Drug development is such a formidable challenge that, some believe, it can only be undertaken effectively by big pharma. Time-consuming and expensive, the classic approach has been to screen vast numbers of low molecular weight compounds for efficacy in blocking disease processes and then move through various levels of cell culture, animal evaluation, and human trials to find safe and effective low molecular weight substances of value in disease treatment.
An alternative tactic is to screen tissues for marker proteins that might be the focus of therapeutic intervention. While this approach has allowed the discovery of numerous drug targets, it is laborious, expensive, and highly complex.
In recent years another route to drug discovery has been explored. For many diseases, circulating serum antibodies in patients can be used to detect marker proteins that serve a therapeutic or diagnostic function. These patient sera can be screened on protein microarrays. This approach, which is more accessible to companies with limited resources, was the focus of many presentations at Cambridge Healthtech Institutes Peptalk: The Protein Information Week.
Human Protein Arrays
According the Paul Predki, Ph.D., vp for proteomics research at Invitrogen (www.invitrogen.com), the companys ProtoArray can be used to study a large number of proteins in a novel format. Consisting of 5,000 human proteins, it was designed to identify elevated or decreased levels of circulating antibodies. Dr. Predki outlined the process as involving the collection of a library of cloned genes, expression in baculovirus as open reading frames, high-throughput purification, and, finally, the building of the microarrays. The proteins run the gamut from kinases, transcription factors, membrane proteins, nuclear proteins, to signal transducers.
The proteins expressed from the collection are printed in duplicate on glass slides and can be screened for interactions with the antibodies rapidly. The proteins were generated as GST-tags and purified under conditions designed to maintain maximum quality. The purified human proteins are printed under controlled conditions and quality controlled for irregular spot morphology and other distribution problems.
Potential biomarkers are identified by probing with sera from diseased patients and comparing with sera from healthy controls. The rationale being that patients harboring a disease state will generate antibodies against critical proteins associated with the condition. Pilot experiments demonstrate that the serum-profiling application protocol for detection of human antibody-protein complexes on protein arrays is specific and highly sensitive.
By comparing serum samples from melanoma patients with healthy controls, the Invitrogen team was able to identify over 100 novel putative biomarkers, including several known to be autoantigens for melanoma. The Invitrogen array technology can also identify cross reactivity of antibodies by screening candidate monoclonals against the proteins present in the microarrays.
Domesticating the Proteome
A major challenge in proteomics is the presence of over-abundant proteins in samples of tissue or biological fluids such as serum. Indeed, 21 highly abundant proteins comprise 90% of serum proteins, and albumin represents one-third of this total. This can severely complicate the task of selecting rare and interesting proteins from a vast collection of stultifying common representatives.
Lee Lomas, Ph.D., director of biological research and development at Ciphergen (www.ciphergen.com), discussed a novel technology for equalizing concentrations of different protein species. We seek to compress the dynamic range of protein concentration using an affinity column based on a peptide combinatorial library, he said.
The peptide library of high diversity is generated synthetically (not through the use of phage display) on a bead support using a classical split, couple, and recombine combinatorial approach, with each bead containing a single unique ligand.
The protein equalizer beads form a large collection of millions of different ligands. When serum is washed through the column, the proteins present in great excess quickly saturate the available binding sites and all nonbound material is washed away. Conversely, rare proteins are concentrated on their specific ligand and prevented from being washed away. Since all ligands are equally represented in the collection, the result is that those molecules that bind to the column and then are subsequently recovered still reflect the overall complexity of the starting material while simultaneously diluting the abundant proteins and concentrating the rare ones.
The eluate can then be coupled to Ciphergens protein arrays, and the recovered proteins further fractionated before being detected through mass spec, using Ciphergens PCS4000 mass reader. In this process, the protein eluates from the column bind to array elements containing a solid-phase extraction media such as ionic, hydrophobic, or IMAC media. Under defined binding conditions, proteins with specific physical-chemical properties can thus be selected before mass spec detection.
Ciphergens Protein Equalizer Technology is not limited to serum, but can be applied to a variety of biological fluids, including urine, saliva, cerebrospinal fluid, bacterial extracts, or cell culture media. When fully automated through robotics, the system can be used to detect, isolate, and characterize new biomarkers for disease by comparing large numbers of samples from normal and diseased individuals.
Robert Negm, Ph.D., vp of GenTel BioSciences (www.gentelbio.com), asserted the need for timely risk assessment and specific anticoagulant therapies tailored for individual care for cardiovascular disease. To meet this challenge GenTel is designing a multiplex immunoassay to measure a panel of markers associated with thrombophilia to identify a molecular signature in blood that can be used to aid in early detection and personalized therapy.
GenTels approach involves printing microspots of specific antibodies at unique addressable locations and subsequently measuring their analytical mass simultaneously using a sandwich antibody configuration.
The companys PATH protein microarray slides consist of a nonporous, ultrathin nitrocellulose surface that achieves high immunoassay sensitivity, since the surface chemistry delivers significantly less fluorescent background compared to conventional nitrocellulose slides, resulting with higher signal-to-noise immunoassay measurements.
GenTels assays consist of a sandwich ELISA, where a biotinylated detector antibody specific for the protein target and a Streptavidin-fluorophore conjugate result in a system that measures 1020 proteins in blood with over 5 logs of dynamic range and a sensitivity down to 0.05 pg/mL.
While the original PATH configuration has the dimensions of a conventional microscope slide, the PATH surface chemistry is also available on PATH HTS. The high-throughput configuration allows for 96 distinct antibody microarrays to be processed in parallel on plate-handling robots.
In a collaboration with Haematologic Technologies (www.haemtech.com), GenTel has evaluated tens of clotting factors associated with thrombophilia. To date GenTel has validated matched antibody pairs, specific for prothrombin, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, and Protein C and demonstrated that they bind their cognate antigens without cross-talk (nonspecific binding) to the alternative antigens and these measurements are reproducible.
We aim to partner with Decision Biomarkers (www.decisionbiomarkers. com) and configure our multiplex immunoassay COAG chip for use with their automated instrument, Dr. Negm said.
Decision Biomarkers Multimark Express automates multiplex immunoassays, with a hands-free, lab-on-a-chip process, leaving the user free from fluid transfer, reagent bottles, and contamination issues.
Quantitative Protein Microarrays
Miniaturization of biological assays requires dispensing liquids in the submicroliter range of volumes, according to Walter Niles, Ph.D., scientific fellow at Aurora Discovery (www.auroradiscovery.com). The company concerns itself with construction of microarrays and to this end has faced the challenges of signal variability through the use of inkjet dispensers instead of pin tools.
Inkjet dispensing allows for dramatic decrease in variance, superior to conventional methods, explained Dr. Niles. The liquid is dispensed by application of direct pressure to the liquid vapor interface and is independent of the balance between the liquids wettability on the surface as opposed to the target surface.
The liquid is ejected completely so the precise volume is delivered, and small volumes (500 pL drops) are dispensed to tightly adhere to the target surface. Dr. Niles and his team use a noncontact liquid handler, PicoRap TR, a robotic device capable of rapid transfer from the source to the destination.
The piezo-dispensing technology offered by Aurora Discovery provides new levels of miniaturization. Using either 360- or 96-well ELISA plates, it is possible to apply nine spots per well in the 360-well plate and 25 well-defined, individual spots per well in a 96-well plate.
These configurations could serve a variety of functions, such as screening a single patient sample simultaneously for either 9 or 25 different markers.