Specificity of animal-free antibodies in immunocytochemical stainings

Abstract

As the use of mixed cell cultures in research is becoming increasingly more common, it is essential that the antibodies used in the immunocytochemical stainings of these types of samples do not exhibit non-specific binding across cell types. Traditionally, control over antibody specificity has been limited by the need to use animals in their production. Advancements in biotechnology have enabled the invention of a variety of new methods for creating antibody-like affinity reagents without the need to use animals. A well-established method for creating animal-free antibodies includes the use of antibody libraries based on phage-display technology, which can be used to manufacture recombinant antibodies with more control over their specificity, affinity and batch-to-batch variation.

In this study I explore the specificity of animal-free recombinant antibodies compared to the traditional animal-derived antibodies in three different cell types, for the purpose of testing their suitability for use in mixed cell cultures. First, I tested antibodies targeting three different proteins (α-smooth muscle actin, platelet-derived growth factor receptor-β and cluster of differentiation 31) typically exhibited by mesenchy-mal stem cells and endothelial cells for non-specific binding to neural cells. In the second part of the study, I tested two neural cell protein (β-tubulin III and neurofilament heavy chain) targeting antibodies for non-specific binding with mesenchymal stem cells and endothelial cells (hASCs and HUVECs, respectively).

The mesenchymal and endothelial cell antibodies tested did not exhibit non-specific binding with the neural cells. From the neural cell antibodies both the animal-derived and animal-free antibodies targeting β-tubulin III exhibited non-specific binding with hASCs.

The results of this study highlight the importance of testing for cross-binding of antibodies used in mixed cell cultures regardless of the manufacturing techniques used in their production. Animal-free antibodies can offer solutions for further improving the specificity and reliability of antibodies for mixed culture uses.