Archive for the ‘Elisa Assay Questions’ Category
Last Updated on Thursday, 24 June 2010 10:44 Written by Administrator Thursday, 24 June 2010 10:44
Question by GAUTAM B: what is a fourth generation anti-hcv immunoassay?
innogenetics anti-hcv fourth generation assay, elisa
Best answer:
Answer by dr.hashemfyasin
you need a simple answer, but I don’t have one.
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Tags: antihcv, fourth, generation, immunoassay | Posted under Elisa Assay Questions | 1 Comment
Last Updated on Thursday, 24 June 2010 01:11 Written by Administrator Thursday, 24 June 2010 01:11
Question by heyheyhey: Which component is colored in ELISA (Enzyme-linked Immunosorbent Assay)?
also, In an ELISA used to detect antibody, which component is linked to the enzyme?
Best answer:
Answer by spasmodeus
The color comes from the chemical alteration of an enzyme substrate, such as 3,3-diaminobenzidine (DAB). Conversion of DAB by an enzyme like horseradish peroxidase (HRP) produces a brown color which can be detected using a spectrophotometer.
The way it typically works is a multiwell assay plate is coated with an antibody against the desired antigen for detection. Samples are loaded into the wells, and the antigen allowed to bind to the antibody coating the plate. Unbound material is washed away, and a secondary antibody conjugated to an enzyme (like HRP) is loaded into the well. This antibody binds to the antigen already immobilized in the well by the coating antibody, forming a so-called “sandwich”. Finally, after unbound secondary antibody is washed away, the enzyme substrate (such as DAB) is introduced. The amount of enzyme still present in the well is directly proportional to the amount of antigen captured in the well and cross-linked to the enzyme by the secondary antibody. The amount of colored enzyme product is also proportional to the amount of enzyme. So, one can compare the amount of product produced from well to well over time, and from this value quantitate the amount of antigen present in a particular sample.
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Tags: assay, colored, component, elisa, EnzymeLinked, Immunosorbent | Posted under Elisa Assay Questions | No Comments
Does anyone know which antibody pairs work with Enzyme-Linked ImmunoSorbent Assay (ELISA for short)?
Last Updated on Monday, 21 June 2010 05:33 Written by Administrator Monday, 21 June 2010 05:33
Question by Diddy: Does anyone know which antibody pairs work with Enzyme-Linked ImmunoSorbent Assay (ELISA for short)?
ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. It utilizes two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme.
I need like a list of antibody pairs that can work with it.
We are working with rats that were injected with Saline & LPS.
Best answer:
Answer by Phillip R
You can check with: http://www.antigenix.com/Merchant2/merchant.mvc?Screen=CTGY&Category_Code=MP
There is a list of antibody pairs there that they sell.
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Tags: antibody, anyone, assay, elisa, EnzymeLinked, Immunosorbent, know, pairs, short, work | Posted under Elisa Assay Questions | 1 Comment
Last Updated on Sunday, 20 June 2010 01:01 Written by Administrator Sunday, 20 June 2010 01:01
Question by msako: Why would you use the same isotype of antibody in ELISA?
Situation: ELISA assay. Microtitre plate –> wells coated with trout serum. Have two antibodies (unknown A and B from mouse). Have to determine which one is the anti-trout. (the other one is anti-catfish). The antibodies are both of the same isotype. Secondary antibody is anti-mouse Ig.
Why do we use the same isotype of antibodies? Is it for negative control?
Best answer:
Answer by N E
It is a control in one sense, but there are several reasons you are using the same isotype. The primary antibody is almost always an IgG isotype, and this is true in your case also (because the secondary antibody is an anti-mouse IgG). The primary antibody is a mouse anti-trout antibody and is more specifically a mouse anti-trout antigen IgG antibody. The secondary antibody (used to detect whether or not there is any primary antibody bound to any antigens) is an antibody against mouse IgG (called anti-mouse IgG antibody). The reason you use the same isotype antibody in all cases as the primary is so you can reduce the complexity of the experiment and reduce the complications you have to worry about. For instance, if you used an IgG and an IgM in different dishes, and a secondary specific for each, then you would have to worry about different binding specificities to the antigen as well as for the binding of the secondary antibody and so you couldn’t directly compare the results. If you use the same IgG and same secondary in all cases, then you can generally directly compare your results, so the only changing factors are the amount and source of the antigen. It is also cheaper and more convenient this way.
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Tags: antibody, elisa, isotype, same, would | Posted under Elisa Assay Questions | No Comments