Single colour reflectometry – short SCORE – can be used to analyse the binding characteristics of hundreds to thousands biomolecular interactions without the need for labels. In the past years, label-free kinetic analysis used to be available only for a few lead candidates at the end of a pharmaceutical development process. But with a high throughput technology like SCORE it now is affordable to screen a high number of candidates at the beginning of the development of therapeutic antibodies. Its advantage is not only the gain of binding information like specificity and binding strength, but also the savings in time and costs due to the high throughput set up and the elimination of labelling steps. Further characterization like epitope mapping and binning can also be integrated into the analysis process.

Biomolecular interactions

On a molecular scale, all essential processes in living organisms go back to the complex interactions of a variety of biomolecules. Knowledge about this so called interactome gives access to understanding how cells work and how they communicate. This knowledge is also used modern drug design, as pharmaceuticals are designed to interact with a specific target to interfere with one specific process. At the same time, unwanted interactions of drugs with off-target molecules need to be avoided. In addition, the functionality of a drug molecule is determined by its binding properties towards its target – usually specific receptors, channels or enzymes. Therapeutic antibodies, for example, are usually designed to recognize a predefined part (epitope) of the target molecule and bind it with high specificity and affinity.

The development of therapeutic antibodies and its challenges

The process of developing therapeutic antibodies usually starts with the generation of a cell line library. Each cell line will produce one type of monoclonal antibody against the selected target molecule. These antibodies will vary in their specificity and affinity towards the target structure. Different antibody candidates (paratopes) will even recognise different structures on the target molecule (epitopes). To screen antibody properties in this early stage of development, different bioanalytical methods are employed and the most promising candidates to move along in the development process are selected. Selection criteria include affinity maturation, humanisation or a set of antibodies to combine into bispecific antibodies, that can be produced in cell lines.

Apart from reaching a high throughput, it can be challenging to identify the most suitable candidates with the analytical methods employed today. One possible cause for misinterpretation are the properties of the employed cell lines, which produces antibodies in different concentrations. By switching to label-free analytical methods this source of errors can be avoided. Label-free methods offer the advantage, that they are not limited to showing which antibody binds the target, but also give access to affinities and the rate constants of the underlying binding process. With these additional characteristics, the most promising antibodies can be selected, even from low concentration samples.

Label-free microarrays

Microarrays have been established as a valuable high throughput technology in the past years. Thousands of biomolecules can be immobilised (spotting) onto one microarray. The binding of a component to the biomolecules is then evaluated using fluorescence labelled reagents. However, this technology does not give information on affinity and rate constants of the binding events. Only the combination with SCORE read-out allows label-free high throughput screening of microarrays with real time analysis of the complete binding process. Due to time resolved observation of all binding events on the microarray simultaneously, association and dissociation can be analysed. This gives access to kinetic and thermodynamic characteristics of all binding reactions on the microarray in a fast and simple setup.

Label-free microarrays are a perfect tool to tackle the challenges in the development of therapeutic antibodies. All antibody candidates in a screening campaign can be immobilised directly from cell culture supernatants by making use of established microarray printing processes and IgG (Immunglobulin G) coated capture surfaces. Label-free screening of the interaction of the antigen against each antibody spot gives the user an easy way to identify and rank each antibody according to its binding characteristics. Farther along the development process the label-free microarray technology can be used for epitope mapping and binning assays. These are of great interest to identify the epitope that is bound by the antibodies. Binning experiments allow the layout of epitope maps. Furthermore, linear epitopes can be identified using peptide microarrays.

Figure 1: Epitope binning can be performed in a streamlined workflow. All antibody candidates in a library are printed in an array format and screened against each individual antibody (A). Analysis of the binding pattern (B) yields the binding matrix for the antibody library (C) and an epitope map (D).

Figure 1A shows a schematic of how a binning experiment with different antibodies are conducted. In the first step, replicates of all antibody candidates are printed to produce the microarray. Using the array, one measurement is performed for each antibody. In each of these, the antibody is incubated with a surplus amount of antigen before the mixture is flushed over the microarray. By observing the binding pattern of such mixtures with the antibody array (figure 1B), one can find antibody pairs which do not hinder each other by binding the epitope on the antigen. The result is the so called binning matrix (figure 1C), which is used for epitope mapping (figure 1D). Antibodies identified during the screening process can also be analysed for cross reactions with similar antigens or off-target binding partners using label-free microarrays. To this end, peptide microarrays featuring the antigen as well as numerous other protein epitopes from the cell interactome can be employed.

In addition to therapeutic antibodies, peptide based drugs are promising candidates to treat various diseases. They can be synthesised in large variety of structures for a screening campaign. Peptide microarrays are now used to provide customer specific peptide libraries. The classic method to produce peptide arrays is the printing of pre-synthesised peptides to a surface. Novel methods of in situ peptide synthesis directly on a chip surface are further accelerating the progress in this area.  In combination with label-free detection via SCORE it is now possible to analyse identify target binders in peptide libraries and characterise them in the same measurement.

Figure 2: The binding of an antibody against a peptide microarray was analysed with SCORE (left) and standard fluorescence methods (right).

Figure 2 shows the binding of an anti-FLAG antibody to an array of 1485 peptides measured with SCORE in comparison to a standard fluorescence based measurement. In addition to the binding information, which can be attained with fluorescence labels, SCORE allows the time resolved observation of the binding process from the complete microarray. This gives access to kinetic and thermodynamic parameters (association rates ka, dissociation rates kd and affinity KD) of the biomolecular interactions (Figure 3A+B) on all spots. In order to find the targets with highest affinities data can be visualised as an affinity heatmap.

Figure 3: Binding curves from hundred up to thousands candidates against a target are recorded in real time in one run (A). Analysing binding at different analyte concentrations yields affinities as well as association and dissociation rates for all binding pairs.


Label-free microarrays will improve and accelerate the development of biologics like therapeutic antibodies. In combination with peptide and protein microarrays, SCORE technology will help to identify the best drug candidate earlier, with high accuracy and decrease time and costs of the biologics screening process.


Read the German version of this article published in LABO.

This article was written by
Günther is CEO and co-founder of Biametrics. He has a background in biology and holds a PhD in physical chemistry. He is one of the inventors of SCORE technology and always on the lookout for the next groundbreaking application of label-free microarrays.