Simultaneous detection of a number of molecular interactions in a single sample could enable researchers to relate such interactions to one another for autoimmunity research and similar applications. In the past decade, several groups have implemented multiplexed assays for autoantibody detection. With these studies, though, either the autoantibody or the protein must be labeled, which can interfere with the binding sites and possibly produce false negatives.Surface plasmon resonance (SPR) imaging with miniaturized and parallelized immunoassays in microarray format can help address this challenge. In the Nov. 14 issue of Journal of the American Chemical Society, researchers with the University of Twente in Enschede and with Radboud University in Nijmegen, both in the Netherlands, reported using an SPR imaging analysis system with a novel SPR dip angle scanning principle to monitor multiple biomolecular interactions on microarrays simultaneously with great precision.Researchers have reported a surface plasmon resonance imaging system that utilizes SPR dip angle shifts to monitor biomolecular interactions on microarrays. Such simultaneous detection of multiple interactions could benefit autoimmunity research and a variety of other studies.Most SPR imaging systems use a single fixed incident light angle, which makes it difficult to monitor numerous biomolecular interactions in parallel on a microarray. The most reliable way to circumvent this problem is to measure SPR dip angle shifts, which correspond to mass changes linearly. By gauging such shifts for all spots on a microarray separately, investigators can compare the magnitude and affinity of biomolecular interactions with confidence.The researchers achieved this using a scanning SPR microarray imaging system made by Ibis Technologies BV of Hengelo, the Netherlands, which allows simultaneous imaging of up to several hundred spots and allows accurate detection of angle shifts by using innovative scanning optics. They monitored the interactions between citrullinated peptides and serum antibodies from 50 rheumatoid arthritis patients and were able to detect various anticitrullinated protein antibodies at the same time. (The citrulline amino acid has been shown to be an important part of the antigenic determinants targeted by autoantibodies specific to rheumatoid arthritis; citrullinated peptides are commonly used as antigenic targets in ELISA diagnoses.) Richard B.M. Schasfoort, the principal investigator of the study and chief scientific officer at the company, described several advantages of the system beyond its ability to image many spots at once. Because of the imaging feature, he said, investigators can check the quality of the surface, and automation of the sample delivery to the sensor surface is performed using liquid-handling procedures.“Because the instrument detects refractive index changes in the evanescent field of the sensor surface, it also detects nonspecific binding events very easily, but this is compensated in the instrument by measuring both specific and nonspecific interactions simultaneously,” Schasfoort said. “This is the advantage of measuring many label-free biomolecular interactions enabling referencing, correcting, compensating for optimal accuracy and reliability of the data.”He noted that the sensitivity was slightly lower than can be achieved with existing ELISA tests, even excluding the nonspecific binding events from the results or referencing them with another spot with no specific binding. “[But] replacing well-known ELISA technology is not our goal,” he said, adding that label-free detection and the determination of molecular affinity are more important considerations.Using the system with panels of tests with greater than 100 spots, clinicians working with autoimmune diseases could obtain complete profiles of related autoantibody interactions, including references, blanks, positive and negative controls and more, allowing them to test new therapies and to monitor disease progress.The researchers continue to test the system’s potential, however. “The company wants to improve the specifications (noise levels, detection limits, etc.) in order to improve application areas other than autoimmune diseases,” Schasfoort said.Contact: Richard B.M. Schasfoort, University of Twente and Ibis Technologies, both in the Netherlands; e-mail: r.b.m.schasfoort@tnw.utwente.nl