Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses.

This study presents new type of the lateral flow immunoassay (LFIA) for multi-target analysis. A test, named alarm-LFIA, has an essentially new function that consists in notice (signaling the danger) about the presence at least one target from the controlled list without identification. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies, and multi-specific conjugate that binds the several targets. The alarm test is based on the novel conjugate with broaden specificity due to the immobilisation of a mix of antibodies, specific to several structurally different targets, on the surface of gold nanoparticles. For proof of concept, multi-specific conjugate to five important potato viruses (potato virus X, -M, -S, -Y and potato leaf roll virus) was fabricated using five antibodies with different specificity. The alarm-LFIA was developed for rapid detection of the total infection caused by up to five viruses. Detection limits of the viruses in potato leaf extracts are from 10 to 30 ng/mL. The alarm-LFIA was successfully used for viruses' detection in potato leaves; results were confirmed by enzyme-linked immunosorbent assay. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples.

How to Improve Sensitivity of Sandwich Lateral Flow Immunoassay for Corpuscular Antigens on the Example of Potato Virus Y?

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL-1 to 5.4 ng∙mL-1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.

Development of a lateral flow immunoassay for rapid diagnosis of potato blackleg caused by Dickeya species.

Early detection of potato infections is essential for effective disease management. The aim of this study was to develop a lateral flow immunoassay (LFIA) for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani. Polyclonal antibodies specific to different strains of Dickeya were obtained from rabbits after immunization with bacterial cells of D. dianthicola and D. solani. Enzyme-linked immunosorbent assay testing with use of a wide range of bacterial species showed that the polyclonal antibodies detect closely related strains of D. dianthicola and D. solani. Cross-reactivity with widespread pathogenic bacteria (nine species) and saprophytes of healthy potato plants was not detected. The LFIA based on the obtained antibodies and gold nanoparticles with average diameter of 20 nm was developed. Under optimized conditions, the LFIA method enabled the analysis of potato extracts within 10 min, with a visual limit of detection of 1 × 105 CFU/ml for leaves and 4 × 105 CFU/ml for tubers. The assay was tested on potato stem and tuber extracts, and the results of the LFIA were confirmed in 92.1% of samples using the real-time polymerase chain reaction. The findings confirmed that the developed LFIA could be used for monitoring blackleg infection without the need for special equipment or skills. Graphical Abstract The developed lateral flow immunoassay is an efficient tool for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani.

Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens.

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 10(4) cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. Graphical abstract Location of binding zones in the developed multiarray on a test strip (MATS) for simultaneous detection of eight pathogens.