We initially had good results with avidinylated-SAP. It combined well with biotinylated antibody to produce extremely potent cytotoxic materials, and had low toxicity itself. However, the weaknesses of avidin are well-documented. Probably the most severe is its high isoelectric point that has been suggested to cause nonspecific binding. As we produced more batches of avidinylated-SAP and completed comparative studies, we in fact, found this to be the case.
I use avidinylated-SAP to demonstrate that my antibody internalizes. It worked quite well for me.
A couple of months ago we received two reports from customers that they were seeing that, even in batches that had performed well in quality control testing, there was a non-specific cytotoxicity with some cells and/or cell lines. Since a major use of this material is to demonstrate internalization of the biotinylated targeting agent, this was an unacceptable situation. We changed to streptavidin to overcome these specificity issues. As shown in the figure above, streptavidin-SAP has an excellent capacity to transform a biotinylated reagent into a potent cytotoxic targeting vehicle, while streptavidin-SAP alone has no detectable cytotoxicity.
KNRK cells are plated at 2500 cells/well and incubated overnight. Streptavidin-SAP is premixed with Biotinylated-IB4 in equimolar concentrations or added to a plate alone. Saporin, IB4-SAP, and the Streptavidin-SAP + Biotinylated-IB4 mixture are added in 10-µl volumes and plates incubated 72 hrs. PMS/MTS is added and the plates are incubated 15-30 min, then read at 490 nm.