NIJMEGEN, The Netherlands—A new "lab-on-a-chip" method promises rapid in-office profiling of autoantibodies in rheumatoid arthritis (RA) without the need for fluorescent labels or preprocessing. The researchers, led by Richard Schasfoort, MD, of the University of Twente in Nijmegen, The Netherlands, reported proof-of-principle data on 50 RA patients and 29 controls in the November Journal of the American Chemical Society.¹

"Detection of serum anti-CCP [anti-cyclic citrullinated peptide] autoantibodies has been carried out directly from diluted serum. In a single step without using labels or conjugates, the test can be run which is much more complicated in an ELISA system," Dr. Schasfoort told Musculoskeletal Report, noting that the new method provides a faster, more flexible way to monitor serum autoantibodies than conventional techniques. Dr. Schasfoort is head of the biochip group at the University of Twente and is chief scientific officer of IBIS Technology Corp. The research was supported by the Dutch Technology Foundation within a project called, "proteomics on a chip for monitoring autoimmune diseases."

Fast way to detect CCP-positive sera

This proof-of-principle experiment used a 24-spot microarray containing human IgG and two different linear citrullinated peptides plus the corresponding control peptides spotted on an N-hydroxy-succinimide preactivated polycarboxylate-coated gold sensor surface using a noncontact spotting instrument. Assays compared sera  from 50 RA patients and 29 controls (10 normal healthy subjects, 10 osteoarthritis (OA) patients, and 9 systemic lupus erythematosus (SLE) patients). The serum reactivity was quantified as the ratio between the surface plasmon resonance (SPR) angle shifts observed for the citrullinated peptide and the control peptide (C/R) after binding of serum antibodies. RA sera were also tested with ELISA for CCP positivity.

The C/R ratios were

• 8.6 for RA sera that were CCP-positive by ELISA
• 0.96 for RA sera that were CCP-negative by ELISA
• 1.01 for normal controls
• 1.15 for SLE controls
• 1.09 for OA controls

The technique is based on SPR, which allows simultaneous real-time monitoring of hundreds of biomolecular interactions on microarrays. SPR is an optical method for measuring the refractive index of material adsorbed onto a thin metal layer, usually gold. Photons of P-polarized light interact with free electrons of the metal, inducing wavelike oscillation of the free electrons and reducing the reflected light intensity; this creates the SPR dip where there is no reflection at all. [Figure 1]

The method developed by Dr. Schasfoort's group uses miniaturized and parallelized immunoassays in a microarray in combination with scanning SPR imaging [Figure 1]. Each chip can be reused up to 50 times by clearing the sensor surface with two 30-second incubations with 10 mM glycine-HCl pH1.5. The process is also much faster and technically simpler than enzyme-linked immunosorbent assay (ELISA), which requires testing separately for each type of autoantibody.



"The assay is extremely simple," Dr. Schasfoort said. "Insert the chip and several diluted (100*) serum samples in the rack of the autosampler. The instrument injects the serum sample in the flow cell, and the responses of all the spots are measured immediately and saved. The instrument shows the responses directly on the screen." A full analysis of interactions can be done in about 1 hour.

"[T]he specificity of autoantibody responses highlights their potential as important tools for improved diagnosis, disease classification, and prognosis. Miniaturized multiplex assays can deliver a fingerprint of a patient's autoantibody repertoire requiring only a limited amount of patient material," Dr. Schasfoort writes.

"Here, we showed that SPR imaging of protein/peptide microarrays provides a method that allows a one-step multianalyte detection of autoantibodies in patients' sera, which does not require additional reagents to visualize antibody binding. The ability to measure in a fully automated fashion with liquid handling procedures increases reproducibility, and once an experiment is started it can continue unattended," the authors write.

The investigators will next study the relationship between serum autoantibody profiles and RA disease activity. "This could lead to more personalized treatment in the future," Dr. Schasfoort said.

SPR technology is expected to move quickly from bench to bedside. Dr. Schasfoort noted that the basic all-purpose SPR instrument costs about 179,000 Euros ($250,000), but that he expects cheaper SPR instruments to become available for use in general hospitals for screening of autoantibodies in serum.

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