Towards Lateral Flow Quantitative Assays: Detection Approaches.

Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology for lateral flow (immunochromatographic) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qualitative conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quantitative information about the content of a target compound in a sample mixture. This review describes the main options for detecting, processing, and interpreting immunochromatographic analysis results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examined.

Gold nanoparticles of different shape for bicolor lateral flow test.

Spherical gold nanoparticles are the most commonly used marker in lateral flow assays. However, the widespread practice of using identical coloration for the test and control zones of test strips can lead to erroneous interpretations of the assay's results. We propose an immunochromatographic test strip with lines of different colors. For this purpose, gold nanoparticles of different shapes were used, namely blue nanoflowers in the test zone and red gold nanospheres in the control zone. A detailed synthesis procedure for nanoparticles and their conjugates is considered and design parameters for optimal results are described. For the first time, nanoparticles of different shapes have been combined in the test strip with indirect labeling of specific antibodies (via their interaction with labeled secondary antibodies). Using the T-2 toxin (T2T) as an example, an instrumental detection limit of 30 pg/ml and a working range 0.06-0.9 ng/mL were achieved in an analysis of water-organic corn extracts.

Multiplex highly sensitive immunochromatographic assay based on the use of nonprocessed antisera.

The format of an immunochromatographic multiassay is first proposed with native antisera and a universal conjugate of antispecies antibodies with gold nanoparticles. This format allows (1) the exclusion of purification and conjugation stages for specific antibodies and (2) significant reduction of the concentration of specific antibodies in the system. The independent use of specific antibodies and a conjugated marker provided a low detection limit and high signal intensity. The proposed format was implemented for the simultaneous detection of two herbicides. The instrumental limits for the detection of atrazine and chlorsulfuron were 0.1 and 0.7 ng/mL, respectively, and the analysis time was 20 min. The suitability of the test system for monitoring these herbicides in nontreated apple and blackcurrant juices is shown. The assay technique is simple, sensitive, and easily transferrable to any other antigen. Graphical abstract The proposed format of the immunochromatographic multiassay is based on the use of native antisera and a universal conjugate of antispecies antibodies with gold nanoparticles. In this way purification and conjugation stages for specific antibodies are excluded, and the concentrations of specific antibodies and the conjugated marker can be varied independently to obtain a low detection limit.

"External" antibodies as the simplest tool for sensitive immunochromatographic tests.

The scheme of immunochromatographic competitive analysis with "external" specific antibodies is offered for the first time. The test strip includes all the necessary components for analysis (membranes, protein antigen conjugates, colored nanoparticles), except specific free unconjugated antibodies that are transferred to the buffer used for sample dilution. Thus, the concentrations of the antibodies and markers can be chosen independently, and dilution of the sample with buffer (a routine procedure for any immunochromatographic test) ensures pre-incubation of the antibodies with the sample. Both factors increase the sensitivity of the test. The proposed scheme has been proven to be effective for the determination of deoxynivalenol and T-2 toxin; detection limits of 500pg/mL and 50pg/mL, respectively, were achieved. This is an order of magnitude lower than the limits of detection of traditional tests using the same reagents. The analysis was conducted in water-organic extracts (20% methanol); the duration of the analysis is 10min (it's the same time as for a traditional test). The proposed approach is universal, and it can be used to detect a variety of compounds.

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

OBJECTIVE: To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. RESULTS: Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml-1 at 15 min of assay duration. CONCLUSIONS: The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

A new kind of highly sensitive competitive lateral flow immunoassay displaying direct analyte-signal dependence. Application to the determination of the mycotoxin deoxynivalenol.

A new kind of competitive immunochromatographic assay is presented. It is based on the use of a test strip loaded with (a) labeled specific antibodies, (b) a hapten-protein conjugate at the control zone, and (c) antibodies interacting with the specific antibodies in the analytical zone. In the case where a sample does not contain the target antigen (hapten), all labeled antibodies remain in the control zone because of the selected ratio of reactants. The analytical zone remains colorless because the labeled antibodies do not reach it. If an antigen is present in the sample, it interferes with the binding of the specific antibodies in the control zone and knocks them out. Some of these antibodies pass the control zone to form a colored line in the analytical zone. The intensity of the color is directly proportional to the amount of the target antigen in the sample. The assay has an attractive feature in that an appearance in coloration is more easily detected visually than a decoloration. Moreover, the onset of coloration is detectable at a lower concentration than a decoloration. The new detection scheme was applied to the determination of the mycotoxin deoxynivalenol. The visual limit of detection is 2 ng·mL-1 in corn extracts (35 ng per gram of sample). With the same reagents, this is lower by a factor of 60 than the established test strip. The assay takes only 15 min. This new kind of assay has wide potential applications for numerous low molecular weight analytes. Graphical abstract Competitive immunochromatography with direct analyte-signal dependence is proposed. It provides a 60-fold decrease of the detection limit for mycotoxin deoxynivalenol. The analyte-antibody-label complexes move along the immobilized antigen (control zone) and bind with anti-species antibodies (test zone).

"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 0.6ng/mL compared to 11ng/mL with an ordinary test strip. For zearalenone, a test strip was created with visual detection limit of 6ng/mL with reagents inefficient in the traditional test strip (which is not able to detect even 9µg/mL of zearalenone). Thus, the proposed approach allows obtaining 'dry', multi-stage, immunochromatographic test strips, providing a highly sensitive detection method.

Direct immunosensing by spectral correlation interferometry: assay characteristics versus antibody immobilization chemistry.

A 3-channel biosensor based on spectral correlation interferometry (SCI) has been adapted for direct optical detection of antigens by measuring changes in thickness of a biolayer on functionalized glass slips employed as affordable single-use sensor chips. The instrument is insensitive to the bulk refractive index of a solution under test and provides signals in metrological units (pm or nm). Using real-time monitoring with the SCI, protocols for fabrication of sensor chips with different functional (epoxylated, carboxylated, and biotinylated) surfaces for antibody immobilization have been developed and optimized to minimize chip-to-chip variations and achieve better limit of detection (LOD), shorter assay time, and longer shelf life. The optimized coupling surfaces have been compared for detection of human serum albumin (HSA) used as a model agent of medical significance. The dynamic ranges for measuring the HSA concentration were 0.07-20, 0.12-30, and 0.25-10 µg/ml, and the assay durations were less than 20, 15, and 30 min for the epoxylated, carboxylated, and biotinylated chips, respectively. The advantages of each type of sensor chip have been shown, namely, the carboxylated chips feature the shortest assay time, the epoxylated ones demonstrate the best LOD, and the biotinylated chips exhibit the longest shelf life in an unprotected environment. The developed protocols of antibody immobilization can be used in different biosensors and assay techniques including those based on fluorescent, magnetic or plasmonic labels, etc. The SCI is well compatible with various partially transparent layers used in biosensing and with microarrays for multi-analyte detection.

Rapid multiple immunoenzyme assay of mycotoxins.

Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

Rapid immunoenzyme assay of aflatoxin B1 using magnetic nanoparticles.

The main limitations of microplate-based enzyme immunoassays are the prolonged incubations necessary to facilitate heterogeneous interactions, the complex matrix and poorly soluble antigens, and the significant sample dilutions often required because of the presence of organic extractants. This study presents the use of antibody immobilization on the surface of magnetic particles to overcome these limitations in the detection of the mycotoxin, aflatoxin B1. Features of the proposed system are a high degree of nanoparticle dispersion and methodologically simple immobilization of the antibodies by adsorption. Reactions between the immobilized antibodies with native and labeled antigens are conducted in solution, thereby reducing the interaction period to 5 min without impairing the analytical outcome. Adsorption of immunoglobulins on the surface of magnetic nanoparticles increases their stability in aqueous-organic media, thus minimizing the degree of sample dilution required. Testing barley and maize extracts demonstrated a limit of aflatoxin B1 detection equal to 20 pg/mL and total assay duration of 20 min. Using this method, only the 3-fold dilution of the initial methanol/water (60/40) extraction mixture in the microplate wells is necessary. The proposed pseudo-homogeneous approach could be applied toward immunodetection of a wide range of compounds.