Biosensor for the detection of tick-borne encephalitis
Siberian scientists have developed a bioluminescent probe to detect viruses
Scientists from the Institute of Chemical Biology and Fundamental Medicine SB RAS together with colleagues from the Institute of Biophysics SB RAS (Krasnoyarsk) have created a biosensor capable of recognizing tick-borne encephalitis.
The bioluminescent probe is a recombinant protein consisting of two parts. One binds to the virus, and the second serves as a signaling molecule, which makes it possible to detect its presence. "The first fragment of the biosensor is a variable domain of specific monoclonal antibodies that contacts directly with the virus protein," explains Vera Vitalievna Morozova, a senior researcher at the IHBFM SB RAS, Candidate of Biological Sciences. – The second is a luminescent protein (luciferase) obtained in IBF."
"The mentioned luciferase (in this case, soft coral Renilla muelleri) is a small single–stranded protein, it was cloned by our photobiology laboratory, and we introduced 7 mutations into it, as a result of which the stability of the enzyme significantly increased," explains Lyudmila Alekseevna Frank, a leading researcher at IBF SB RAS, Doctor of Biological Sciences. It is known that the quantum yield of bioluminescent reactions (catalyzed by specific enzymes – luciferases) is much higher than that of conventional chemiluminescent (glow accompanying some chemical processes – ed.) reactions. That is, the amount of light emitted as a result of luciferase reactions is greater, and thus, luciferases used as signaling molecules make it possible to identify targets with higher sensitivity.
Both components are combined using genetic engineering methods, resulting in a plasmid – a genetic construct containing the combined genes of two proteins. Then it is placed in bacterial cells, in which a hybrid, fairly stable artificial protein is synthesized – it can be isolated from cells and work with it further.
"Technically, the whole testing process looks like this: a tick is taken, ground, an aqueous part is extracted from it, which may contain a virus, then an aqueous suspension is introduced into cells containing antibodies," explains Ivan Konstantinovich Baykov, a junior researcher at the IHBFM SB RAS, Candidate of Chemical Sciences. – In the case when the latter have linked the tick-borne encephalitis virus, its accumulation occurs, which is then detected by the addition of a bioluminescent probe and the subsequent chemical reaction of luminescence. If there is no tick-borne encephalitis virus, then the antibodies remain empty, there will be no signal."
It is more difficult to detect tick–borne encephalitis in the blood, it is present there for a short time, and then penetrates into the nervous system, so the main use of the probe is an express check of ticks carrying the disease.
If we talk about the hardware, then a luminometer is needed to perform the analyses, with the help of which radiation quanta from the luciferase part of the biosensor are recorded.
Scientists tested ticks previously infected with a virus with a certain titer simultaneously in commercial test systems and using a created probe. "The advantage of this development is a higher sensitivity, it allows you to detect even a small amount of tick–borne encephalitis virus," Ivan Baykov notes. "To put it into practice, we need to conduct a number of more experiments, identify which impurities can interfere, optimize the sample preparation protocol and the prototype of the test system itself."
By the same principle, it is possible to develop probes for the detection of other viruses: in the same way, take the right antibody and combine it with a bioluminescent protein using an already known technology.
"This also applies to bacterial or fungal agents," Vera Morozova comments. "Now we are trying to get test systems to detect pathogenic fungi that are often found in immunosuppressive patients."
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12.04.2016