Originally Published IVD Technology April 2005
Anniversary Essays
3. Assay development
Changes in the development of rapid assays since 1995.
Kevin D. Jones and Klaus Hochleitner
 |
 |
Kevin D. Jones, PhD, is technical marketing manager, diagnostics, at Whatman (Florham Park, NJ). |
The historical image of the development scientist has always been one of the lone researcher, working in glorious isolation in his lab. Before 1995, this was actually the case for people developing rapid assays: no real vehicle existed to allow communication between research labs or individual researchers. In the late 1980s, several companies had R&D groups performing rapid-assay work. Many of these original companies have gone on to spawn new businesses. At the time, communication between scientists was rare, and was very much the exception rather than the rule.
During the early 1990s, the basic text that a lateral-flow developer would have had in hand, aside from the technical information derived from patent documents, was the S&S guide, “Optimization of nitrocellulose-based membrane- based immunoassays,” and maybe a copy of “The Latex Course.” This meant that in each of the labs where development was being performed, solutions to common problems were rediscovered many times. Every test was an adventure into the unknown, and therefore development times were long, with many false leads that had to be followed.
At the time, scientists had to rely on membranes and pad materials that were originally intended for filtration applications, and which were in no way optimized for rapid diagnostic test purposes. The equipment available for dispensing proteins onto membrane surfaces was, in most cases, developed either for applying reagents to thin-layer chromatography matrices or for the application of ink to paper. The result, again, was that a great deal of development effort had to be invested in optimizing specific application technologies. This nonideal situation resulted in much frustration and heartache for the original developers of lateral-flow assays.
In 1995, the launch of IVD Technology gave developers a chance to communicate with one another. For the first time, diverse researchers were able to learn and share. IVD Technology, therefore, became a driving force for an amazing number of technological improvements on both the materials and equipment sides of rapid-test development and manufacturing.
Over the years, there have been articles written on almost every aspect of the development and production of rapid assays—from the optimization of membranes and reagents to the development of complete assays and manufacturing equipment. Making this information available to a wide audience has significantly enhanced the knowledge base of developers. Anyone entering the field today can learn the basics of developing an assay without rediscovering what these basics entail.
Technical Innovations
It was not only the expanding base of publicly available information that pushed in vitro diagnostics forward. A number of important early technical improvements were made commercially available by a range of equipment and materials manufacturers.
One of these—the development of lateral-flow assays whose membranes exhibit lateral-flow characteristics, not the vertical-flow qualities of earlier filter membranes—was essential. Several membrane manufacturers realized the need for membranes that were optimized for reproducing protein binding, transporting liquid parallel to the membrane surface, and creating mechanical stability to facilitate automated manufacturing processes. This led to the development of what are today known as direct-cast membranes, in which a nitrocellulose layer is manufactured directly on top of a polyester film. The vast majority of developers now routinely use these direct-cast membranes as the workhorse of lateral-flow development. Almost all new assays start with a direct-cast membrane.
It was also around this time that manufacturers actively searched for better conjugate release materials. While the basic materials have remained unchanged, there has been a continuous drive toward the adoption of easier-to-use materials, which has ultimately resulted in the development of pretreated pads onto which conjugates simply need to be sprayed.
An even more important improvement was introduced at the sample application site. For tests that detected antibodies against infectious agents or the agents themselves in human blood, the blood itself could not be used as a sample in most cases, because hemoglobin was leaching from the red blood cells (RBCs), causing an unacceptably high background on the membranes. Therefore, serum or plasma had to be prepared, more or less restricting infectious-disease diagnostics to hospital/laboratory environments where respective equipment for preparing serum and plasma was available. The introduction of so-called blood separation pads, which retain the RBCs in the pad matrix, was a big step toward truly rapid infectious-disease diagnostics. The new pads made this type of diagnostic test available even in very remote regions, leading to a major improvement in healthcare in countries with a less-developed health-care infrastructure.
Detection Technologies
 |
Paramagnetic lateral-flow assay by BBInternational Ltd. (Cardiff, UK). |
A number of cutting-edge detector technologies are just entering the marketplace. The most important one is probably the use of paramagnetic particles. These particles, for the first time ever, permit the detection and quantification of conjugates retained at the test and/or control lines deep within the membrane matrix, in places where they could not contribute to signals that are to be detected by optical means.
The equipment used in the manufacturing of rapid assays has also improved over the last 10 years. While the basic design of this equipment has largely remained unchanged, improvements in areas such as the consistency and accuracy of liquid deposition have been significant.
One major improvement is the control that can be achieved in the printing of the capture line. In 1995, there were some commercial systems available; however, many developers still had to build custom-made equipment for production purposes.
Many attempts were made to adapt existing printing equipment to diagnostic applications, with varying degrees of success, and a number of companies tried to convert airbrush or ink-jet technology to the production of diagnostic strips. It quickly became apparent that many of these systems did not offer the control required to allow the deposition of a controlled line onto a membrane. While airbrush technology is still used for applying colloidal particles or blocking strips, it failed the more demanding application of the capture line.
More promising was the use of microsyringes to slowly pump the solution through a print head. This basic system has been used for almost every successful application system over the last 10 years. There are a number of commercially available systems today in which microsyringes still control the flow of liquid; the only change is that there are more ways for the liquid to be applied to the membrane. A lot of work has been done in order to improve the accuracy and reproducibility of line-dispensing processes, controlled drying of materials, and high-volume manufacturing capabilities. The equipment considered state of the art is able to dispense liquids, if necessary, in subnanoliter drop sizes, at a price and with a consistency that developers could not even dream of a decade ago.
 |
The XY3050 dispense platform with Biojet Quanti dispensers from BioDot Inc. (Irvine, CA). |
During the last 10 years, there have also been considerable jumps in the quality and availability of the antibodies and detection reagents used. In the early 1990s, it was common to raise specific antibodies as the need to use them arose. Today, there are catalogs of standard antibodies for routine tests.
The conjugates used have also improved immeasurably. The quality of commercially available colloids (both gold and latex) has improved in both quality and consistency. It is now common for colloids to be manufactured in batches of between 20 and 100 L; 10 years ago, it was difficult to produce batches much larger than 100 ml with any degree of consistency or accuracy. There is also a much greater understanding of the possibilities offered through the use of other colloidal systems. For instance, magnetic beads, quantum dots, and up-phosphors are all now being investigated for use in lateral-flow systems.
A Focus on Quantitation
 |
40-nm gold colloid by BBInternational Ltd. |
One of the most significant changes has been the desire to develop truly quantitative assays. While all assays possess a certain degree of quantitation, the move from semiquantitative (or qualitative) assays to fully quantitative ones has been a major challenge for assay developers. The levels of consistency and reproducibility required in quantitative assays push the limits of conventional materials; however, there exists a range of different technologies for reading a quantitative assay. The more-traditional techniques simply use a colored colloid (such as the familiar gold or dyed latex). More recently, a range of systems have been developed that use magnetic labels and fluorescent particles. The sensitivity of these alternative techniques makes the development of truly quantitative and highly sensitive assays a realistic possibility.
Conclusion
Today, information about developing a rapid assay is readily available—every membrane manufacturer now organizes courses teaching people about binding protein to a membrane or about new technology trends. In addition, membrane and equipment manufacturers have joined forces to provide test developers with packages of optimized membrane and manufacturing technologies, accompanied by superior technical support including hands-on training in dedicated application laboratories, contract assay development, and small-scale contract manufacturing. Over the last 10 years, the breadth of tests available has increased dramatically. These range from simple pregnancy tests to infectious-disease and cancer tests. Strip tests can be qualitative and simple, such as the ubiquitous pregnancy test, or can provide sensitivity and consistency similar to that seen on analyzers costing hundreds of thousands of dollars.
So, how will lateral-flow assays develop over the next 10 years? Predicting future changes is always a touchy business; still, the basic platform has remained largely the same 16 years after its inception. The only significant changes in assay development have been in the number of disease states that can be detected and the volume of tests produced.
The sensitivity that can be achieved has improved significantly, where needed, and the increasing use of traditional colloidal beads as well as the more exotic fluorescent and magnetic markers has allowed for the development of highly sensitive, quantitative assays.
The focus of many developers has shifted from the West to the emerging markets of Asia and Africa, where an inexpensive and robust test technology still has a great deal to offer. Perhaps the greatest influence that the next decade will have on the development of lateral-flow assays will emerge from the current round of patent litigation. Until that is resolved, the development of lateral-flow assays, at least in the West, is merely marking time.
Even 10 years after its initial launch, IVD Technology remains one of the main conduits for communicating new technologies to the rapid-assay development market. Congratulations to IVD Technology for what it has helped accomplish during the past decade. We’re looking forward to continuing to push the technology boundaries, and to another successful 10 years.
Copyright ©2005 IVD Technology
