At Protagen Jens Beator, Ph.D., protein biochips director, and his team have developed the UNIarray program to systematically look for autoantibodies in patient sera.
“Autoantibodies are constitutive and dynamic components of the immune system,” Dr. Beator explained. “And in good alignment with the definition of a biomarker, they can change specifically with the development of diseases as it is already established for several autoantibodies. Further, autoantibodies are stable in serum or plasma for years, which makes both retro- and prospective studies easily possible.”
The development goal at Protagen is to create indication-specific biochips for clinical screening projects containing panels of (e.g., 20–200) diagnostic biomarkers that differentiate patient populations. The case study presented at the “Biomarker World Congress” highlighted the profile of autoantibodies that were developed for multiple sclerosis (MS) to differentiate relapsing/remitting MS from primary progressive MS and secondary progressive MS. The company defined a panel that shows promise to differentiate subgroups of therapeutic responders and nonresponders.
For development of the MS biochip, Protagen started the project by screening a human recombinant protein-expression library of >10,000 different proteins. Candidate biomarkers were confirmed using an antigen-verification biochip, which is a protein biochip printed with greater than 330 affinity-purified biomarker candidates for the disease state. The company also printed 1,968 other affinity-purified recombinant human proteins, including autoantigens from previous studies. Human serum samples from some 100 diseased and 100 healthy patients were analyzed with these chips to verify disease-specific antigens. To enable the determination of autoantibody titers in the test samples, a serial dilution of an IgG calibrator is spotted on the chip for standardization.
The result of this work is the first biochip prototype for serum-based diagnosis and therapeutic monitoring of multiple sclerosis, according to Dr. Beator.
The focus of in vivo toxicology studies at Merck Research Labs is on toxicity biomarker endpoints as defined as necrosis and degeneration, according to Katerina Vlasakova, research associate in the systems toxicology and safety assessment laboratories. These differ from other ADME endpoints in that they directly correlate with and diagnose an adverse event. Specifically, the scientists at Merck have developed multiplexed assays that target tissue toxicity biomarkers for kidney, heart, skeletal muscle, liver, and acute phase proteins.
“We work with urine for determining kidney toxicity by measuring clusterin, albumin, and Kim-1 levels. We use plasma samples for measuring toxicity in the other organs, looking at cardiac Troponin I, skeletal Troponin I, A2M, AGP, GST-a and other biomarkers,” shared Vlasakova. “We work with accessible biomarkers from plasma and urine sources. If there was an organ injury, those biomarkers will be detectable in plasma or urine.”
With the use of multiplex analysis the Merck scientists can monitor for all target organ toxicities in case the source of toxicity is unknown or just focus on one panel (e.g., kidney) if kidney toxicity is expected.
Taking a multiplex assay approach is key so that multiple endpoints can be obtained from one assay in a given sample. It also maximizes information capture where samples are limiting. Proteins have a different half-life so having multiple markers ensures that toxicity will be detected even when timing of sampling was not optimal for some.
As with the biomarkers used by Biotrin, these Tox biomarkers are tissue-specific and not compound-related, so that they are applicable for general use in all compound screening.
Merck Research Labs uses a Meso Scale Discovery Sector Imager 6000. The Meso Scale technology is similar to ELISA in that it uses antibodies for analyte capture and detection. It differs in several ways, though. First, the capture antibodies are adsorbed to a carbon surface instead of plastic used for ELISA. This enables a “denser” coverage of capture Ab. Second, detection antibodies are conjugated to an electrochemiluminescent label that emits light when electrochemically stimulated. This method of detection is more sensitive and results in wider assay detection range than can be achieved with ELISA, according to the company. The plate contains electrodes, and a CCD camera that measures luminescence. Individual carbon spots within a well allow for multiplexing assays.
The general protocol for an MSD assay is similar to an ELISA protocol; samples are diluted and pipetted on a blocked plate. Typically there are two 1–2 hr incubations (first with sample and second with detection antibody) with a wash after each step. After the last wash, Read buffer is added, and the plate is read on the Sector Imager 6000. Throughput is limited at the first step where individual samples have to be handled and diluted. The plate read itself, only takes about 1–2 minutes.
The main advantage is multiplexing; Meso Scale has commercialized a kidney toxicity 6-plex. In a 96-well plate format a Merck scientist can run 40 samples in duplicate, in addition to a standard curve. At the end of the day, data output in the form of light units can be captured from 240 measurements in a single plate. The use of a standard curve enables the conversion from light units to concentration.
“We are always looking to optimize the current antibody assays and searching literature for new promising biomakers,” noted Vlasakova. “The biomarkers we are developing tend to be more sensitive and more specific than classical biomarkers of tissue toxicity.”