluorescence this can lead to difficulties with fluorescers with a small Stokes shift. Fluorescence may not be easy to resolve from the exciting wavelength. Another problem is associated with scattering of the incident light to the detector, especially when samples are somewhat turbid.

Why ELISA cant be fully automated

Can you tell the difference between how many marks are in each box? Sensitivity of Absorbance Measurements Spectrophotometry: Luminescence

Sensitivity of Luminescence Measurements Can you tell the difference between how many marks are in each box? 0 40 Spectrophotometry: Luminescence

Immunoassay Signal Generation ENZYME COLOUR DETECTION ENZYME FLUORESCENCE DETECTION TIME RESOLVED FLUORESCENCE DETECTION RADIOACTIVITY (I 125 ) CHEMILUMINESCENCE DETECTION Minimum Detection Limit (moles of tracer) Detection Method Sensitivities of different tracers VITROS ECi DETECTION SYSTEM Vitros ECi

What you want Accuracy Precision Speed Sensitivity Specificity

Immunoassays: Radioimmunoassay (RIA, IRMA) Elisa (enzyme linked immunosorbant assay) Immunofluorometric assay Immunluminometric assay

Immunoassays basics ( virus?)

Physical Concepts in immunoassays: Radioactivity Spectrophotometery Fluorescence Luminescence

The wavelength of light determines how it interacts with matter. We use these interactions as a probe to obtain chemical information about samples. Spectrophotometry is the use of “light” in chemical measurements Spectrophotometry 400 nm750 nm IR UV IR typically referred to by wavenumber (10-12,500 cm -1 ) Blue - Orange Blue-green - Red Green - Purple

Fluorescence FITC + UV light

Phosphorescence

Chemiluminescence

Bioluminescence

Antigen HRP label 2nd Antibody Immunometric Assay on Streptavidin Coated Well Substrate Light 1st Antibody Biotin tag Solid Phase Plastic Well Streptavidin Biotin spread evenly over well

Ferritin Antibody: Passive Adsorption

Ferritin Antibody: Biotinylated on Vitros Coated Well

Basic requirements for immunoassay Standards Specific antibodies Labelled antigen or antibody Separation system Quality control

Advantages of 2-site immunometric assays Increased sensitivity Increased precision Better specificity Greater assay range Shorter assay times

Disadvantages of 2 site immumetric assays Need for large quantities of pure antibody (monoclonal antibodies usually employed) 2 antibody binding sites required (limit range of analysis) High dose “hook” effect Need for multiple washing steps Non specific interference due to heterophyllic antibodies

Advantages of isotopic labels Simple coupling reactions Label properties do not alter on coupling No background signal Efficient/convenient detection systems No additional cost to detect signal Very useful for research assays

Non isotopic labels - advantages No radioactivity –safety aspects –disposal Extended life of label Speed of detection Ease of automation Theoretical increase in sensitivity Possibility of homogeneous assays Simple/safe label preparation

Non isotopic labels - disadvantages Safety aspects of labels/substrates Serum/buffer effects Extra manipulations in detection Inefficient detection in some cases No recounting possible in some systems Limitation of separation system “Dedicated” instruments Commercial pressures

Hormone assay in the future Dominated by immunoassay techniques ? GCMS Increased sensitivity Better automation –computers –robotics –non isotopic labels Near patient testing devices

Immunoassay automation Non isotopic labels Microprocessor power Improved robotics Better antibodies  faster reaction times

Immunoassay Analysers Immunological reagents Precision usually good Wide variations in sensitivity, specificity and accuracy between analysers Careful definition of assay requirements Whether any one analyser will satisfy all requirements

Types of automated system Work simplified batch systems Automated batch systems Total automation (black box) - random access Portable (bedside biochemistry) instruments

 Intellicheck Technology – advanced instrument intelligence - monitors and verifies:  Patient sample quality  detects short samples, clots, bubbles, viscosity  Sub-system performance  Sample & reagent volumes in reaction well  Reports issues to the operator Advantages of automation

 Single Use Disposable Tip Sampling  No sample carryover  Bubble, Clot, Low & High viscosity detection  Auto dilution facility using Disposable Tip Ensures sample integrity Advantages of automation

Intellicheck - Sample Metering Verification  Patented Pressure Level-Sensing technology  Bubble, Clot & viscosity detection Normal Aspiration Abnormal Aspirate bubble detected Pressure +ve/-ve

Intellicheck™ Technology Sample + Reagent Verification  Verification of correct sample and assay reagent volume dispensed into the Microwell  Level sensing technology  Eliminates the potential for a misreported result by not processing an assay when an exception is detected  Detection with automatic recovery  Ensures result integrity Aspirate Probe Level sense detection extension integrated with Aspirate Probe Dispense Probe Sample + Reagent Well Wash Probe Assembly Microwell

Advantages of automation Increased precision Work simplification Versatility Less contact with samples Rapid turnaround time

Disadvantages of automation Lack of reagent choice Total reliance on manufacturer Lack of range of analytes Little use for “research” assay Increased cost