Principles and application of elisa biology essay
A cut-off point may be determined by comparing it with a known standard. A technique to accomplish this was published by Wide and Jerker Porath in To procure monoclonal antibodies it is a tedious process in case of matched pairs and are more expensive than polyclonal antibodies.
Types of elisa
With the standard readings a standard curve is plotted and the line of best fit is obtained. A color producing substrate, like Tetramethylbenzidine TMB , in staining. Herein, the after addition of a suitable substrate the treatment with streptavidin-enzyme conjugate is done. Minimum precursor requirement makes it less error prone. The lack of secondary antibody minimises signal amplification, that reduces the sensitivity of the assay. Due to the heterogeneity of epitopes, polyclonal antibodies can be powerful tools for the thorough detection of an antigen. Other variants are well and well plates. Polyclonal antibodies Polyclonal antibodies are complex antibody pools containing various epitopes of a single antigen. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of well plate. Disadvantages Optimization in terms of antibody becomes problematic due to cross-reactivity issues. Advantages Increased sensitivity, since more than one labeled antibody is bound per primary antibody. It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood.
Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens.
There is also a contribution made by the spatial arrangement of the whole complex. Avidity is a more complex term that accounts for the total stability of the antibody-antigen interaction.
High affinity antibodies produce the greatest number of stable immune complexes and therefore provide the most sensitive detection. This method gives an S-shaped data curve that distributes more of the data points into the more user friendly sigmoidal pattern.
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