Affinity maturation of tacrolimus antibody for improved immunoassay performance.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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 characteristics, such as affinity, specificity, or stability, into previously isolated and otherwise desirable antibodies to enhance assay performance. These in vitro selections can also be performed under conditions meant to mimic the assay in which the reagent will ultimately be used, to increase the likelihood of successful assay development.
Siegel
Robert W
RW
Core R&D, Diagnostic Division, Abbott Laboratories, Abbott Park, IL, USA. siegelro@lilly.com
Baugher
Wade
W
Rahn
Tanya
T
Drengler
Susan
S
Tyner
Joan
J
D000818:Animals; D000915:Antibody Affinity; D002460:Cell Line; D022801:Complementarity Determining Regions / Q000235:genetics; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism; D007118:Immunoassay; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007166:Immunosuppressive Agents / Q000032:analysis* / Q000276:immunology; D051379:Mice; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D016559:Tacrolimus / Q000032:analysis* / Q000276:immunology
D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007166:Immunosuppressive Agents; D011993:Recombinant Fusion Proteins; D016559:Tacrolimus