It depends on the purity of antibodies. You can try 5,10 and 20 microgram/ml with EDC and NHS. However, for the application of EDC and NHS you have pretreat the surface and produce carboxyl group on SPCE. You can do it by using some chemicals like carboxymethyl dextran or using anodic treatment with NaOH.

Thank you.

I pretraet the SPE surface with 0.1M H2SO4 at 1600mV during 180s. I use 15µg/ml. My signal saturates immediately even for very low concentrtion antigen.

OK you are welcome, could you let me know that whether you have used EDC/NHS or passive immobilization? In addition, which techniques you applied to meausre current response and how long you have incubated HRP-labelled antibody along with TMB and H2O2?

I use EDC/NHS. Each incubation time is 15 min. I use 2mM hydroquinone as redox mediator in presence of H2O2

One protocol is attached.

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