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Lec # 2 ELISA

Lec # 2 ELISA. (Enzyme Linked Immunosorbent Assay) . What i s ELISA?. diagnostic method determining protein concentrations Purpose to determine if a particular protein is present in a sample and if so, how much. 

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Lec # 2 ELISA

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  1. Lec# 2ELISA (Enzyme Linked Immunosorbent Assay)

  2. What is ELISA? • diagnostic method • determining protein concentrations • Purpose • to determine if a particular protein is present in a sample • and if so, how much.  • samples in solution (i.e., biological fluids, culture media or cell lysates) • blood plasma, serum or cell/tissue extracts in a multi-well plate format • Advantages • ELISAs are quick and simple to carry out, and since they are designed to rapidly handle a large numbers of samples in parallel, they are a very popular choice for the evaluation of various research and diagnostic targets

  3. Antibodies specific for the protein of interest are used to probe the plate. • Background is minimised by optimising blocking and washing methods (as for IHC), • specificity is ensured via the presence of positive and negative controls. • Detection methods are usually colorimetric or chemiluminescence based.

  4. Types • you can determine how much antibody is in a sample, • or you can determine how much protein is bound by an antibody. • The distinction is whether you are trying to quantify an antibody or some other protein • Direct Elisa • Indirect Elisa • Sandwich Elisa

  5. Direct ELISAs • involve attachment of the antigen to the solid phase, followed by an enzyme-labeled antibody. This type of assay generally makes measurement of crude samples difficult, since contaminating proteins compete for plastic binding sites. • Indirect ELISA • involve attachment of the antigen to a solid phase, but in this case, the primary antibody is not labeled. An enzyme-conjugated secondary antibody, directed at the first antibody, is then added. This format is used most often to detect specific antibodies in sera. • Sandwich ELISA • The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a capture antibody to a solid phase support. Samples containing known or unknown antigen are then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection.

  6. ELISA PROTOCOL

  7. Microtitre plate • flat plate • typically has 6, 24, 96, 384 or even 1536 sample wells • Made of : most common is polystyrene, used for most optical detection microplates. • Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate. • This process occurs though hydrophobic interactions between the plastic and non-polar protein residues.

  8. Positive control – sample containing protein • Negative control- sample not containing protein • Standard A sample containing a known concentration of the target protein from which the standard curve can be obtained. 

  9. ELISAs begin with a coating step, where the first layer - either an antigen or an antibody - is adsorbed to a polystyrene 96 well plate. (Adsorption is the passive attachment of a liquid to a solid surface creating a thin film.) • Coating is followed by blocking and detection steps • Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage

  10. sample Blank 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Negative standard Positive control

  11. ELISA Machine • the entire plate is placed into a plate reader • optical density (i.e. the amount of colored product) is determined for each well. • The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells

  12. ELISA Results • The ELISA assay yields three different types of data output: • 1) Quantitative:ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples. • 2) Qualitative:ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen. • 3) Semi-quantitative:ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.

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