USES OF TECHNOLOGY
Detection of tiny amounts of biological molecules (analytes) was first made possible by radioactive reagents. However, radioactivity poses a health threat and adds significant cost to ensure proper handling and disposal of materials. The development of organic fluorophores opened the door to the development of entirely new assays with greater safety and ease of use. Demand for fluorescent technology has continued to increase due to the enormous growth in high-throughput techniques for studying biomolecules. These techniques use automation to increase speed and miniaturization to reduce costs, but miniaturization requires high sensitivity to detect very small quantities of analytes. Fluorescence uses excitation by light, avoiding radioactivity as well as more cumbersome procedures of other methods, but its low sensitivity has impeded miniaturization and made some assays impossible or too costly to run. Despite its current limitations, fluorescence has become the cornerstone of high-throughput research. Researchers in the field believe more sensitive technologies are needed in this large market.
Lanthanides are attractive candidates for use as reagents because of their unique luminescent properties. Lumiphore has superior lanthanide detection reagents through an exclusive license to patents granted in 2002 by the University of California at Berkeley. This technology is based on a proprietary shell around the lanthanide molecule that confers exceptional luminescence, brightness, stability and versatility. Lumiphore’s patented lanthanide technology offers numerous advantages over current fluorescent systems:
- High signal-to-noise ratio results in assay sensitivities significantly better than current fluorophores.
- Multiple lanthanide fluorophores (four different colors) enable up to four simultaneous assays.
- More linear response to different concentrations.
- Resistance to photo bleaching allows archiving of results for improved quality control and for comparisons between old and new data.
The use of fluorescent reagents can be divided into three broad categories: nucleic acid analysis, protein analysis and small molecule analysis. These applications are employed across the entire drug development and discovery pathway.
||How Fluorescence is Used
||Benefits of Lumiphore Reagents
|High- Throughput Screening
||High-Throughput Screening (HTS) is an empirical method for identifying compounds that inhibit or augment the activity of specific proteins believed to be involved in a disease process. Companies screen libraries of hundreds of thousands of compounds against a single target to find small molecules with the greatest potential to become pharmaceuticals. Many different fluorescent assays are used in HTS, including fluorescence energy transfer (FRET), time-resolved fluorescence (TRF), and fluorescence polarization (FP). FRET and TRF rely on energy transfer between two fluorescent dyes, usually Cy3/Cy5 or Cy5/Cy7, to detect the reaction of interest. FP detects the change in polarization of a dye as a result of binding between a ligand and receptor or other reaction of interest.
||Automation and miniaturization have made HTS economically feasible. Current costs are about 50 cents per assay, but companies would like to reduce this down to 5 cents per assay. The most promising avenue for reducing costs is further miniaturization, but more sensitivity is required.
||High-Content screening (HCS) uses cell-based assays to produce multi parameter or functional information about the interaction of a compound with a specific target. As with HTS, fluorescence is used to detect the reaction of interest.
||High sensitivity is extremely important in these studies. Additionally, Lumiphore's technology enables kinetic studies because the lanthanides do not photo bleach.
||Antibodies are used to detect specific antigens. The most common labeling method is fluorescence, used in both research and diagnostic applications.
||Not all proteins in a sample are expressed at high enough levels to be detected with current methods. Increased sensitivity is needed to study many important proteins that are tightly regulated by cells. The ability to multiplex means multiple analytes can be detected from a single patient sample.
||There are two types of protein microarrays. Capture protein microarrays are conceptually similar to DNA microarrays. They capture target proteins from a tissue sample and provide a quantitative measure via a label, usually fluorescence. Interaction protein microarrays are used to query protein-protein, protein-lipid, and protein-small molecule interactions. They are also used to query protein function. (A kinase, for example, interacts with what it phophorylates.) In both types of protein microarrays, the captures or interactions can be detected with a variety of methods, but the most common is fluorescence.
||Proteins cannot be amplified, and tissue samples are often small, so many proteins expressed at low levels cannot be detected on current protein microarrays. These proteins could be the key agents in disease processes but are beyond the capabilities of current detection methods.
||There are over one million proteins in the human body. A variety of technologies are used in their analysis, including gel electrophoresis, mass spectroscopy and cellular based methods. Gel electrophoresis is critically important in proteomics because it permits the separation of important proteins from samples for further analysis.
||Proteins that are important in disease processes are often present in small amounts that are not detectable with current technology. Lumiphore's technology will lower the limits of detection and enable multiplexing to improve process throughput.
||The microscope is a reliable and established technology for examining fluorescent signals from tissue samples. One of the most common signals is from a labeled antibody that binds specifically to a protein target of interest. This type of experiment is termed immunocytochemistry, and can be used to determine which cell types express a protein of interest in a tissue containing a wide range of distinct cell types.
||The resolution possible with fluorescence microscopy, and the amount of information obtained, depend on the optics of the microscope and the sensitivity of the fluorescent label.
||Primarily used to study gene expression by assessing the level of mRNA in a sample or by comparing levels between normal and diseased tissues. Oligonucleotides or cDNA strands are attached to a surface and used to capture specific genes in the sample tissue. Because the sample genes are labeled with a fluorescent dye, usually Cy3 and/or Cy5, the binding events can be detected with laser excitation and read quickly by a scanner.
||Current detection methods can identify about 80% of the genes in a typical sample. Identification of the remaining 20% is possible via amplification, but this process is not usually linear, interfering with accurate quantification of expression levels. Identifying and quantitating the remaining 20% will require more sensitive detection methods.
||This method uses the polymerase chain reaction (PCR) to quantitate levels of gene expression. An oligonucleotide probe is labeled with both a fluorescent dye and a quencher. When the probe is intact, prior to cleavage by DNA polymerase, there is no fluorescence. During each PCR cycle the reporter dye is cleaved from the probe, producing fluorescence. The intensity of fluorescence increases proportionally as the DNA is amplified. Used in research and diagnostics, including gene expression profiling and SNP analysis.
||The key to quantitative PCR is quantifying the levels of the gene during the linear phase of an otherwise geometric expansion. The sooner the signal can be detected over background noise, the larger the linear range. Lumiphores's technology can expand this range and provide more accurate quantitation. The availability of different complexes permits multiplexing, which is difficult with current technology.
||The genomes of humans vary from individual to individual. One way to study this variability is to sequence entire genomes from a wide range of people, but this is prohibitively expensive. A more practical method is to first identify the differences, and then study how the differences affect the predisposition to disease or the response to drug treatments. Differences can be as small as single nucleotide polymorphisms (SNPs). There is a range of methods to identify and study SNPs, but most involve fluorescent oligonucleotide probes of some sort.
||Again, it comes down to miniaturization. To make SNP testing economically viable, most experts estimate that the costs per SNP will have to be on the order of a tenth of penny, but they are currently on the order of several dollars. Miniaturization is required to bring costs down further, and this will only be achieved with more sensitive detection methods, such as those Lumiphore can offer.
Immunoassays are a significant segment of the $20 billion in vitro diagnostic market accounting for nearly 40% of total sales. Key applications are endocrine testing ($1 billion worldwide), cancer markers ($700 million), blood processing ($600 million), therapeutic drug monitoring ($400 million) and drugs of abuse testing ($300 million). Increased use of automation and the introduction of rapid immunoassays are driving demand for homogenous, highly-sensitive tests that do not rely on radioactive detection methods. Additionally, the identification of new disease markers is spurring growth in this market.
Sensitivity can pose a major challenge in diagnostic assays because diagnostic proteins can exist at levels too low to be reliably detected with current technology. Radioactive detection methods can be used but these systems are expensive and hazardous to use. Lumiphore’s reagents are the ideal solution for low-level protein detection. Continued efforts to automate diagnostic tests require reagents that function well in homogeneous formats, and Lumiphore’s lanthanide compounds are ideally suited to homogeneous assays. Lumiphore’s compounds can be multiplexed, enabling the detection of multiple analytes from a single clinical sample, leading to faster test times at lower cost.
Over the last ten years, pharmaceutical discovery has focused on screening large libraries of chemical compounds against disease targets of interest to find potential drug leads for further testing. In a single HTS facility, hundreds of thousands of compounds are repeatedly tested each year against about 50-100 targets. Current total spending on HTS, including equipment and consumables, is estimated to exceed $2 billion. Consumables alone are expected to account for nearly $1 billion in sales by 2005. Fluorescent reagents have been rapidly embraced by the HTS industry as a means to reduce its reliance on radioactive assays and to enable new assay methods. However, as the number of targets and compounds being screened has increased, the industry has begun to look for ways of lowering screening costs by reducing assay volumes. Miniaturization reduces the amounts of compounds and reagents used in the assays. But progress in miniaturization has slowed in recent years because of limits in handling small liquid volumes, and because of limits in the sensitivity of detection systems. Many of the predicted advances from 384-well microplates to 1536-well microplates have been delayed because of these problems with liquid handling. Overcoming these problems has not been a priority, however, since in the absence of better detection methods, the benefits of such small volumes cannot be utilized. Lumiphore’s technology will push miniaturization over this plateau and dramatically reduce screening costs.
Lumiphore’s lanthanide compounds can be used with the current instrumentation that companies already have in place. For example, instruments such as Amersham’s LEADseeker and Farcyte, and PerkinElmer’s Victor (II and V) and Envision, are widely used in HTS facilities and perform time-resolved measurements. Interestingly, several companies have developed time-resolved instruments for lanthanide fluorescence detection without offering the reagents.