Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies.

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

Application of egg yolk IgY on carboxylated polypyrrole films for impedimetric detection of PfHRP2 antigen.

This paper described an impedimetric immunosensor for detecting Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Antibodies from egg yolk (Ab-PfHRP2, IgY type) were linked covalently to the screen-printed gold electrodes (SPGE) surface modified with a thin film of Poly-pyrrole-pyrrole 3 carboxylic acid (P(Py-Py3COOH) to develop the sensing platform. The fabrication steps were followed by microscopic (scanning electron microscopy), spectroscopic (RAMAN spectroscopy and Energy-dispersive X-ray spectroscopy), and electrochemical (electrochemical impedance spectroscopy (EIS) and cyclic voltammetry) techniques. The determination of Ag-PfHRP2 was performed by EIS, and the BSA(bovine serum albumin)/Ab-PfHRP2(IgY)/P(Py-Py3COOH)/SPGE immunosensor recorded a linear response at 100-1000 ng mL-1 concentration range, with a limit of detection (LOD) of 27.47 ng mL-1. Its performance was confirmed by Enzyme-Linked Immuno-Sorbent Assay. The fabricated device uses a simple strategy of IgY immobilization, showing high sensitivity and good selectivity, and can be considered an alternative for carrying out malaria tests.

One-step enzyme-free dual electrochemical immunosensor for histidine-rich protein 2 determination.

In the present work, we describe a novel one-step enzyme-free dual electrochemical immunosensor for the determination of histidine-rich protein 2 (Ag-PfHRP2), a specific malaria biomarker. A gold electrode (GE) was functionalized with the PfHRP2 antibody (Ab-PfHRP2) using dihexadecyl phosphate (DHP) polymer as an immobilization platform. The Ab-PfHRP2/DHP/GE sensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The developed immunosensor was employed for indirect Ag-PfHRP2 determination by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The linear range was 10-400 ng mL-1 and 10-500 ng mL-1 for EIS and DPV, while the limit of detection was 3.3 ng mL-1 and 2.8 ng mL-1, respectively. The electrochemical immunosensor was successfully applied for Ag-PfHRP2 determination in human serum samples. Its performance was compared with an ELISA test, and good correspondence was achieved. The coefficients of intra- and inter-assay variations were less than 5%. The electrochemical immunosensor is a useful and straightforward tool for in situ malaria biomarker determination.