New approachesin improvingsensitivity and specificity ofPCR(immunePCRaptamer PCR).



Immuno-PCR is a group of hypersensitive methods for detecting antigens that combine the specificity of antibody recognition with antibodies and the sensitivity of PCR.

The method of immuno-PCR (IPPC) was first described by Sano in 1992. This method has the advantages of polymerase chain reaction (PCR) and enzyme immunoassay (ELISA), which provides detection of the analytes present in the samples under study at extremely low concentrations

The method is based on the use of monoclonal antibodies linked to DNA. The antibody binds to the antigen, and then the amplification of the polymerase chain reaction exponentially amplifies the signal.

In order to increase the reliability of immuno-PCR, two different antibodies can be used that identify different epitopes of the same antigen. In this case, each of the antibodies is connected along a DNA chain with a different sequence of nucleotides, but they are nevertheless complementary to each other in a small area. These complementary regions serve as primers for the DNA polymerase in the first stage of the polymerase chain reaction.

The technology was created in 1992 and since then it has been repeatedly modified. For example, antibodies can be replaced with aptamers. In addition, unlike such analytical methods as ELISA and immunoimmunoassay immuno-PCR assay, in one experiment to determine many different antigens at once, because after binding of antibodies to antigens, these antibodies can easily be identified by unique oligonucleotide DNA sequences attached to antibodies.

Another important advantage of the immuno-PCR method is that the technology does not require working with dangerous radioactive material as in radioimmunoassay, does not require complex or expensive equipment, and at the same time it achieves the highest sensitivity, which allows recommending it to oncologists for early diagnosis of precancerous states. So, one of the types of immuno-PCR, called agglutination-PCR (ADAP), allows to detect from zepto- (10-21) to atto (10-18) moles of antibodies in 2 microliters of the sample. What makes it possible to identify, for example, autoantibodies of anti-thyroglobulin from human blood plasma with a 1000-fold increase in sensitivity in comparison with the current radioimmunoassay method.

Equipment required for mastering these methods, like SlipChip, can be made even in handicraft production, and even a cell phone with a built-in camera can be used for recording and evaluating results. Therefore, the technology can be implemented even in rural hospitals.

Aptamers are short (20–80mer), single-stranded DNA or RNA sequences or proteins that bind to target molecules with high affinity and specificity through their 3-dimensional structures. RNA sequences make up the majority of nucleic acid aptamers, perhaps because they can be synthesized by in vitro transcription in the laboratory and, with a 2′-OH, would potentially provide more diverse secondary structure than single-stranded DNA molecules. Nucleic acid aptamers are often identified using an iterative enrichment technique, where oligos or proteins with increased affinity and specificity to a target molecule are isolated from a sequence pool after several rounds of selection. Nucleic acid aptamers are selected in vitro based on affinity for the target molecule, which might be a protein, virus, or cell. SELEX (Systematic Evolution of Ligands by EXponential enrichment; see the sidebar) is one of the most common iterative enrichment methods used to Identify nucleic acid aptamers.

 

Status andprospects of usingaptamersin Veterinary Medicine

To obtain aptamers with specified properties, the SELEX technology was proposed (systematic evolution of ligands by exponential enrichment.) As a starting material in this method, an oligonucleotide library is used in which each oligonucleotide has common 3 'and 5' flanks in length 17-25 nucleotides, and the middle regions of 20-60 nucleotides in length are unique.This library can be synthesized as a single preparation of an oligonucleotide, in which in the middle part in each position it is equally probable Each of the four nucleotides is incubated with this molecule, which is usually fixed on a solid carrier, and the oligonucleotides that do not bind to the target are removed and the bound ones are amplified by PCR using common 3 'and 5' flanks. This cycle is repeated several times, as a result of which enrichment occurs with sequences having an affinity for the target molecule.

Finally, the aptamer molecules are cloned in the plasmids and individually tested for their properties. This technology allows to obtain aptamers for periods from two weeks to several months. The experience of using SELEX has shown that aptamers can be obtained for almost any target: proteins, polysaccharides, small organic molecules, viruses and whole cells.

Aptamers can be considered as analogs of monoclonal antibodies. Moreover, they have a number of important advantages over antibodies. Their preparation is much simpler, cheaper and faster than the preparation of monoclonal antibodies. They have a much smaller size, and therefore easier to penetrate into tissues and cells, may have higher affinity and specificity.

The results obtained using aptamers indicate the possibility of their use in biotechnology for the controlled expression of genes, as well as for diagnosis and therapy.

One of the most interesting achievements in this field of research is the creation of ribozymes with allosteric properties. In such constructions, the catalytic domain of the ribozyme is coupled to sequences of aptamer receptors interacting with regulatory molecules. In the presence of low molecular weight regulators, specific activation or inhibition of the enzymatic activity of the ribozyme can occur. Such hybrid structures are called aptazymov. Aptazim, which has RNA ligase activity, was activated 105 times in the presence of specific low molecular weight effectors. In a similar approach, ATP-dependent ribozymes were constructed.

Important results can be obtained by screening in vitro aptamers among a pool of fragments of natural RNA. Natural sequence libraries can be used to search for domains of nucleic acids that interact specifically with known molecules of biogenic origin, for example, proteins of retroviruses for which the target host RNA targets are unknown.

 


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