May 1, 2016 (Vol. 36, No. 9)

Richard A. A. Stein M.D., Ph.D.

Immunoassays Are Incorporating Brighter Signals and Multiplex Views

Interdisciplinarity emerges as a defining feature of the research that catalyzes the development of new immunoassays, with nanomedicine, genomics, engineering, and protein biochemistry being only a few of the increasingly vital contributing fields.

“As engineers, we like to define problems at the outset,” says Hadley D. Sikes, Ph.D., assistant
professor of chemical engineering at MIT. “We think about who will be using an assay and what their needs are.”

One of the major research efforts in Dr. Sikes’ lab focuses on the development of low-cost immunoassays that can be used in areas lacking elaborate public health infrastructures. An existing challenge in serological testing is the limited availability of immunoassays in resource-poor settings, such as mobile healthcare units and countries with less-developed infrastructures.

According to Dr. Sikes, major limitations in the field include “waiting on antibody production, figuring out how to integrate them into stable, affordable devices, and performing standardization to ensure that different users get the same results.” These limitations, however, may be addressed with new technology.


Polymerization-Based Colorimetry

For the first time, Dr. Sikes and colleagues developed a colorimetric approach that integrated a paper-based immunoassay with a visible light-induced polymerization-based amplification reaction to generate a color response on paper-based devices. This strategy addresses two shortcomings that have been consistently reported for many immunoassays.

One problem involves visual cues. The color response obtained from enzymatic reactions or nanoparticles may sometimes be faint. Also, the lack of high contrast between negative and positive samples makes it difficult for users to clearly identify true positive reactions.

“The other problem is that for many enzymatic reactions, there is a key time window where the user has to look at the assay to obtain the correct result,” notes Dr. Sikes. False-positive readings may result if too much time has elapsed until reading the test, and there is also the danger of false-negative results if tests are read to early.

“We are trying to get brighter signals to make it easier for end users to interpret the tests,” asserts Dr. Sikes. “And we are also trying to inexpensively automate timing.”

The technique developed by Dr. Sikes and colleagues allowed the detection of proteins in complex mixtures, promising utility for a broad spectrum of diagnostic applications. In a validation study that detected a Plasmodium protein, this immunoassay required 100 seconds for polymerization after capturing the analyte, and it provided flexibility in performing the polymerization and visualization steps. The results were easy to read and interpret.


Pock-Marked Nanoparticles

“We are working on immunodiagnosis tools to develop assays,” says Chandra K. Dixit, Ph.D., a postdoctoral research fellow in the laboratory of James Rusling, Ph.D., a professor of chemistry at the University of Connecticut, Storrs. “The major focus is on prostate cancer detection.”

A widely used biomarker for prostate cancer detection is the prostate-specific antigen (PSA). One of the limitations of diagnostic biomarkers is that for many conditions, the use of one or few biomarkers leads to suboptimal sensitivity and specificity. For example, while an increased PSA level is often indicative of prostate cancer, it can also be the result of benign prostate conditions, and elevated levels are not necessarily predictive of malignancy.

“We are not relying on a single biomarker,” informs Dr. Dixit. “Instead, whatever we are trying to develop involves multiple biomarkers.”

Dr. Dixit and colleagues previously developed an array of eight prostate cancer biomarkers, a strategy that increases the sensitivity, specificity, and predictive value of the test. “This assay is a step toward building a multiplex immunodiagnostic system for cancer detection,” explains Dr. Dixit.

Immunoassays involve multiple steps, such as capturing, blocking, and detection, and variables at each of these steps influence the overall quality and the performance of the test. For example, different combinations of monoclonal and polyclonal antibodies may be used for antigen capture and detection.

“Any of these combinations affects the sensitivity of detection drastically,” insists Dr. Dixit. “Using the best combination is crucial.”

A key effort by Dr. Dixit and colleagues focuses on developing artificial antibodies that can be used for immunological diagnosis. These antibodies are imprints of a protein that can be detected on a solid support by virtue of the very specific antigen-antibody binding.

The artificial antibodies rely on silica nanoparticles and a layer of polymer that coats a solid support with a soft matrix on which the antigen is placed. After removal of the antigen, the nanoparticle remains decorated with spots that specifically detect only one type of specific antigen. Individual nanoparticles can present thousands of such pockets and, thus, mimic thousands of antibodies distributed all over the particle.


Photoelectrochemical Detection

A fluorogenic substrate is nonfluorescent, but when it is acted upon by an enzyme, it may produce a fluorescent compound. Accordingly, fluorogenic substrates have been put to use in many immunoassays. These immunoassays, however, have their limitations. For example, they may rely on enzymatic fluorogenic substrates that are unstable and expensive.

To address such limitations, scientists at CIC BiomaGUNE, a nonprofit research organization in San Sebastian, Spain, have been working on photoelectrochemical detection methods. “We managed to produce and optimize an immunoassay based on quantum dot enzymes,” says
Valery Pavlov, Ph.D., a principal investigator at the institution. In an article recently published in Biosensors and Bioelectronics, Dr. Pavlov and colleagues reported that they had developed a process in which cadmium sulfide quantum dots were enzymatically generated in situ, based on a reaction catalyzed by alkaline phosphatase.

In this reaction, sodium thiophosphate is hydrolyzed to hydrogen sulfide, which in the presence of cadmium ions forms cadmium sulfide quantum dots, or semiconductor nanoparticles. Graphite electrodes, sensitized by a polyvinylpyridine-bearing osmium complex, are used to measure the current that originates from the nanoparticles, which arise from the photooxidation of 1-thiolglycerol that occurs when the nanoparticles are  illuminated with a standard UV light source.

To validate this approach for immunoassays that frequently detect antibodies in liquid phase, Dr. Pavlov and colleagues optimized this system for an enzymatic assay measuring bovine alkaline phosphatase. Specifically, the investigators developed a new immunoassay that relied on detecting the interaction between bovine serum albumin (BSA), rabbit anti-BSA antibodies, and alkaline phosphatase-labeled anti-rabbit IgG antibodies.

“Our assay has better detection limits than comparable assays that use fluorogenic substrates,” asserts Dr. Pavlov. The lowest amount of antibody that could be detected with this system was 2 ng/mL, and detection showed linearity up to 20 ng/mL. This assay was able to detect amounts of substrate that are five times lower than what can be currently detecting using standard, chromogenic ELISA, for which currently the limit is around 10 ng/mL. This method provides new opportunities for fast and less expensive photoelectrochemical methods that can be used in immunoassays.

Previously, Dr. Pavlov and colleagues developed a self-replication assay in which site-directed mutagenesis was used to generate mutant versions of human and mouse prethrombin-2 that were able to convert autocatalytically into alpha-thrombin.

“We modified prethrombin so that it was able to cleave its fluorogenic substrate and produce another molecule, alpha-thrombin, initiating a self-replicating system,” explains Dr. Pavlov. In these mutants, the factor Xa cleavage site was replaced with a thrombin cleavage site.

Alpha-thrombin, a serine protease that selectively cleaves a bond in fibrinogen, is a key coagulation cascade enzyme. Its measurement is essential to characterize pathology that affects the coagulation cascade. Further assessment of the cascade involves sensitive and reliable methods to quantitate plasma prethrombin and thrombin activities.

In the self-replicating system, activation of mutant prethrombin-2 by active alpha-thrombin initiated an autocatalytic reaction that led to the formation of more active thrombin. This reaction, in combination with conventional assays to detect human plasma prothrombin, amplified the readout signal by two orders of magnitude.


Researchers based at CIC biomaGUNE reported on a photoelectrochemical (PEC) process that makes use of a graphite electrode modified with an electroactive polyvinylpyridine-bearing osmium (Os) complex. The system relies on the in situ enzymatic generation of cadmium sulfide (CdS) semiconductor nanoparticles (NPs). When the NPs are irradiated with ultraviolet light, photooxidation of 1-thioglycerol is mediated by the Os complex on the surface of the graphite electrode. The PEC methodology was combined with a specific ELISA to develop a novel immunoassay.

Pathogenic/Nonpathogenic Contexts

“In infectious diseases, immunoassays usually work well for pathogens that are well recognized by the immune system and are always associated with disease,” says Martin Rottman, M.D., Ph.D., professor of clinical microbiology at the University of Versailles. For example, immunoassays frequently used in virology detect the presence of antibodies against hepatitis B, hepatitis C, or human immunodeficiency virus.

These serological tests are relatively easy to perform and interpret. In addition, depending on the response profile, they are informative about the presence of the virus in an individual.

“The problem is that many infections are caused by microorganisms that are normally present in people, such as Staphylococcus aureus or Staphylococcus epidermidis,” notes Dr. Rottman. “It is challenging to detect the profile of antibodies that are directed against some of these antigens in the context of disease.”

Dr. Rottman recently led a study that reported on the development of a multiplex antibody detection-based immunoassay for helping diagnose prosthetic joint infections. The study described how antigens from Streptococcus agalactiaePropionibacterium agnes, and several Staphylococcus species were selected by means of comparative immunoproteomics, which was used to evaluate patients with prosthetic joint infections and normal hosts.

“We screened 100 antigens between patients who were infected as compared to noninfected ones,” details Dr. Rottman. “We synthesized some of these antigens and used them for the immunodetection of the clinical infection.”

In a prospective study on 455 consecutive patients, Dr. Rottman and colleagues revealed that this new multiplex immunoassay provides information that cannot be captured using routine serological inflammation markers, providing opportunities to noninvasively evaluate patients with suspected prosthetic joint infection. This assay, called BJI Inoplex, was developed by the French company Diaxonhit. It is a CE-IVD test that promises to be particularly helpful in patients with normal serological inflammatory markers and in those with inconclusive bacteriological results.


Researchers point out that in infectious diseases, immunoassays usually work well for pathogens that are well recognized by the immune system and are always associ-ated with disease. [iStock/poba]

Atypical Autoantibody Profiles

“We have discovered novel targets in specific subsets of patients with autoimmune responses,” says Peter D. Burbelo, Ph.D., staff scientist at the National Institute of Health/National Institute of Dental and Craniofacial Research. This statement reflects a potentially fruitful concept: distinct autoantibody profiles might exist in different patients with the same disease. Besides adding subtlety to the monitoring of autoimmune diseases, this concept could explain how patients with the same condition may show different clinical manifestations.

For example, some patients with systemic lupus erythematosus present predominantly with kidney pathology, whereas others mostly develop lung disease. For a long time, it has been known that some patients with herpes zoster develop postherpetic neuralgia. Although immune suppression has been implicated, the underlying mechanisms have not been understood. In a recent study, Dr. Burbelo and colleagues reported that in a subset of patients with postherpetic neuralgia, anticytokine autoantibodies could be responsible for the uncontrolled reactivation of the virus and the subsequent nerve damage that leads to this chronic pain condition.

One of the limitations in understanding the pathogenesis of autoimmune diseases is that autoantibodies usually exist up to a decade before patients present with clinical manifestations, but very little is known about the prognostic implications of autoantibody profiles in asymptomatic individuals. To fill this gap, several studies are examining the presence and the dynamics of autoantibodies prior to the onset of clinical signs and symptoms. “There is a huge niche that serology can fill for autoimmune diseases,” concludes Dr. Burbelo.


Some antibodies may be consistent with pathogenic or nonpathogenic response profiles. To resolve such ambiguities, researchers are combining multiplex assays with immunoproteomic analysis. [iStock/extender01]

ADC Complexity Requires Large, Small Molecule Bioanalytical Expertise

Antibody drug conjugates (ADCs) have grown increasingly popular because they combine the therapeutic effectiveness of cytotoxic drugs and the specificity of monoclonal antibodies. The chemistry required to link the antibody and drug components can result in a complex sample with a varying drug-to-antibody ratio (DARs). The DAR has profound therapeutic implications: a low DAR reduces efficacy, whereas a high DAR increases toxicity.

The DAR also impacts ADC bioanalysis. For example, DAR influences ADC quantitation because it affects how the ADC binds to the immobilized recognition antigen in a ligand-binding assay (LBA). AIT Bioscience recommends creating ADC reference materials for every possible DAR level to determine the LBA format that results in the most accurate quantitation of the ADC sample, per American Association of Pharmaceutical Scientists guidelines.

Given the heterogeneous nature of ADCs, the bioanalysis of ADCs requires both large molecule expertise and small molecule expertise. In particular, assessments of exposure, safety, and risk will be incomplete without input from liquid chromatography-tandem mass spectrometry (LC–MS/MS). To form a well-rounded ADC assessment team, a sponsor and a contract research organization (CRO) should allocate personnel to fulfill the following roles: overall lead, bioanalytical scientist(s) with LBA and LC–MS/MS expertise, pharmacokineticists, a drug safety representative (if nonclinical), and a clinical pharmacologist (if clinical).

























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