Hi, I'm using a BioVision kit for nivolumab.

When I first ran the kit, I spiked in a certain concentration of the drug, nivolumab. I was able to get that concentration. I followed the protocol with twice the incubation periods.

When I performed this kit again and again, I wasn't able to get the spiked concentration. Every time afterwards, I get a 4x concentration than what I originally spiked in. The spiked in samples kept having saturated abs values.

I tried the followings things,

-changing incubation times again (tried different ones, such as the original protocol and 2x longer)

-soaking wells with wash buffer

-the drug I spiked in with sits in plasma (due to nature of the project). I tried using DPBS in case IgG in plasma is causing non specific binding.

-using a completely new kit

-thoroughly mixing the reagents, substrates, standards and samples

Currently, I am trying to remake the drug I use for spiking. In addition, I will also dilute down one of the standards for a spike as well.

I just don't get how when I first ran it, everything work as intended. The next few days when I repeat it, everything seems to be off by a systematic factor...

Thank you very much