During the past five years, the pace of changes in IVD assay developers’ working practices has accelerated significantly. Three of the trends that were discussed in an article published five years ago in IVD Technology’s 10th anniversary issue have continued to have a direct positive impact on the development of IVD assays, which are the following: the improvement of communication among researchers; the improvement in and development of detection technologies; and the continued development of equipment and techniques for researchers. This article will highlight the key changes that have emerged during the past five years in the ways that assays are developed.

What has also been seen in the last five years is not only a significant improvement in communication among researchers, but also researchers all over the globe now have better and faster access to resources. This trend has had a direct impact on assay development in a number of different ways. The amount of information that can be obtained quickly and easily about the key materials that are used in assays has greatly increased.

Also, the biological components of tests can be rapidly and unequivocally identified. In the past, such materials had often been somewhat mysterious and a little scary. For example, target identification or the production of antibodies and the characterization of those same materials had been done by a few skilled individuals and academics. That information has been made available on a number of databases containing biological information and in scientific journals, and is accessible to many researchers through computerized searches over the Internet. The ability to identify assay target molecules and compare them with other materials that have already been identified has simplified the entire development process.

Trends and Advances
The amount of information that can now be gathered on any diagnostic target in a relatively short time frame would have been unimaginable only a few years ago. For example, the advances in proteomic techniques that allow rapid target identification and characterization are changing how target compounds are found. This is perhaps one of the biggest changes in assay development in recent times: the bioinformatics revolution. Having such directed development is a huge boon for everyone involved in IVD. The ability to predict the structure of a target antigen on a computer screen and then design specific binding domains in a synthetic material could potentially revolutionize assay development.

By using standard proteomic techniques, thousands of possible target molecules can be screened, and those with the greatest potential can be found. Having such focused processes will make assay development a faster and less risky endeavor, especially in the biomarker area. The ability to identify, characterize, and utilize such target biomolecules more rapidly should significantly shorten the development cycles. A process that has historically been hit or miss and extremely time consuming and expensive will be completed in a matter of a few years instead of decades.

The means by which scientists communicate among themselves has expanded considerably. The traditional peer-reviewed journals are still significant and have an important role to play. However, with more research being performed by the IVD industry, the relevance of some of those academic journals is being questioned. At the same time, the availability of those same journals for online searching provides instant research and access to articles that may have been missed in the past. This instant access saves many hours that would have been spent delving and looking through journal abstracts in library basements.

The advent of blogging and alternative communication means also allows individuals or IVD companies to get their messages out more rapidly. However, that can lead to ideas or data gaining general acceptance in the scientific community or by the population at large without an adequate review of the results and experimental design. That can be a threat to the integrity of the assay development process. Overall, this increased speed and level of communication will only continue to benefit researchers as they can freely share their experiences, both good and bad, easily and quickly. Such communication can still be improved. For example, after finding an interesting paper, getting a response from an author can still take a long time. But the improvements seen in communication so far have had a positive impact on assay development.

During the past five years, detection methodologies have evolved. In particular, the challenges of multiplexing and microfluidics have forced IVD companies to innovate in order to overcome the problems with the physics that are involved in handling small volumes of liquids.1 The extension of novel detection techniques into IVD assays has been seen in many areas; for example, the research on metalized DNA as a detection technique, and the continued research into surface plasmon resonance as a label-less technology, especially for biosensors.2,3 In 
the area of rapid diagnostics, the trend has been to move to more sensitive quantitative labels, such as time-resolved luminescence, or the expanded use of paramagnetic particles, particularly in the very convenient lateral-flow formats that allow more sensitive assays to be used in CLIA-waived environments.4,5

The accuracy and availability of robotic systems has enabled researchers to speed their assay development efforts, with simple, reliable machines taking over many of the routine lab processes and allowing scientists to innovate rather then perform mundane tasks. Until recently, the standard of lab automation in many diagnostic labs would probably have been a multi-pipettor. However, the advances in robotics and miniaturization have allowed many research labs to include such systems in their workflow. The improvements in the accuracy and control of microsyringes and electrostatic depositions have enabled such systems to routinely dispense liquids in almost any volume, down to sub-nanoliter quantities. Such increased control of reagents is perhaps something that will become increasingly important during the next five years.

Opportunities and Challenges
The point-of-care (POC) testing market still offers opportunities, although it has not developed as rapidly as many people in the IVD industry had hoped. The biggest selling tests in the POC market, at-home blood glucose and pregnancy testing, broke through in the general consumer market almost 20 years ago. However, very few POC assays have broken through in this manner as their developers had hoped. But even though POC tests have failed to penetrate the market as desired, many IVD companies still believe in the huge potential for simple, rapid assays in the POC market. The sensitivity, ease-of-use, and sheer number of assays available in POC systems have grown significantly.

While the medical community is still resistant to change, all medical professionals are realizing that the pace of life is becoming faster and their patients are expecting immediate clinical answers. The biggest driving forces that will encourage the introduction of more POC testing will be the increasingly hectic lifestyles and the growing impatience of patients who want to be diagnosed and start treatments during initial consultations with their physicians. Physicians will respond to such forces, and they will need to get the results of diagnostic screening while their patients are still in the examining room.

The desire to move traditional diagnostic testing into POC settings has naturally led IVD companies to work on reagent-less systems, or more accurately, systems in which all of the required reagents for the assays to be performed are dried onto the substrates being used. Users simply have to add the test sample, which then rehydrates the reagents to run the assay. Tests that have only a sample-application step minimize any potential mistakes that could have been made in the past.

Lateral-flow test strips have been improved through the use of self-indicating papers and new design concepts, and the number of simple-to-use assays have been supplemented by a series of reagent-less biosensors.6-9 The original reagent-less system, the urine test strip, is still a very important part of the diagnostic lab. However, IVD companies are increasingly trying to encourage users to read these test strips with an electronic device that adds value to the technology and records the results directly into a lab information system (LIS), which collates and sends out all laboratory results.   

High-volume tests, particularly those that fit the automation models adopted by hospitals of all sizes, and testing that confirms the results of POC systems will likely keep the traditional central testing lab in business. In comparison, POC tests and systems will be increasingly used for situations in which instant results are required (e.g., initial screenings in emergency rooms or ambulatory clinics) and to facilitate the goal of personalized medicine.

Biomarkers have also not achieved the level of significance in the IVD industry that many people expected. The greatest reason for this is perhaps the lack of specificity that most biomarkers exhibit. The medical community expects IVD tests to provide unequivocal answers. But when there is an elevated level for a biomarker, physicians often do not know exactly what to do since elevated levels for many biomarkers can be due to a number of reasons. Even biomarkers that at first appear familiar can have multiple interpretations.

An example of this problem is the very familiar hormone human chorionic gonadatrophin (hCG). Most people correctly associate elevated hCG levels with pregnancy. However, it is also an important biomarker for several forms of cancer (e.g., germ cell and islet cell cancers, and even testicular cancer in men). This lack of specificity has led some people to resist the use of many biomarkers in diagnostics, and even successful biomarkers such as carcinembryonic antigen (CEA) or prostate specific antigen (PSA) have received mixed responses.

In order for biomarkers to be effective, the way that results from biomarker tests are interpreted needs to change. Instead of providing an instant diagnosis of a condition, many biomarkers are better used as an annual check-up; the significance of the biomarker would be a change in the level of the biomarker that is being expressed. This variation in measured levels would indicate that there has been a change in a patient’s condition, and other, more invasive screening tests (e.g., biopsies, colonoscopies, CT scans, etc.) could identify the exact cause that resulted in the observed change in the biomarker level. Using biomarkers as an additional screen to aid in the selection of individuals who need more extensive follow-up would increase their use significantly.

It is also well known that some cancers will express up or down the level of a number of biomarkers. True diagnosis requires that the levels of all the biomarkers should be evaluated at the same time. This multivariate technique will perhaps allow greater differentiation between those cancers. While biomarkers have been proven to be a useful aid in diagnosing a number of conditions, they have yet to find their true niche in the IVD industry. The advances in the development of complex multivariate assays will perhaps provide doctors with the simple diagnosis that they require, and will enable biomarkers to have a more significant role in the practice of medicine.

Conclusion
While certain areas of the IVD industry have significantly changed during the past 15 years, much has remained the same. The sale of hematology, immunology, and chemistry analyzers to central labs still accounts for the largest part of the IVD market. Many of the more exciting and exotic technologies that were predicted to revolutionize the industry (e.g., microarrays, biomarkers, and genetic testing) are still not generally used outside research environments, with a few noticeable exceptions. The POC market is still largely focused on the same areas it was five years ago: blood glucose, pregnancy testing, coagulation, blood gas, and cardiac markers. The recent flu scares did increase the demand for POC tests that were able to identify respiratory tract infections. But in many other areas, the uptake of POC assays has been very slow.

Where do the IVD industry and assay development go from here? There are some distinct directions that most people would agree are safe bets. The drive for devices and systems to be more reliable and easier to use will continue. POC testing will become more significant as the desire for faster results and personalized medicine pushes POC into new areas. The increase in POC testing will contribute to the continued decline of the central lab business. But the central lab business will continue to have an important place in the market. And most significantly, the Internet will continue to speed assay development by enabling collaborations among research groups in a way that was unimaginable only a few years ago.

References
1. R Park, “Advances and Challenges in Detection Technology,” IVD Technology 15, no. 4 (2009): 24-29.

2. R Park, “Evolving Detection Technology Methods,” IVD Technology 14, no. 4 (2008): 24-29.

3. RF Day, “Surface Plasmon Resonance for Biosensors,” IVD Technology 13, no. 2 (2007): 45-50.

4. X Song, “Lateral-Flow Assays Using Time-Resolved Luminescence,” IVD Technology 15, no. 4 (2009): 31-37.

5. B O’Farrell and J Bauer, “Developing Highly Sensitive, More-Reproducible Lateral-Flow Assays, Part 1: New Approaches to Old Problems,” IVD Technology 12, no. 5 (2006): 41-49.

6. SMZ Hossain, et al., “Reagentless Bidirectional Lateral Flow Bioactive Paper Sensors for Detection of Pesticides in Beverage and Food Samples,” Analytical Chemistry 81 (2009): 9055-9064.

7. S Kunzelmann and MR Webb, “A Fluorescent, Reagentless Biosensor for ADP Based on Tetramethylrhodamine-Labeled ParM,” ACS Chemical Biology 5 (2010): 415-425.

8. S Yang, et al., “A Novel Reagentless Biosensor Based on Self-Assembled HRP and Nile Blue Premixed with Poly(styrenesulfonate) Architectures,” Canadian Journal of Analytical Sciences and Spectroscopy 51, no. 3 (2006): 174-179.

9. F Xia, et al., “One-Step Construction of Reagentless Biosensor Based on Chitosan-Carbon Nanotubes-Nile Blue-Horseradish Peroxidase Biocomposite Formed by Electrodeposition,” Talanta 78, no. 3 (2009): 1077-1082.

Kevin Jones, PhD, is chief scientific officer and vice president at EDP Biotech. He can be reached at drkevinjones@me.com