"Multistage in one touch" design with a universal labelling conjugate for high-sensitive lateral flow immunoassays

Immunoreagents with good results in the competitive enzyme-linked immunosorbent assay are often unable to provide the required detection limit in the traditional competitive immunochromatographic assay. The solution may be either the production of new reagents or improving the test strip. In the latter case, the assay is often performed stepwise using additional liquid reagents, but this is a significant drawback for practical use. We introduce a test strip made as a dry chemical device that still provides the two-step immunochemical interactions — formation of a complex of specific antibodies with an antigen and its detection by a conjugate of antispecies antibodies with a nano-sized label. Analysis with this test strip is similar to that with ordinary test strips and requires no additional reagents and manipulation. The use of specific antibodies and marker as two separate components allows to improve the analytical parameters. The new test significantly lowers the limit of detection, making it possible to use antibodies previously ineffective in immunochromatography. The proposed approach was tested by determining zearalenone and aflatoxin B1 mycotoxins. The visual limit of detection for aflatoxin B1 decreased to 0.6 ng/mL compared to 11 ng/mL with an ordinary test strip. For zearalenone, a test strip was created with visual detection limit of 6 ng/mL with reagents inefficient in the traditional test strip (which is not able to detect even 9 μg/mL of zearalenone). Thus, the proposed approach allows obtaining 'dry', multi-stage, immunochromatographic test strips, providing a highly sensitive detection method.