Dual-reporter feature allows for data generation in half the time

The NMI Natural and Medical Sciences Institute at the University of Tübingen in Germany is highly regarded for their applied research and early adoption of multiplexing alternatives to singleplex immunoassays. It is no surprise that the NMI scientists were the first in the world to bring our new xMAP INTELLIFLEX^® System into their laboratory to see how its novel features could benefit them.

Since the installation of this system in 2021, scientists at NMI and their collaborators have been adapting xMAP^® assays to take advantage of the xMAP INTELLIFLEX^® System’s unique dual-reporter feature, which makes it possible to interrogate two parameters per analyte. In a new publication in JoVE, they describe the results of a dual-reporter serological test for antibodies to Borrelia bacteria responsible for Lyme disease, a tick-borne infectious disease.

Interrogating two parameters per analyte with xMAP INTELLIFLEX^® DR-SE System’s dual-reporter feature

The paper highlights the importance of correctly identifying the infectious agent via antibody testing since Lyme disease can be caused by several members of the Borrelia genus. This requires testing for antibodies to many different antigens, ideally all at once to streamline the workflow and produce results quickly. In addition, the scientists note that testing for both IgG and IgM responses is useful because these antibodies tend to increase at different times as the disease progresses.

Generally, testing for Lyme disease — also known as Lyme borreliosis — is based on serology assays run after clinical symptoms are found. “In both Europe and North America, diagnostic guidelines recommend a two-step testing series consisting of an enzyme-linked immunosorbent assay (ELISA) with an immunoblot to evaluate antibody response against Borrelia specific antigens,” Häring et al. write. “However, this approach lacks sensitivity and is suboptimal, particularly in the early phase of infection when seroconversion may be incomplete and anti-Borrelia IgG and IgM titers are too low.”

Developing multiplexing alternatives to singleplex immunoassays

In this project, the scientists developed a multiplex alternative to traditional ELISA testing. They first designed single-reporter xMAP assays that could detect either IgG or IgM antibodies against Borrelia infection and then combined them into a dual-reporter assay that can measure both isotypes at once. The test utilizes four antigens representing “the most common pathogenic Borrelia species native in Europe … and North America,” the authors note. “This allows decisive pathogen identification and the ability to discern early IgM and later, more durable, IgG immunoreactivity in patient samples.” (While the assay reported in this study utilizes four antigens, the team later expanded it to eight for a more comprehensive analysis.)

Conserving time and resources through dual-reporter assays

The team thoroughly evaluated the performance of both the single-reporter and the dual-reporter assays. In the latter, they found no significant cross-reactivity between the two antibody systems used to detect IgG and IgM simultaneously. Inter- and intra-assay precision studies showed high reproducibility with excellent results across a range of sample concentrations. “This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements,” the scientists report. “This assay allows essentially double the serological information to be generated from a blood sample in half the time.”

They also note the flexibility and scalability of this approach, since multiplex assays can be run on 96-well or 384-well plates, allowing for high-throughput and automated testing when needed. In addition, the scientists note, “a major advantage of this bead-based Borrelia multiplex assay is the ease with which the assay can be modified or expanded to evaluate different or additional analytes, [such as] antibodies against antigens of further Borrelia species.”

For Research Use Only. Not for use in diagnostic procedures.

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