Immuno-RCA for highly sensitive detection of the antigen-antibody complex in the blood group antigen model

The problem of detecting tiny quantities of analytes by immunochemical methods has been tried for a long time. One approach is to use nucleic acid amplification methods to amplify the signal from a single antigen-antibody interaction. An amplification method suitable for microarrays is the rolling circle amplification reaction. The principle of the method is usage of a conjugate of a detecting antibody with a primer and subsequent isothermal amplification. The generation of a huge single-stranded reaction product starts after adding the necessary components for amplification reaction: circular oligonucleotides, which serves as a template for amplification and phage phi29 polymerase with the other components. This reaction product consists of a lot of repeats of a nucleotide sequence, that is complementary to the circular template. The fluorescent DNA probe can hybridize to each repeat on the product molecule, resulting in a significantly higher level of fluorescence than with fluorescently labeled antibody or streptavidin development systems. In addition, the reaction product remains immobilized on the surface, allowing usage of this approach for the detection of antigen-antibody interactions in other solid-phase analysis systems, such as microarrays. A common problem with such approaches is the nonspecific sorption of components of the immunochemical reaction or amplification reaction, leading to a high background. It is obvious that no matter how highly sensitive the analysis is in theory, a high background will reduce the entire potential of the method to nothing. Herein, we have developed a method that makes it possible to detect small amounts of antibodies to glycans in blood serum and in swabs from tumor cells in a microarray format using a model of blood group antigens. It was possible to obtain a 30 to 70-fold increase of fluorescence level from a specific interaction compared to the use of fluorescently labeled streptavidin. The method we are developing is promising, as it allows us to significantly increase the signal from the specific antigen/antibody interaction in the glycochip format, which will make it possible to detect antibodies to glycans in samples with a very low concentrations of antibodies, for example, in washes from tumor cells.