Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

Abstract

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

“…As an extension, we also demonstrated that mCherry‐AzF in cell lysates was efficiently immobilized onto a DBCO‐functionalized well in a plate via SPAAC, indicating general feature of the technique developed here. As reported in literature, the site‐specific immobilization feature of this technique would provide additional advantages, such as orientation control and retained activity, over conventional immobilization techniques. For large‐scale applications, several issues need to be addressed in future.…”

“…Protein immobilization onto solid supports has been widely utilized for research and industrial processes . Immobilization of antibodies and receptors plays an important role in constructing sensitive biosensors for diagnostics and drug screening . Furthermore, protein immobilization is critical in generating protein arrays for proteomics .…”

“…Although several non‐covalent attachment techniques were developed for direct immobilization of proteins, immobilization of non‐target proteins occurred and it was not ideal for a long‐term operation . Conventional covalent attachment techniques using primary amines or sulfhydryl groups in a target protein usually resulted in poor selectivity toward a target protein, poor orientation control, and significant activity loss due to chemical modification at critical sites . Emerging site‐specific immobilization techniques addressed some of these issues .…”

“…Conventional covalent attachment techniques using primary amines or sulfhydryl groups in a target protein usually resulted in poor selectivity toward a target protein, poor orientation control, and significant activity loss due to chemical modification at critical sites . Emerging site‐specific immobilization techniques addressed some of these issues . Certain enzymes, such as farnesyltransferase and N‐myristoyltransferase, were used to incorporate a bioorthogonal chemical handle into a specific site of a target protein, followed by immobilization of a target protein onto supports .…”

Fundamentals of affinity cell separations

“…Changes in pH, temperature, and salt concentration can potentially denature affinity proteins; monoclonal‐affinity cell separations are significantly impacted by these environmental changes, whereas pAbs are more robust . Furthermore, the orientation of the antibody affinity ligand and the hydrophobicity of the capture surface also affect cell separation performance .…”