Posts Tagged ‘sensitive’
Last Updated on Saturday, 10 July 2010 04:04 Written by Administrator Saturday, 10 July 2010 04:04
Development of a highly sensitive inhibition immunoassay for microcystin-LR [An article from: Analytica Chimica Acta]
This digital document is a journal article from Analytica Chimica Acta, published by Elsevier in 2004. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.
The development of a rapid one-step antigen-immobilized inhibition ELISA for microcystin-LR is described. For microplate coating a microcystin-biotin conjugate was synthesized. Using the commercially available monoclonal antibody MC10E7 in our newly established assay, IC”5″0 values of 0.045@mgl^-^1 have been achieved. The detection limit for microcystin-LR was 4ngl^-^1. Considering the guidelines proposed by the world health organization (WHO) for microcystin-LR in drinking water (1@mgl^-^1) the sensitivity of our test is more than sufficient. The period of assay processing could successfully be shortened to about 3h without any loss in sensitivity. The suitability of the newly developed assay was evaluated with microcystin-LR spiked environmental water samples. Recovery rates for microcystin-LR between 60 and 165% were obtained in the linear range of the test format. The antigen-immobilized test format provides a highly reproducible, easy, and fast to perform detection system for microcystin allowing an internal retrospective quality control of the assay.
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Last Updated on Tuesday, 15 June 2010 05:59 Written by Administrator Tuesday, 15 June 2010 05:59
Question by boggle: Why is the qualitative method ELISA assay very sensitive?
Answer by realme
It is very sensitive because an antibody binds to the antigen, and an ezyme can be used to develop visible signal. The test was improved by use of fluorogenic substrates rather than chromogenic substrates (visible colors).
Fluorogenic methods are typically several orders of magnitude more sensitive than the older chromogenic methods.
The fluorogenic substrate produces fluorescent molecules where the antigen and antibody binds together. By using a spectrophotometer, the amount of fluorescence can be quantitated.
The test is simple and very fast (compared to other test methods) and needs no special lab.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be measured.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
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