Mathematical Model of Serodiagnostic Immunochromatographic Assay

This article describes the mathematical model for an immunochromatographic assay for the detection of specific immunoglobulins against a target antigen (antibodies) in blood/serum (serodiagnosis). The model utilizes an analytical (non-numerical) approach and allows the calculation of the kinetics of immune complexes’ formation in a continuous-flow system using commonly available software, such as Microsoft Excel. The developed model could identify the nature of the influence of immunochemical interaction constants and reagent concentrations on the kinetics of the formation of the detected target complex. On the basis of the model, recommendations are developed to decrease the detection limit for an immunochromatographic assay of specific immunoglobulins

Measurement of (Aptamer–Small Target) <i>K</i>_D Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropy

Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. On the basis of FA registration, the binding constants of the aptamer–OTA-Flu and the aptamer–OTA complexes were found to be equal to 245 + 33 and 63 + 18 nM, respectively. The value for the aptamer–OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80 ± 9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer–ligand complexes

Silver-enhanced lateral flow immunoassay for highly-sensitive detection of potato leafroll virus

<p>Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.</p

Correlation between the results of ELISA and immunochromatographic assay.

<p>Estimation of human IgE in the standard solutions by the QD-based immunochromatographic assay is strongly correlated with the results obtained using a commercial ELISA kit (R² = 0.9989).</p

Selecting the optimal concentration of anti-IgE-QD.

<p>The figure shows the relationship between fluorescence intensity in the test zone of the immunochromatographic assay and the concentration of anti-IgE-QD conjugate. A standard solution of IgE with a concentration of 200 kU/L was used as a sample.</p

Correlation between ELISA and immunochromatographic assay results for samples from 95 human subjects.

<p>The immunochromatographic assay is able to estimate total IgE in the serum of human subjects, with good correlation kit (R² = 0.9884) to the results of a commercial ELISA.</p

Quantum-Dot-Based Immunochromatographic Assay for Total IgE in Human Serum

<div><p>To rapidly quantify total immunoglobulin E levels in human serum, we developed a novel quantum-dot-based immunochromatographic assay that employs digital recording of fluorescence. It can detect IgE levels of 5–1000 kU/L, with a coefficient of variation ranging from 2.0 to 9.5%. The assay can be processed in 10 min. The developed assay was tested on 95 serum samples. The correlation coefficient between the IgE values obtained by the proposed assay and those obtained by a commercial ELISA kit was 0.9884. Our assay thus shows promise as a new diagnostic tool for IgE detection.</p></div

Fluorescence intensity in the test zone of an immunochromatographic test strip at different combinations of antibodies against human IgE.

<p>Fluorescence intensity in the test zone of an immunochromatographic test strip at different combinations of antibodies against human IgE.</p

Estimation of total IgE by immunochromatographic assay and ELISA: mean (n = 8) and standard deviation.

<p>Estimation of total IgE by immunochromatographic assay and ELISA: mean (n = 8) and standard deviation.</p

Change in fluorescence intensity with analysis time.

<p>The figure shows the relationship between the analysis time and the fluorescence intensity in the test zone of the immunochromatographic test system. A standard solution of IgE with a concentration of 200/L was used as a sample.</p