Elisa basics[2]
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Elisa basics[2]

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Elisa basics[2] Elisa basics[2] Presentation Transcript

  • ELISABASICSIVDWSeptember 29, 2005Courtesy of:Brett RobertsAgdia, Inc. agdia
  • ELISAEnzyme Linked Immuno-SorbentAssayELISA – an immunological test, using an enzymeas a label to determine presence of target protein.•The enzyme linkage or labeling allows you tofollow your target protein and if present (qualify)and at what amounts (quantify).•An enzyme conjugate is an enzyme bound orjoined with an antibody which binds with yourtarget protein. This enzyme labeling is a safe andeffective way to track your antibody.
  • ComponentsAntigen• Any substance that stimulates animmune response.• The antigen is your target proteinwhich comes from your sampleextract. Example: Bt protein in cornsample. The antigen binds to theantibody.
  • ComponentsAntibody• An antibody is a proteinmade in response to anantigen.• Each antibody binds only toits antigen.
  • Producing Antibodies Animals are Serum is drawn and injected over a antibodies are period of weeks separated and with purified purified antigen (ex. Bt protein)
  • ComponentsEnzyme ConjugateAn enzyme conjugate (EC) is anantibody joined with an enzyme.Enzyme labeling allows theresearcher to follow the antibody.This joining of the enzyme toantibody is often called conjugation.
  • Enzyme FunctionEnzymes are proteins that speed up the rateof a chemical reaction without being used upand usually react only to particular substrates.The rate of this reaction is proportional to theamount of enzyme present. In the case ofnon-competitive ELISA, the more bindingyou have of the enzyme conjugate to theantigen, the stronger your color developmentwill be.
  • Substrate FunctionA substrate is a compound orsubstance that undergoes change.Substrates bind to active sites onthe surface of enzymes and areconverted or changed. In ELISAthe specific substrate usedchanges color.
  • ELISA Formats (Sandwich)Direct sandwich ELISA – antibodies (Ab)are coated to micro wells. Antigen (Ag) isadded and binds with antibody. Excessantigen is washed away. Enzyme conjugate(Ab-E) is added and binds with antigen toform the double antibody sandwich. Wells arewashed to remove any excess (Ab-E).Substrate is added and color development isobserved. The enzyme conjugate binds‘directly’ to the antigen. Ab + Ag + Ab-E
  • Double Antibody Sandwich (DAS)1. Plates are typically coated with antibody by vendor. DAS Direct2. Sample extract is added and if present, target proteins bind to antibodies. Incubate 1 to 2 hours.3. Plate is washed. Any unbound material is washed away.4. Enzyme conjugate* is added. Incubate 1 to 2 hours.5. Plate is washed. Any unbound material is washed away.6. Substrate is added.7. Stop solution (optional) is added.8. Plate is read visually or © Peter Sforza, Virginia Polytech by plate reader.
  • PLATE READER•Machine which measures color density in platewell.•A specific wave-length of light is chosen(depending upon the color you expect to capture).•Light is cast from the bottom of the well throughthrough the sample.•No color change wells absorb very little light;wells with color change absorb more light; this isthe optical density or OD.
  • Reading plates•Select proper wave-length on machine.• Carefully wipe bottom of plate to removeexcess moisture. Plates that are wet maydiffuse your light source, giving inaccuratereadings.•Mix the plate. This will distribute colorevenly. Some machines have a ‘mix’ setting.
  • OD Values – good results 1 2 3 4 5 6 7 8 9 10 11 12A 0.02 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.3B 0.02 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.4C 0.04 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.5D 0.02 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.3 4 control wellsE 0.01 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 0.02 Buffer + Known pos no ECF 0.02 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 0.03 Buffer + ECG 0.03 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.3 Known positiveH 0.02 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 0.04 Known negative A1 – H1 are expected negatives
  • OD Values – unclear results 1 2 3 4 5 6 7 8 9 10 11 12A 1 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.3B 0.9 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.4C 0.9 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.5D 2.4 2 1.5 2 1.5 1.5 0 1.5 1.5 1.5 1.5 1.3 4 control wellsE 0.01 2 1.6 1.6 0 1.6 1.6 1.6 1.6 1.6 1.6 2 Buffer + Known pos no ECF 2 1.2 1.2 1.2 1.2 1.2 0 1.2 1.2 1.2 1.2 0.9 Buffer + ECG 1.2 0 1.2 1.2 1.2 2 1.2 1.2 1.2 1.2 1.2 1.3 Known positiveH 2.1 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1 Known negative A1 – H1 are expected negatives
  • Standard Curve Example 2 OD 1 .Values . 0.5 . 0 0 10 20 30 40 50 Sample concentration ng/ml Curve set using dilutions: 40ng/ml, 30ng/ml, 10ng/ml
  • Questions?
  • Thank You agdia