College of American Pathologists
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  PCR and ELISA adaptations—will they fly?






July 2007
Feature Story

William Check, PhD

Are you ready to explore the final laboratory frontier—measuring ultralow concentrations of protein analytes? Are you ready to boldly go where laboratorians have never gone before—beyond the nanomole, the picomole, and the femtomole, into the territory of the attomole, the zeptomole, and the elusive yactomole? Methods have been developed that make it possible to detect protein antigens at levels 1,000 to 10,000 times lower than conventional assays. These methods are being used now in research settings, and expert opinion differs on how likely it is that technical and commercial considerations can be overcome to bring them into the diagnostic laboratory. But the techniques themselves are ingenious and hold great promise.

Of one of the more innovative, promising, and problematic methods, immuno-PCR, James Versalovic, MD, PhD, associate professor of pathology at Baylor College of Medicine, says, “For ultrasensitive quantitation of proteins, immuno-PCR is beginning to enter a maturation phase, with many potential applications that go beyond infectious diseases. It combines amplification with protein detection, which you can’t do with ELISA or other immunoassays. So there is a reason for pursuing this strategy.” However, Dr. Versalovic cautions, “It is really an open question at this point when or whether this method will become a mainstay in the clinical arena. We have been studying its potential applications in a diagnostic laboratory setting during the past year and are moving ahead cautiously.” Asked about the time frame for implementing immuno-PCR, Dr. Versalovic speculates, “It is not yet ready for prime time. I am not sure if we are going to see it become a routine diagnostic test in the next two years. We need a leap in user-friendly reagents—that will be a key step—and the price [of kits and reagents] will need to drop.”

Niel Constantine, PhD, professor of pathology and director of the clinical immunology laboratory at the University of Maryland School of Medicine, Baltimore, has been working with immuno-PCR for seven years. His evaluation is somewhat more sanguine. “Immuno-PCR is at the stage where it is difficult to perform,” Dr. Constantine acknowledges. But, he adds, “There have been a number of papers published, and it’s clear that the method can work and that it can be used for a variety of applications.” Dr. Constantine sees its great advantage in detecting proteins that are present in very small quantities in blood. He and his colleagues have been developing immuno-PCR assays for prion protein and for biothreat agents such as botulinum toxin and ricin toxin. “You need to detect those in the environment and in blood,” he says. They have also published on the early diagnosis of HIV infection through detection of very low levels of p24 antigen.

To put the challenge of immuno-PCR in perspective, Dr. Constantine reflects on PCR in the late 1980s and early 1990s. “People said that PCR would never be applicable to clinical labs,” he recalls. “They said it had too many problems, particularly contamination. Now it is used ubiquitously, including screening of 15 million blood units per year in the U.S. and in clinical labs everywhere, even in developing countries.” The same thing could eventually happen with immuno-PCR, he suggests—“if we could get some manufacturers involved.

“They are the people who generally standardize assays,” he says. “If a manufacturer came in today who was 100 percent committed to getting this method into clinical labs, it would take probably a year or so.”

At the same time, the stodgy old ELISA is not going gently into that good night. It too is entering the attoworld. Mavanur R. Suresh, PhD, professor and associate dean of graduate studies and research faculty of pharmacy and pharmaceutical sciences at the University of Alberta, Edmonton, has devised an ultrasensitive ELISA technique. Dr. Suresh’s method uses bispecific antibodies obtained from quadromas—fusion products of two hybridomas, each with a different specificity—as its sandwich reagent. His group has applied this technique to many analytes—from infectious agents such as Ebola, the SARS coronavirus, and Western equine encephalitis to tumor markers such as prostate-specific antigen and CA-125. In one application, this approach detected one to two cells of E. coli in about 100 mL of water. Dr. Suresh is now starting to look for a corporate partner.

A less radical approach to increasing the sensitivity of ELISA specifically for HIV p24 antigen has been taken by Jorg Schupbach, MD, professor of virology at the University of Zurich. Dr. Schupbach identifies two problems in detecting p24 antigen for diagnosing and monitoring HIV infection. First, there are specific antigen-antibody complexes that make it difficult to capture the antigen. A rheumatoid-like factor also occurs, which can non-specifically link the capture antibody with the tracer antibody, causing false-positive reactions. Second, traditional antigen tests are simply not sensitive enough, Dr. Schupbach says. His method addresses all these complications.

Ron Wacker, PhD, marketing manager at Chimera Biotec GmbH, Dortmund, Germany, the company formed to commercialize immuno-PCR, described, at the November 2006 meeting of the Association for Molecular Pathology, the development of immuno-PCR over the past 15 years. (For a review, see Niemeyer CM, et al. Trends Biotechn. 2005;23:208–216).

Dr. Wacker noted that PCR gives exponential signal amplification and real-time detection but cannot be used directly for antigen detection. ELISA, the standard technique for protein detection, gives linear signal amplification and endpoint detection and is adaptable to any protein; it is robust and sample tolerant. However, ELISA’s sensitivity is not adequate for some purposes, such as discriminating levels of inflammatory mediators like TNF-alpha, which is crucial to many disease conditions, from arthritis to sepsis. A commercial ELISA kit for TNF-alpha may have a lower limit of detection of 0.5 pg/mL, about 2 million molecules. A tweaked assay was reported to detect as low as 0.2 pg/mL (Petrovas C, Daskas SM, Lianidou ES. Clin Biochem. 1999;32:241–247).

But much greater sensitivity—580 molecules of bovine serum albumin, 10,000 times less than with a standard ELISA—was achieved by combining ELISA with PCR into immuno-PCR, as initially reported in 1992 (Sano T, Smith CL, Cantor CR. Science. 1992;258:120–122). The innovation was to link the detection antibody to DNA, rather than to an enzyme, and to exponentially amplify the antibody-DNA conjugate with PCR. Linkage was accomplished by using biotinylated DNA and joining it to the detection antibody with a streptavidin-protein A chimera, which possesses tight and specific binding affinity both for biotin and immunoglobulin G. (This method was issued a U.S. patent in September 1997. Chimera Biotec has a nonexclusive license to commercialize the assay in the United States.) Using a refinement of this method, Chimera Biotec’s Imperacer kit detects 0.01 pg/mL (12,000 molecules) of TNA-alpha.

To reach such high sensitivity and specificity, Dr. Wacker says, “the conjugate is the key.” One research group modified the assay to use covalent coupling (Hendrickson ER, et al. Nucleic Acids Res. 1995;522–529). However, Chimera has moved to multimeric conjugates for greater resolving power (Adler M, et al. Biochem Biophys Res Commun. 2003;308:240–250). “Coupling is the most critical step in immuno-PCR,” Dr. Wacker told CAP TODAY. He acknowledged that protein-DNA coupling is quite difficult and appears to be the greatest obstacle to broader use of the assay. “I can’t argue this question,” he said. Chimera scientists have developed a proprietary method that allows much easier coupling, which Dr. Wacker declines to describe. “We are interested in being the company with the most experience as designers of immuno-PCR,” he says. “Our main focus is that we are a service provider for assay development. And establishing a new customized immuno-PCR assay takes us only two to three months.”

They offer to advise clients who want to generate a specific application for immuno-PCR. To get that advice, clients have to agree to buy Chimera’s reagents, mainly the antibody-DNA conjugates. Chimera also markets optimized buffers for the immuno-PCR. “We teach [clients] to develop their own assays, but they have to use our reagents,” Dr. Wacker says. In 2006, about 20 companies signed contracts with Chimera for these services, according to Dr. Wacker. Half were large pharmaceutical companies; none were clinics or hospitals. “We are focused on research institutes and pharma and biotechnology companies,” Dr. Wacker says. Some contracts were with companies that develop assays.

In his AMP presentation, Dr. Wacker described the successful application of immuno-PCR to detection of several protein analytes at very low levels:

  • Free PSA with a lower limit of detection of 480,000 molecules, 100 times lower than with an ELISA (Lind K, Kubista M. J Immunol Methods. 2005;304:107–116).
  • Detection of amyloid beta 40+42 with a lower limit of detection of 0.1 pg/mL.
  • Norovirus capsid proteins at a level of 100 particles (10 fg) (Tian P, Mandrell R. J Appl Microbiol. 2006;100:564–574).
  • Rotavirus particles from stool at 100 particles/mL, 1,000 times lower than a standard ELISA (Adler M, et al. Biochem Biophys Res Commun. 2005;333:1289–1294).

Chimera offers research-use-only kits for detecting TNF-alpha, interleukin-6, and HBsAg.

In a clinical research application, an immuno-PCR assay optimized to detect as little as 50 zeptomol (approximately 100 fg/mL) was used to measure drug concentrations in a phase one trial of the antitumor protein aviscumine (Schoffski P, et al. Ann Oncol. 2004;15:1816–1824).

Dr. Constantine’s experience illustrates the challenges that an independent scientist working with immuno-PCR can expect to encounter. He estimates that it took four to five years to get his first assay working. “There are three parts to immuno-PCR,” Dr. Constantine said in an interview with CAP TODAY, “and they can all interact. That’s what causes the problems. Multiple components need to work in harmony.” He compared the difficulty of synchronizing the three components of immuno-PCR to using parts from a pickup truck, a VW Beetle, and a Jaguar. Dr. Constantine has found that “a lot of unwanted interactions can occur because of the bridging portion” of the assay, the antibody-DNA conjugate.

Overcoming these unwanted interactions can be achieved through persistence, in Dr. Constantine’s view. His motto: “Persistence overcomes resistance most of the time.” He says, “Some people have given up after a few months. Fortunately, we had funding for our work on prions,” their first project with immuno-PCR. Dr. Constantine’s group uses the published method whereby a detecting antibody is conjugated via avidin to a biotinylated DNA reporter. The group’s major challenge was background noise, generated if the biotinylated antibody-DNA conjugate attaches to the support or other molecules in the reaction, giving amplified background. Most of the development time was spent overcoming this problem.

Dr. Constantine’s first target was applying immuno-PCR to detecting prion proteins. “There is currently no test to detect infectious units [of prion agents] in the blood,” he says. It might be used for screening blood units—three cases of human transmission of variant Creutzfeldt-Jakob disease, thought to be transmitted by prions in blood, have been reported. (A pathologic form of the prion protein is thought to transmit this and other fatal neurodegenerative diseases.)

Prion proteins are detectable in the tissues of animals with diseases related to vCJD, such as sheep with scrapie, but not in their blood. But, because blood can transmit infection, abnormal proteins must be in blood in very low concentrations, Dr. Constantine and others have reasoned. Calculations predicted that an assay that detects about 5 fg/mL—about 86,000 molecules/mL—would be needed. Dr. Constantine and colleagues showed that immuno-PCR could reliably detect 1 fg/mL—one million times more sensitive than the corresponding ELISA—and occasionally 100 attograms/mL (Barletta JM, et al. J Virol Methods. 2005; 127:154–164). (“Femto-” indicates 10-15, with each succeeding prefix—atto-, zepto-, and yacto—being 1,000 times less.)

The highly sensitive quality of immuno-PCR offers an opportunity to detect HIV infections even earlier than PCR, shortening the latent, undetectable period from 12 days and potentially eliminating some cases of blood-transmitted infection. Detecting 75,000 molecules of p24 antigen is equivalent to 50 copies/ mL of HIV RNA, the current lower limit of detection of most PCR assays. By performing a standard dose-response curve on samples from HIV-infected people with known viral loads, Dr. Constantine showed that immuno-PCR can detect the presence of infection when RNA testing indicates “not-detected” (at 50 copies/mL). Among 52 samples from patients with a viral load of less than 50, immuno-PCR detected p24 antigen in 22 (42 percent) (Barletta JM, et al. Am J Clin Pathol. 2004;122:20–27). This could make immuno-PCR useful in monitoring the course of infection in treated patients. The assay was sensitive down to less than 3 fg/mL of p24, which is less than 50 copies of HIV RNA, the desired goal.

In contrast to the achievements of Dr. Constantine and his colleagues, Dr. Versalovic at Baylor has not yet been able to make a working assay. “It is not just a little bit more challenging [than ELISA or PCR]—it is significantly more challenging,” Dr. Versalovic says. “Linking DNA with protein adds a level of complexity. We are having difficulties optimizing the assays in our lab and we consider ourselves to be fairly sophisticated.” Certainly the difference in outcomes to date reflects that fact that Dr. Versalovic has been working to devise an assay for less than a year, a project that took Dr. Constantine several years. On the other hand, how much time can a diagnostic laboratory devote to making a new assay? For immuno-PCR to make inroads into the diagnostic market, Dr. Versalovic says, “it will require the development of panels of reagents and kits that will make it much easier for diagnostic labs to implement this procedure.”

Dr. Suresh’s efforts to modernize the venerable old ELISA also have at their core a novel coupling agent—bispecific monoclonal antibodies. Traditional monoclonal antibodies are made by fusing a B lymphocyte with a myeloma. “To make bispecific antibodies,” Dr. Suresh says, “we fuse two hybridomas that have different specificities.” One hybridoma makes a monoclonal against the antigen and the other against an enzyme used in ELISA, horseradish peroxidase, or HRP. A quadroma, which is essentially the fusion product of two hybridomas, has the unique property of producing bispecific antibodies with all three combined specificities—antigen/antigen, HRP/HRP, and antigen/HRP—through the random assembly of heavy chains from the progenitor cells’ immunoglobulin genes. “We have developed a method to isolate bispecific antibodies by an affinity column,” Dr. Suresh says. A quadroma is tetraploid, so it has innate genetic instability. After several passages, stable cells can be isolated that constitutively produce diagnostic bispecific tracer antibody “almost in unlimited amounts,” Dr. Suresh says.

For a standard ELISA, complex chemical reactions are used to bind the detection antibody to the amplifying enzyme (HRP). “We don’t know how many HRPs are bound per antibody,” Dr. Suresh says. “There may be antibodies that are not labeled with HRP at all. It is a heterogeneous mixture of molecules that we use in today’s diagnostic world.”

But the bispecific antibody ensures that every molecule has an HRP molecule bound, and at distinct sites that have been selected not to interfere with antigen binding. “In addition to this clean tracer, another advantage is that we achieve the highest theoretical specific activity of the probe, because every antibody molecule has a bound HRP,” Dr. Suresh says. “We just add a little excess of HRP and every antibody is labeled.” And because the tracer antibody is homogeneous, it gives a very clean background. Dr. Suresh calls an ELISA using a bispecific monoclonal antibody a “Velcro” or “molecular zipper” assay.

He has developed bispecific antibodies or complete molecular zipper ELISA assays against Marburg and Ebola viruses, E. coli 0157, PSA, CA-125, Western and Venezuelan equine encephalitis viruses, and the SARS coronavirus (see, for example: Shahhosseini S, et al. J Virol Methods. 2007;143:29–37). “The same strategy can be applied for rapid qualitative detection,” Dr. Suresh says. For Bordetella pertussis, his group has developed qualitative and quantitative assays that were able to detect “literally a few bacteria, say five to seven bacteria” (Tang XL, et al. Clin Diagn Lab Immunol. 2004;11:752–757).

Dr. Suresh is confident that other laboratories can develop assays using his technology. “You need a different quadroma for every antigen that you want to detect,” he says. The hybridoma for HRP is standard from application to application. All that differs is the hybridoma for the antigen. You label the two hybridomas with two dyes, fuse them in a standard way with polyethylene glycol, and separate the double-labeled quadroma using flow cytometry. His affinity column can be used to separate the bispecific antibody on an industrial scale.

In a more standard adaptation of an ELISA, Dr. Schupbach has dealt with interference by immune complexes and by nonspecific antibodies by diluting plasma or serum samples, then boiling for five minutes. “All capability of antibodies to bind is abolished,” he says. He introduced this step in 1993. To increase sensitivity, he uses a Perkin-Elmer signal amplification procedure using biotin tyramide-mediated amplification. “It is inexpensive and very simple and can be applied to all kinds of tests,” Dr. Schupbach says. This approach makes it possible to detect p24 antigen down to about 0.5 pg/mL, 10 to 20 times more sensitive than the traditional antigen test.

Later, Dr. Schupbach discovered that the buffer in the antigen kits did not sufficiently dissociate aggregated p24 molecules if they are present in viral particles. So he developed a better buffer that fully releases particle-associated p24 from aggregates. When he compared results of this method to PCR, he found that antigen levels correlated well with HIV RNA levels and that p24 could even be detected in one-third to two-thirds of HIV-infected individuals with long-term undetectable levels of HIV RNA (Schupbach J, et al. J Acquir Immune Defic Syndr. 2005;40:250–256; Schupbach J, et al. J Med Virol. 2006;78:1003–1010). “We would like to see if we can correlate the concentration of remaining p24 with T-cell activation,” Dr. Schupbach says. According to one hypothesis, the more viral antigen that is present, the higher the activation of CD8 cells.

While this modified ELISA is not as sensitive as PCR for diagnosis, it would make an attractive alternative to viral load testing for monitoring antiretroviral treatment. Dr. Schupbach and his colleagues have demonstrated that it is as well correlated as RNA levels to long-term progression to AIDS or death (Ledergerber B, et al. J Infect Dis. 2000;181:1280–1288; Sterling TR, et al. J Infect Dis. 2002;186:1181–1185). He and others have also shown that it is comparable to PCR for diagnosis of pediatric HIV infections in third-world countries and can be used on dried blood spots (Patton JC, et al. Clin Vaccine Immunol. 2006;13:152–155; Knuchel MC, et al. J Acquir Immune Defic Syndr. 2007;44:247–253). Changes in the concentration of p24 by the more-sensitive ELISA were better correlated to short-term changes in CD4 counts than viral RNA (Schupbach J, et al. J Acquir Immune Defic Syndr. 2005;40:250–256; Brinkhof MW, et al. J Acquir Immune Defic Syndr. 2006;41:557–562). “And p24 is much more stable than viral RNA; the ELISA is much easier to perform and much less expensive,” Dr. Schupbach says. “It is medical and economical nonsense that we pay five to 10 times more for measuring viral RNA and that this marker is not more studied.”

Today, individuals can venture out of Earth’s atmosphere aboard private rockets by paying a hefty fee and being willing to undergo the physical discomforts of weightlessness. Whether space travel will ever become standard outside of science fiction stories is an open question. Development of new assays using immuno-PCR is also limited to scientists who have considerable money or time and are strongly motivated. Whether immuno-PCR or the molecular zipper ELISA or Dr. Schupbach’s modified ELISA ever become standard techniques in clinical diagnostics is also something that only the future will show. But all of their work illustrates the fecund imaginations of scientific explorers and the need for laboratorians to remain alert and informed about novel entries in the captains’ logs—or scientific journals, as they are better known.

William Check is a medical writer in Wilmette, Ill.