ELISA

[seminoma]

What determines if you should use direct ELISA or indirect ELISA? The tests are slightly different, but I don't understand what advantage one has over the other... except that maybe direct ELISA is faster/easier since it's only a single step vs 2 steps with indirect.

If you're testing for the presence of an antigen both direct and indirect ELISA seem to tell you the same thing, whereas only indirect ELISA can test for a specific antibody. Is that the only time you would use it indirect instead of direct (i.e. you simply can't use direct to detect an antibody)?

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[aspiringmd1015]

^direct looks for the antibody bound to the antigen, you need both. But the purpose of indirect as im sure you know is that its more specific. I dont know why you'd want to use direct, but i guess bc it is quicker, so its probably cheaper or something.

 

[sloop]

Direct ELISA—You DIRECTLY bind an immunofluorescent dye-labeled antibody to that antigen. Positive immunofluorescence indicates the presence of antigen.
Indirect ELISA—You have an antigen, add (usually) serum to the antigen, directly binding serum antibodies to the antigen. You then add an immunofluorescent dye-tagged anti-IgFc antibody which binds to the the Fc region of bound antibody, thereby INDIRECTLY binding your probe to the antigen. Positive immunofluorescence indicates the presence of antibody.

Hope this helped.

 

[sloop]

^direct looks for the antibody bound to the antigen, you need both. But the purpose of indirect as im sure you know is that its more specific. I dont know why you'd want to use direct, but i guess bc it is quicker, so its probably cheaper or something.

This is not correct. First, direct ELISA looks for antigen while indirect ELISA is the one that looks for antibody bound to antigen. Additionally, indirect ELISA is not more specific. An example of this is detection of HBsAg in a patient's serum. This test is specific enough that if you find (via direct ELISA—testing for antigen) HBsAg in a patient's serum, you can rest assured that they have Hep B.

If someone is anti-HBsAg (+), as determined by indirect ELISA, this is also specific but for a different thing: prior exposure to HBsAg.

The difference between these tests is what they look for and how they are done. Sensitivity and specificity will depend on the particular antigens and antibodies you are looking for and your immunofluorescence cutoff point for a positive result.

 

[aspiringmd1015]

sorry, not sensitivity, but apparently direct is less sensitive, and indirect is more sensitive. According to Kaplan Immunology

 

[sloop]

sorry, not sensitivity, but apparently direct is less sensitive, and indirect is more sensitive. According to Kaplan Immunology

They're wrong. It depends on the test. A simple example: if the antigen you're looking for is poorly immunogenic, the indirect ELISA would never be more sensitive. Depending on what you're looking at, it is not necessarily more specific either—as illustrated by the hepatitis example, indirect ELISA would not be more specific for active Hep B because the presence of the antigen, not the antibody, is the best marker of active infection with that virus.