title: Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.
abstract: The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Della Ducata
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Jaehrling
authors.affiliation: QIAGEN Lake Constance GmbH, Stockach, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Hänel
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Satzger
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Wolber
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ostendorp
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pabst
authors.affiliation: MorphoSys AG, Martinsried/Planegg, Germany Bodo.Brocks@morphosys.com.
authors.forename: Bodo
authors.initials: B
authors.lastname: Brocks
mesh_terms: D000906:Antibodies / Q000032:analysis*; D000915:Antibody Affinity; D002457:Cell Extracts / Q000737:chemistry; D003217:Conductometry; D017077:Culture Media, Conditioned; D055664:Electrochemical Techniques / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis*; D007074:Immunoglobulin G / Q000032:analysis*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D000095983:Bacterial Lysates
chemical_list: D000906:Antibodies; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; C030259:Broncho-Vaxom; D000095983:Bacterial Lysates
keywords: affinity screening; electrochemiluminescence; equilibrium dissociation constant; high throughput; solution equilibrium titration