Catch and measure
A new tool has been created for the diagnosis and prognosis of the development of autoimmune pathologies
MIPT blog, Naked Science
Scientists from the A. M. Prokhorov Institute of General Physics of the Russian Academy of Sciences and the Moscow Institute of Physics and Technology have developed a new method for diagnosing and monitoring autoimmune pathologies. In just 25 minutes, the biosensor they created not only measures the concentration of autoantibodies in human blood extremely accurately, but also determines their activity for the first time in the world. The combination of these parameters will make it possible to develop new diagnostic criteria for autoimmune diseases, as well as new approaches to their treatment.
The work was published in Biosensors and Bioelectronics, the highest–rated scientific journal in the field of biosensor technologies and analytical chemistry. Autoantibodies produced by the immune system mistakenly perceive the body's own cells and organs as targets to be destroyed. This is how various autoimmune pathologies arise. About 80 such diseases are known: from rheumatoid arthritis, psoriasis and lupus to type I diabetes and multiple sclerosis.
Many of them require monitoring and treatment throughout life to alleviate the condition of patients. Autoantibodies are detected in the blood of about 10 percent of the population. The widespread prevalence of autoimmune pathologies has an extremely negative impact on the economy, which in a number of countries significantly exceeds the damage from cancer. Autoantibodies appear in the blood long before the onset of symptoms of the disease, and their characteristics can predict the severity of the disease.
The treatment of autoimmune diseases is currently significantly complicated by the inconsistency of test results from different manufacturers. "Depending on where and by what method you do the analysis, the results of determining the concentration of autoantibodies in the same patient sample at the same time may differ by 10 or more times," says one of the authors of the work Alexey Orlov, senior researcher at the Biophotonics laboratory of the IOF RAS, a researcher at the laboratory Nanobiotechnology MIPT, graduate of MIPT 2010.
– In fact, until now it was impossible to talk about the concentration of autoantibodies as an objective numerical parameter that can be used, for example, when evaluating the effectiveness of treatment." Such inconsistency of test results from different manufacturers is explained by the fact that autoantibodies are a very complex, unusual object. This is a set of many heterogeneous molecules that interact with each other and with the target in completely different ways. Until now, there was no methodology and tool that would allow taking this factor into account.
In addition, existing and widely used clinical methods did not allow to evaluate the activity of autoantibodies – an indicator of how quickly they contribute to the destruction of tissue. The authors of the published work have created a tool that solves both problems simultaneously: now it is possible to quickly and accurately measure both the concentration and activity of autoantibodies.
Another innovative feature of the development is the simultaneous determination in one sample of the concentration and activity of autoantibodies to several targets at once. This approach significantly increases the diagnostic value of the development. The fact is that different levels of concentrations of autoantibodies to different targets often indicate completely different autoimmune diseases. Correlation analysis of data obtained simultaneously for multiple autoantibodies can significantly improve the accuracy of diagnosis.
"That's why we call our system multiplex or multiparameter," says co–author Averyan Pushkarev, a graduate student at MIPT, a graduate of MIPT in 2018. – Another of our trump cards is the consumable material used for research. This is an ordinary cover glass. The low cost of consumables is especially important for mass medical diagnostics, where it is required to use disposable consumables."
The paper shows the simultaneous characterization of autoantibodies to two targets, but the authors are working to increase their number, since with the help of the so-called microchip technology, up to several thousand targets of about 100 microns each can be applied to such glass.
The difference between the new principle and traditional non-label approaches to determining the kinetic parameters of autoantibodies in blood serum / ©Biosensors and Bioelectronics.
On the surface of the glass, the researchers pass a drop of the patient's blood serum, and its interaction with the target begins. If there are autoantibodies in the serum, they find a target and attach to it, increasing the thickness of the layer on the glass. An interferometric system developed at IOF RAS is located under the glass. This unique optical recorder allows you to measure the thickness of the molecular layer at any point of the glass and monitor its change in real time.
"A very important nuance: unlike many other techniques, autoantibodies interact not with stationary targets, but with moving ones," adds Alexey Orlov. "For the first time in the world, we were able to study the interaction of autoantibodies with targets that are exactly as they are in the body, that is, in their natural, natural form and environment."
This is how it is done (see the illustration). After the autoantibody attaches to the target fixed on the surface of the glass, scientists pass a solution of free target molecules along the surface. Here, the authors of the work managed to implement an approach that no one has been able to put into practice to solve this important task until now. Each autoantibody has a recognizing site – the "hand" that it grabs the target, the so-called Fab fragment.
He has not one, but two or more such "hands". One of them holds the antibody to a stationary target, and the other "catches" mobile targets. This process reflects the genuine (native) activity of the antibody, which can now be measured numerically. Moreover, such a scheme allows, on the one hand, to fix autoantibodies from blood serum on the glass, while maintaining their natural shape, and on the other, to avoid as much as possible the addition of other components that can greatly distort the data obtained.
"We have developed not only an effective diagnostic tool, but also a unique tool for studying autoantibodies," says Peter Nikitin, head of the team of authors, head of the Biophotonics Laboratory of the IOF RAS, a 1979 graduate of MIPT. – On the blood samples of patients, it was shown that the quantitative value of the activity of autoantibodies is a parameter that does not depend on their concentration.
Clinicians were able to carry out quantitative monitoring of both important parameters directly during the course of the disease and develop new methods of diagnosis and treatment of autoimmune diseases based on this." The work was carried out with the support of the Russian Science Foundation.
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