Developing phage display tools for use in transfusion medicine

Abstract

GETTING A STARTn surveying the list of the more than 100 past and present NBF awards, what is as striking as the diversity in transfusion medicine topics is the diversity of recipients in terms of both their medical or scientific background and their stage of career ranging from trainee to senior level academician. For each of them, it would appear that funding through the NBF played a particularly personal and unique role in his or her professional development. My case is no exception. Although I had gained much experience in basic science research as a graduate student in biophysics in the late 1970s and early 1980s, my subsequent 7 years of clinical training comprising medical school, a clinical pathology residency, and a blood banking-transfusion medicine fellowship had placed me, in 1991, somewhat out of touch with the world of bench research.During my time away from the lab, everything suddenly became "molecular" and available as a kit. Techniques like polymerase chain reaction (PCR) and DNA sequencing had been invented and became routine laboratory tools, and researchers purchased buffer concentrates instead of making reagents themselves with a balance and pH meter. I was now about to start my academic career and before even considering the notion of applying for federal funding, I was in need of transitional support to afford me the opportunity to become "retooled" as well as to help me define my new area of research investigation.Having devoted my graduate work to the study of the human red cell (RBC) membrane cytoskeleton, 1-3 I decided to stick with the RBC but let my newly kindled I interest in immunohematology lead me to travel approximately 100 Å away from the membrane cytoskeleton through the phospholipid bilayer to the extracellular surface of the RBC where the antigens reside. In particular, I was interested in studying the immune response to RBC antigens as a way of gaining a better understanding of human humoral immune responses, notably those that can distinguish between closely related polymorphic structures. At this point in time, much had been known about the murine immune system because of the ease with which laboratory animal experimentation could be conducted, but ethical issues and other constraints put a damper on the at-will intravenous administration of foreign substances into humans to study the development of humoral immunity. It was apparent though, that the transfusion of allogeneic blood for therapeutic purposes and the undesirable sensitization that can often occur could provide a unique opportunity to ask basic questions about human antibody formation such as clonal relatedness, somatic mutation, affinity maturation, and others.To study human RBC antibodies on a molecular genetic level, I needed to be able to isolate and produce human monoclonal antibodies (MoAbs) from the peripheral blood B cells of sensitized individuals. Technical difficulties inherent in the immortalization of human B cells led me to the use of molecular approaches that obviated the need for cellular trans...

Phage display‐based molecular methods in immunohematology

“…Antibody phage display has been the subject of a number of reviews as an emerging technology in the field of transfusion medicine 8‐10 . At the core of the technology are RBC antigen‐specific monoclonal antibodies (mAbs) which are displayed on the surface of filamentous bacteriophage particles.…”

“…Recently, the development of phage display technology for use in pretransfusion testing has been extended further by exploiting the naturally occurring presence of unique DNA sequences within the particles 10,22 . The concept here is that the phenotype of a cell can be determined by assaying the genotype of the detecting reagent.…”

“…Antibody phage display has been the subject of a number of reviews as an emerging technology in the field of transfusion medicine. [8][9][10] At the core of the technology are RBC antigen-specific monoclonal antibodies (mAbs) which are displayed on the surface of filamentous bacteriophage particles. In contrast to expensive and time-consuming conventional cellular methods for generating mAbs from B-lymphocytes, antibody phage display works by immortalizing the immunoglobulin genes rather than the cells from which they were derived.…”

“…12 Recently, the development of phage display technology for use in pretransfusion testing has been extended further by exploiting the naturally occurring presence of unique DNA sequences within the particles. 10,22 The concept here is that the phenotype of a cell can be determined by assaying the genotype of the detecting reagent. The advantages of this approach ("phenotyping-byreagent-genotyping") are that it exploits the antigen recognition properties of an antibody with the benefits of nucleic acid-based detection schemes (i.e., extraordinary high sensitivity and specificity, requirement for minute amounts of reagents, adaptability to automation, etc.).…”