Detecting Antibodies with Bioluminescence

image of sensor proteins

Light emitted from sensor proteins turned bluer when samples contained higher concentrations of antibodies against three viruses. [Credit: Adapted from ACS Sensors 2020, DOI: 10.1021/acssensors.0c00564]

As the COVID-19 pandemic rages on, antibody tests continue to play a key role in understanding the disease and how it spreads. The presence of antibodies in the blood means that person was infected with coronavirus and had a past immune response.

Most large-scale antibody tests output a simple positive/negative response, even though some applications require a more quantitative answer that specifies the amount of antibodies in the sample. For instance, if a connection exists between the number of antibodies in the blood and immunity against COVID-19, population-wide quantitative testing would prove greatly beneficial.

With this in mind, a research team in Japan has developed a rapid, cost-effective platform for quantitative antibody detection that uses bioluminescent proteins, cotton threads and a smartphone (ACS Sens., doi: 10.1021/acssensors.0c00564). A proof-of-concept study demonstrates the device’s ability to successfully detect HIV antibody levels in pig blood within five minutes.

A simpler test

Antibody quantification is traditionally performed with sophisticated, high-cost analytical instruments based at central laboratory facilities. Technicians must go through significant training to learn the multistep testing process, which involves tasks like sample pretreatment and reagent handling.

Daniel Citterio, professor of applied chemistry at Keio University, Japan, and his colleagues saw the need for a new type of antibody test that overcomes the limitations of standard clinical assays. Given the current pandemic, they sought to design something easy-to-use, portable and fast that could be used anywhere—even in low-resource areas of the world.

“This motivates us to develop devices of high operation simplicity and user-friendliness, where all components required for an assay are integrated, so that the test can be performed within a short time by simply adding the sample to be analyzed,” said Citterio.

Luminescent antibody sensors

The device relies on engineered proteins called luminescent antibody sensors (LUMABS) that combine two optically active subunits. One is the light-emitting molecule known as luciferase, an enzyme responsible for bioluminescence in animals, and the other is green fluorescent protein. The LUMABS also contain two domains that selectively bind to the targeted antibody.

In the absence of the targeted antibody, the close proximity of the luciferase and green fluorescent protein allows energy transfer to occur. The result is a fluorescent emission of green light. However, in the presence of the targeted antibody, blue light is emitted due to the change in 3D structure of LUMABS. The two optically active subunits remain far enough apart to prevent energy transfer, and blue light from the bioluminescent subunit is observed.

Cotton threads served as substrates for the LUMABS within the researchers’ microfluidic analytical device, which only required a single drop of blood to function. They employed a smartphone camera to detect the hue of the emitted light, where a more blueish color indicated a higher concentration of antibodies.

Results in only five minutes

Citterio and his colleagues tested their device using pig blood spiked with HIV, dengue fever and influenza antibodies. The results demonstrated that it could successfully quantify antibodies in only five minutes with a finger prick of blood. For the next steps, they aim to perform an evaluation with clinical samples and antibodies from other diseases.

“[Our collaborators have] already developed further types of LUMABS targeting other antibodies, including therapeutic antibodies that are increasingly used as drugs to treat various cancers, autoimmune diseases and inflammatory diseases,” said Citterio. “While a LUMABS with specificity for COVID-19-related antibodies is not available at this time, its development is theoretically possible.”

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