Characterization of a human pancreatic secretory trypsin inhibitor mutant binding to Legionella pneumophila as determined by a quartz crystal microbalance.

We describe the isolation from a large phagemid library of a human pancreatic secretory trypsin inhibitor (hPSTI) mutant that binds to Legionella pneumophila. To gain further insight into the binding kinetics of the isolated hPSTI mutant, an immunosensing system based on a quartz crystal microbalance (QCM) was used. In contrast to ELISA procedures, k(on) and k(off) rates could be derived from the QCM sensograms. Thus, it is possible to characterize specific intermolecular interactions between proteins and phages isolated from large phage display libraries by QCM.

Determination of phage antibody affinities to antigen by a microbalance sensor system.

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.

Quartz crystal microbalance (QCM) as a device for the screening of phage libraries.

An immunosensing system based on a quartz crystal microbalance (QCM) is presented for the selection of both antigen specific recombinant antibodies and antigen specific human pancreatic secretory trypsin inhibitor (hPSTI) mutants isolated from large phage libraries. The QCM was integrated into a flow injection analysis system for the straightforward analysis of large sample numbers. Measurements were performed using a biotinylated antigen immobilized by streptavidin onto the gold surface of the quartz crystal and phages displaying recombinant antibodies or hPSTI mutants. The results obtained by the QCM were in accordance to those of a well established enzyme linked immunosorbent assay (ELISA). Therefore, the QCM is well suited for the detection of single high affinity clones isolated from large phage display libraries.

Characterization of immobilization methods for African swine fever virus protein and antibodies with a piezoelectric immunosensor.

A direct piezoelectric flow injection analysis immunoassay for the detection of African Swine Fever virus and antibodies is presented. The peptide-specific monoclonal antibody 18BG3 and the virus protein 73 were used for detection with a quartz crystal microbalance. Accumulation of the analyte on the surface of this mass-sensitive biosensor resulted in a shift of the resonant frequency. Highly selective receptor layers were applied on the sensing electrode of the quartz crystal for detection of the complementary analyte. Different immobilization methods proved to be appropriate for coating of the monoclonal antibody 18BG3. A quartz crystal covalently coated with the antibody 18BG3 detected virus protein VP73 samples more than 20 times and was stable for more than 30 days. The coating of virus protein was performed by physisorption. A sensor with a virus protein receptor layer detected antibody 18BG3 samples 10 times within one day. The sensor device was able to perform one measurement cycle including blocking and regeneration within 30 min. With the help of a suitable carrier liquid, measurements with serum samples were performed. The calibration curves for measurements in buffer and in serum could be determined and the detection limits for virus protein detection were 0.31 and 1 microgram/ml, and for antibody detection 0.1 and 0.2 microgram/ml, respectively.

Biosensor-based methods in clinical diagnosis.

Despite the multitude of different parameters currently measured in the clinical laboratory, only a minor part of these are measured by means of biosensor-based methods. Within the group of biochemical sensors, enzyme or metabolic sensors as integrated devices in clinical analyzers are dominating. Economically most important are sensors for measuring blood glucose. The implementation of lactate and urea sensors in commercial analytical systems is just beginning and a few more analytes are being intensively investigated in research laboratories (1). Thus, it seems that metabolic biosensors slowly leave the academic playground.

The Quartz Crystal Microbalance (QCM) as an Immunosensor.

This chapter demonstrates a preliminary experimental approach to detect antiviral antibodies by means of a quartz crystal immunosensor. As an example of the large numbers of immunoassays currently applied in the clinical laboratory, the screening for human immunodeficiency virus (HIV) specific antibodies was presented as a model application. The quartz crystal microbalance belongs to the group of acoustic immunosensors and its principle of operation is based on the propagation of acoustic shear waves in the substrate of the sensor. Phase and velocity of the acoustic wave are influenced by the specific adsorption of antibody molecules onto the antigen-coated sensor surface. This primary measuring allows the continuous monitoring of molecular interactions in liquids.

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.

A scanning tunneling microscope (STM) for biological applications: design and performance.

The design of a scanning tunneling microscope (STM) for biological applications, operating at ambient pressure, is described. The STM is combined with an "auxiliary" light microscope to facilitate finding and identifying specimen areas of interest. The performance of the STM has been tested with evaporated gold films and with graphite. We have evaluated evaporated carbon and platinum/carbon films deposited on glass or mica to be used as specimen supports. First applications to biological material coated with a conducting film of platinum/carbon are described.