Affinity maturation of tacrolimus antibody for improved immunoassay performance

Clinical Chemistry
Robert W SiegelJoan Tyner

Abstract

Organic solvents used for extraction of tacrolimus from whole blood samples lower the apparent affinity of the antibody used in a diagnostic immunoassay, thereby affecting the detection limit. We used in vitro recombinant antibody engineering to screen and isolate clones from diverse libraries with mutagenic complementarity regions (CDRs) from tacrolimus 1-60-46 hybridoma cell line, with improved binding to tacrolimus in the presence of 10% methanol organic solvent solution. We isolated a number of clones with mutations in variable heavy (VH) CDR 2, variable light (VL) CDR 1, and VL CDR 3 with improved binding. Various combinatorial pairings constructed from these individual mutations contained >10-fold improvements in both the dissociation rate and overall equilibrium affinity constants. Selected clones produced as IgG have increased functional sensitivity, with a 3- to 6-fold reduction in the limit of detection relative to the parental tacrolimus 1-60-46 monoclonal antibody in the Architect Tacrolimus immunodiagnostic assay. The recent advent of recombinant in vitro antibody display technologies in general, and yeast surface display in particular, allows the flexibility to engineer new or augment specific analytical character...Continue Reading

References

Sep 11, 1990·Nucleic Acids Research·S Mizushima, S Nagata
Jan 1, 1983·Methods in Enzymology·T L Orr-WeaverR J Rothstein
Dec 1, 1995·Therapeutic Drug Monitoring·J J Fung, T E Starzl
Jun 1, 1997·Nature Biotechnology·E T Boder, K D Wittrup
Feb 12, 1998·Journal of Molecular Biology·B Al-LazikaniC Chothia
Feb 9, 2000·Biotechnology Progress·J J VanAntwerp, K D Wittrup
Jun 17, 2000·Biotechnology Progress·E T Boder, K D Wittrup
Sep 14, 2000·Proceedings of the National Academy of Sciences of the United States of America·E T BoderK D Wittrup
Oct 30, 2001·Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association·J DietemannF Berthoux
Apr 17, 2004·Journal of Immunological Methods·Robert W SiegelMichael J Feldhaus
Apr 30, 2004·Protein Engineering, Design & Selection : PEDS·Christilyn P GraffK Dane Wittrup
Jan 28, 2005·Assay and Drug Development Technologies·Ryan J Darling, Pierre-Alexandre Brault
Sep 28, 2005·Protein Engineering, Design & Selection : PEDS·Jane M Weaver-FeldhausRobert W Siegel
May 30, 2006·Journal of Immunological Methods·Maria GrovesRonald H Jackson
Sep 12, 2006·Journal of Molecular Biology·Béatrice LuginbühlAndreas Plückthun
Dec 19, 2006·Nature Biotechnology·Consuelo Garcia-RodriguezJames D Marks
Apr 5, 2007·Nature Protocols·Ginger ChaoK Dane Wittrup
May 1, 2007·Journal of Molecular Biology·Christian ZahndAndreas Plückthun

❮ Previous
Next ❯

Citations

Jan 1, 2013·World Journal of Biological Chemistry·Zhi-Jian LuPei Jin
Oct 25, 2011·Journal of Molecular Biology·Bruno H MullerFrédéric Ducancel
Nov 6, 2014·PloS One·Li PengWilliam F Dall'Acqua
Jan 15, 2014·Clinical Chemistry·Verena Gounden, Steven J Soldin

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.