•     ANTIBODY IS IMMOBILIZED ON MICRO-PLATE WELLS

•     COMPETITION BETWEEN DRUG IN FORENSIC SAMPLE AND ENZYME LABELED DRUG FOR ANTIBODY BINDING SITES

•     THE UNBOUND MATERIAL IS WASHED OUT.

•     CHROMOGENIC SUBSTRATE ADDED TO DEVELOP COLOR WITH BOUND ENZYME

     RESULTING COLOR IS READ IN A SPECTROPHOTOMETER

    COMPONENTS OF AN ELISA

•      ANTIBODY: IgG fraction of serum purified by affinity chromatography.

•      ENZYME: Horse Radish Peroxidase (HRP) MW 44,000, glycoprotein with  4 lysine residues

•      SUBSTRATE: TMB (3,3’,5,5’,tetramethylbenzidine) The enzyme acts as a catalyst to oxidize    substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow

ADVANTAGES OF ELISAS

•      Heterogeneous Assays: The immune reaction stage in an ELISA is carried out prior to the signal generation stage and the specimen is washed out prior to the addition of the color development reagent. Hence whole blood, tissue homogenates and bile do not quench the assay signal generated.

•      Oriented Antibody: Immunalysis orients the immobilized antibody on the micro-wells, allowing the binding sites to stick out away from the solid polystyrene support. This minimizes steric hinderance and allows most antibody binding sites to bind either drug in the forensic specimen or the labeled enzyme conjugate.

•      Optimal Enzyme conjugates: All enzyme conjugates are matched to the corresponding antibody depending on the specificity or broad cross reactivity requirements. E.g. : The Cocaine Metabolite ELISA is very specific for Benzoylecgonine while the Barbiturates ELISA is geared to pick up a wide range of structurally similar compounds.

•      Small sample size: These assays have been designed to minimize the amount of specimen added to the micro-well, hence greatly reducing interference from some forensic matrices. For a 50 ng/mL morphine cutoff in whole blood, only 10 µl of a 1:10 dilution of blood is used. This is equivalent to a sample size 1 µl of whole blood.

•       Long shelf life: All Immunalysis ELISA kits have a minimum shelf life of one year. In fact, most Immunalysis microplates are stable for greater than 12 months at room temperature provided the foil bags are sealed.

•       “Ready to use” reagents: All Immunalysis ELISA reagents require no Pre-dilution or reconstitution. This greatly reduces dilution errors caused by the need to pre-dilute different enzyme conjugates. The only reagent that requires pre-dilution is  the enzyme conjugate for the Immunalysis Acetaminophen ELISA kit.

•      Non isotopic: There are no radioactive components to these assays removing the need to comply with the NRC regulations associated with radioimmunoassay.

•         No azide or mercury preservatives: Immunalysis ELISA reagents have no sodium azide or Thiomersal preservatives. Sodium Azide inhibits the Horse Radish Peroxidase enzyme used in the ELISA.

      Lends itself to different levels of automation: Different pipetting stations for sample and reagent distribution together with micro-plate washers and readers help to automate the process and increase throughput.