As most of us know by now, the tradeoff of the ease and speed of rapid antigen tests is low sensitivity. But now a team of chemists from York University has figured out how to reduce false negatives without making the test overly difficult for home use.
Lateral flow immunoassays, like COVID-19 or pregnancy home tests, work by placing a liquid biological sample on a strip of paper-like membrane. The sample flows along the strip to display a positive or negative result, generally within a few minutes. The problem is that the test isn’t sensitive enough to pick up early-stage infections or pregnancies, where the biological sample contains little of the virus or hormone.
In the case of COVID-19 for example, up to 40 per cent of infected and symptomatic people test negative the first time. Test again a day or two later after the virus has multiplied, and the result will almost certainly be positive.
“By then, it may be too late for preventing disease spread as the person may have not self-isolated,” says Sergey Krylov, who led the study published in Angewandte Chemie.
Working with the hepatitis B virus, Krylov’s team was able to increase diagnostic sensitivity from 73 to 98 per cent without affecting its 95 per cent specificity. To understand how they did it, it’s necessary to first understand more precisely how strip tests work.
A rapid antigen test involves strips, or membranes, embedded with dried antibodies that tightly bind with the antigen in question, for example, a viral protein. Once the liquid biological sample is added to the membrane, capillary action carries it along. In the case of an infected sample, antigens encounter the dried antibodies and bind with them.
Each antibody is modified with a gold nanoparticle label that gives off a red-wine colour when in solution. As capillary action continues to move the combined antibody-antigen-nanoparticles along, they come to a set of antibodies immobilized or bound to a specific position on the strip. These bound antibodies then capture the combined nanoparticle-antigen-antibodies and hold them.
In doing so, they form structures called “sandwich complexes.” Sandwich complexes are so named because the antigens are sandwiched between antibodies on the membrane and antibodies bound to the gold nanoparticles. If enough of these sandwich complexes bunch up, they will display a red line. The higher concentration of virus in the sample, the higher the concentration of the sandwich complexes, the more solid the line.
So far, so good. But when the viral load in the sample is low, the concentration of the sandwich complexes will be low and the line will be faint, or non-existent. The obvious answer, according to the York chemists, is to boost the concentration of gold nanoparticles per sandwich complex. So that’s what they did.
First, the team designed a second set of gold nanoparticles that bind to the nanoparticles in the sandwich complexes. Then they added these new nanoparticles to the strip.
Since the strip was already wet, capillary action could no longer move the second set of particles along. Instead, the chemists used an ordinary 9V battery to create a strong electric field between the strip ends. This field exerted an electrostatic force on the charged gold nanoparticles, moving them down the wet test strip.
The outcome, says co-author Vasily Panferov, is that “the additional gold nanoparticles react with the sandwich complexes and form multilayer agglomerates of gold nanoparticles. The aggregates have much greater colouration than single nanoparticles.”
Using this approach, the chemists were able to detect more than 60 times lower concentrations of hepatitis B protein as compared to conventional test strip. Krylov says the same concept could be applied to toxin testing in the food and beverage industry.
Krylov stresses that it’s important that the first capillary-action driven step is exactly the same as the conventional strip-based assay. If this first step gives a positive result, then the second step isn’t necessary.
The second step is only necessary if the first one returns a negative result. In this case, Krylov’s team created a portable prototype for generating an electric field that he says would be easy to use.
Clinical biochemist and director of Princeton Biochemicals Inc., Norberto Guzman cautions that while the approach does indeed drastically reduce false negatives, it doesn’t deal with false positives. Any given antibody may cross-react with other molecules in the sample, which means the hepatitis B antibody embedded in the test strip may also capture these other molecules and produce a false-positive result.
In other words, a positive reading doesn’t necessarily mean the patient has hepatitis B. They may have some other illness. Reducing the rate of false-positive results would require the development of better-quality antibodies, which is outside of the scope of this work.
“If everything is provided to you to do the second test at home, it would be fantastic,” says Guzman. “But it will increase the price and the time.”
Krylov and his team recognize the challenges and they’re talking with engineers who might collaborate on refining the system.
“I am also talking to companies aiming to commercialize this within a year or two,” says Krylov. “But maybe starting with veterinary, agricultural, food and beverage, and drug testing.”