Determination of Phage Antibody Affinities to Antigen by a Microbalance Sensor System

Abstract

Over the past decade, phage display has maturated to be a frequently used method for the generation of monoclonal antibodies of human origin. The essential step of this method is the "biopanning" of phage carrying functional antibody fragments on their surface on an immobilized antigen. The screening of large combinatorial gene libraries with this method usually leads to a set of diverse clones specifically binding to the antigen that need to be characterized further. Beside its specificity, the key parameter to be determined is the affinity of the recombinant antibody fragment to its antigen. Here, we present a mass sensitive microsensor method that allows the estimation of antibody affinity directly from the phage supernatant. Binding of phage antibodies to the antigen immobilized on a quartz crystal microbalance (QCM) induced a mass dependent decrease in frequency. This principle was used to determine the apparent affinity of a single-chain (sc)Fv antibody against the RNA polymerase of Drosophila melanogaster presented on the surface of a filamentous phage (M13) from its association and dissociation rates. The apparent affinity obtained is in accordance with the affinity of the scFv fragment as determined by conventional equilibrium enzyme-linked immunosorbent assay (ELISA) and plasmon resonance methods.

Biosensors and biochips: advances in biological and medical diagnostics

“…Another form of transduction that has been used for biosensors is the measurement of small changes in mass [151][152][153][154][155][156][157][158][159]. This is the newest of the three classes of measurements, however, it has already been shown to capable of very sensitive measurements.…”

Quantitative measurement of p53-p53 antibody interactions by quartz crystal microbalance: A modelsystem for immunochemical calibration

“…The quartz crystal microbalance has been shown previously to provide important gravimetric data required to evaluate protein surface concentrations and to evaluate antibody sensitivity and specificity [10]. The method can also yield information on the adsorbed film viscoelastic properties due to vibrational losses within protein film caused by the shear motion of the quartz crystal surface [9].…”

QCM Technology in Biosensors

“…QCM technology has a huge field of applications in biochemistry and biotechnology. The availability for QCM to operate in liquid has extended the number of applications including the characterization of different type of molecular interactions such as: peptides (Furtado et al, 1999), proteins (BenDov et al, 1997), oligonucleotides (Hook et al, 2001), bacteriophages (Hengerer et al, 1999), viruses (Zhou et al, 2002), bacteria (Fung & Wong, 2001) and cells (Richert et al, 2002); recently it has been applied for detection of DNA strands and genetically modified organisms (GMOs) (Stobiecka et al, 2007). Despite of the extensive use of QCM technology, some challenges such as the improvement of the sensitivity and the limit of detection in high fundamental frequency QCM, remain unsolved; recently, an electrodeless QCM biosensor for 170MHz fundamental frequency, with a sensitivity of 67 Hz cm -2 ng -1 , has been reported (Ogi et al, 2009); this shows that the classical QCM technique still remains as a promising technique.…”