Competitive ELISA Explained For Beginners
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- čas přidán 26. 09. 2022
- ELISA is an abbreviation of enzyme linked immunosorbent assay and utilize the bond between an antibody and its specific antigen for it to work. There are 4 main types of ELISA:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
Today we will look closer at competitive ELISA which is carried out in the following manner:
1. First, the sample is mixed with an excess of antibodies specific to the target antigen. These antibodies react with the antigen and form an antigen-antibody complex.
2. Second, the mixture is added to a micro well plate, with wells containing antigens that also are specific to the antibodies that were mixed in with the sample. However, only unbound antibodies can bind to the antigens that the plate is coated with since in the antigen-antibody complex, the antibody has already bonded.
3. Third, enzyme-conjugated secondary antibodies which bind to the first set of antibodies are added.
4. Fourth and finally, a substrate specific for the enzyme which is linked to the secondary antibody is added, and the enzyme converts this substrate into an observable signal.
This means that the more target antigen is present in the sample, the more antibodies are bound by it. As a result, the less free antibodies are left to bind to the antigens coated to the wells. This results in that less enzyme conjugated secondary antibodies can bind and produce a signal, hence resulting in a weaker signal. In other words the more of a color change occurs, the LESS target analyte is present in the sample.
It is also important to note that the way competitive assays are carried out may vary slightly but they all follow a similar structure as the one laid out here. However, sometimes, labeled antigens same as the target antigen are mixed with the sample. In this case the plate has been coated with capture antibodies specific to these and as such, they have to compete for binding spots. The essential idea is still the same and the signal is also in this case inverse.
Please ask if anything was still unclear to you! Most likely more people than you are wondering the same thing!! They are just too chicken to ask!😉
Thank you so much man, I had never properly understood competitive elisa
I'm grateful for being able to help out! Thank you for letting me know you found it helpful!
Now I understand ELISA . Thank you.
Great to hear!
Amazingly explained each type of ELISA!
Thank you so much!🙏
Perfect explanation, thank you!
Thank you for taking the time to let me know you enjoyed it!🙏😇
how is the antigen-antibody complex first made not recognized by the secondary antibodies ? if im assuming correctly they shouldnt be able to recognize them because we only want the antigen-antibody complexes made by the unbound antibodies correct??
Thank you !!
Happy I could help😇👍
this is what I will write for my exams!!!!
Good luck! Remember that I made a mistake here in terms of how antibodies bind.
In the video, the antibody binds to the antigen on its FC region. However it should bind on its FAB regions.
I'll make a corrected video in the future but I wanted to make sure you know this before your exam!
Great explanation! Thank you
Thank YOU for taking the time to comment! I really appreciate it!
This explaination isn't enough tho. Having the conjugated antibody bound to the fixed or the free antigen shouldn't matter. The signal should be the same either way.
You need to explain that a last washing step takes place after the addition of both the conjugated antibody and the free antigen. This will result in some of the conjucated antibody being left in the plate, if it was bonded with the fixed antigen. That's why the reduction in signal occurs.
That's a really great point and I don't know what I was thinking not including the final wash in the video🙈
I will do an improved version in the future and fix it but in the meantime I hope it's okay if I pin your comment?😇
Nice video, but why the heck do you draw the antibody with its binding to the antigen on its FC region and not on its FAB regions, its very misleading imo?
It was a mistake on my part. I will make a new version and remove the old video when I do👍
this confused me even more
I'm sorry to hear that. I try to get better at explaining all the time but I have a long way to go still!
I get it now after watching the first 3 videos on Elisa. Try doing that
@@deidarasan2760 Yeah I try to cover one specific thing per video but striking a balance between repetition and giving context for people who have not watched the previous videos without overdoing it can be quite difficult.