Hi everyone! I´m going to perform a conjugation between a single domain antibody (VHH from llama) and the HRP.
I'm using a commercial kit from Thermo. The datasheet indicates an approx 4-fold molar excess of HRP to IgG. However, the VHH is smaller (just 15 kDa), so I don´t know if this ratio would be the best first option to try with my protein.
What ratio would you recommend me to try? Other datasheets indicate that the ratio can be adjusted between 1:1 to 1:4, but always thinking the reaction using a complete immunoglobulin (150 kDa).
Thanks in advance for your help!
Florencia
The standard ratio is unlikely to be optimal given the relatively low surface area of the antibody and you will just need to try different molar ratios. However, I would actually regard this as labelling of HRP with the smaller antibody. How much Ab you can attach without damaging the enzyme is the unknown here. However, logic would suggest that the ratio switches round in favour of the smaller molecule and you may then get a multivalent product with high avidity.
Thank you for your answer with this new question Nick!
Probably my first try will be with less HRP.
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