Did you ever dream that as a scientist you would be a matchmaker? That’s hardly what we were trained for. But if you want your ELISA to be happy, then you have to hone your skills in the art of l’amour.
Yes, you will need to negotiate a successful matchup between an antibody and an antigen. To find the best signal-to-noise ratio, you will have to determine when there is little connection resulting in a less than robust response. Or you might have an antibody that isn’t ready to settle down with just one antigen and causes high background. But if you know what to look for, you can make a match that will result in a life-long relationship.
Here is what you need to consider when being a matchmaker for ELISA:
The affinity of an antibody refers to how strongly an antibody recognizes its epitope on an antigen. For a good ELISA, you want a strong bond between your antibody and antigen. After all, the more they are attracted to each other, the more they will come together in a short amount of time.
For a successful relationship, the antibody must pair up with only one antigen. An antibody that is promiscuous and flirts with several proteins will never be the one true choice for ELISA.
Pairing up an antigen and an antibody for ELISA might require a little tinkering. You want to make sure the antigen is exposed to just the right amount of antibody, otherwise it could be too overwhelming. Try out your antigen with different concentrations of antibody to find the right match.
Is ELISA direct or indirect? Indirect ELISAs tend to have a more robust signal and can tolerate antibodies with less affinity.
Or perhaps you fancy a sandwich ELISA? Then you have to think about making a couple of matchups: the capture antibody with the antigen and the detection antibody with the antigen. They both need to be able to interact without interfering with one another. And you will also have to make sure the two antibodies don’t fall for each other instead of the antigen.
Do you want sparks to fly for ELISA to be captured in a chemiluminescent reader? Or do you want to see ELISA blush with your very own eyes? The read-out of your ELISA will determine the type of antibody you look for. You can choose from radioactive, chromogenic, chemiluminescent or fluorescent read-outs.
With a little bit of research and some trial and error, you can be a great matchmaker for ELISA!
Photo courtesy of Jed.