Many scientists expressing interest in single domain antibodies have been curious about the best ways to detect single domain antibodies with in vitro assays. When detecting typical IgG antibodies, secondary antibodies with conjugated enzymes or fluorophores are used for detection in a variety of assays. However, single domain antibodies by nature cannot be targeted by secondary antibodies. This is because they are made up of only the variable heavy domain of a heavy chain only antibody, and thus have no constant domain.
To combat this problem, ProSci has tailored our single domain antibody development process to allow for at least one constant region in the antibody. When developing the plasmid, both a 6-histidine tag and a c-Myc tag are incorporated into the sequence. The reasoning is twofold: first, the tags are used for the purification of the antibodies using affinity chromatography, and second, both tags can be targeted by monoclonal antibodies developed against them.
How to Detect sdAbs
Let’s use an ELISA experiment as an example of how to detect ProSci’s single domain antibodies. Imagine you are interested in a protein that you have developed a single domain antibody against. After treating each well with the target protein, you would then treat it with your single domain antibody for detection. Next, you would treat each well with an anti-his tag antibody or anti-myc antibody based on preference as secondary antibody. Finally, you would treat each well with a secondary antibody conjugated with either a fluorophore or enzyme
as a ‘tertiary antibody,’ and visualize the results appropriately.
Though detecting antigens using a single domain antibody may take additional reagents, the principle of targeting antigens in this way can be applied in other assays like immunohistochemistry, immunocytochemistry, and functional assays. Single domain antibodies may be more useful in these applications for detecting proteins in their native conformations by binding epitopes no other antibody can access. By using either this detection method or some variant, you will be able to detect proteins in their native conformations using single domain antibodies with ease.
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