Fluorescein Isothiocyanate Labeling Antigen-Based Immunoassay Strip for Rapid Detection of Acidovorax citrulli

Abstract

A simple and fast immunoassay strip to detect Acidovorax citrulli (Ac) using fluorescein isothiocyanate as a marker was developed. Fluorescein isothiocyanate (FITC) was added to sample culture medium for bacteria incubation, and the bacteria could emit a yellow-green fluorescence under ultraviolet light and become a fluorescent probe. This immunofluorescence strip (IFS) was based on the binding between fluorescent bacteria and the unlabeled monoclonal antibody (McAb) immobilized on the test area in nitrocellulose membrane. The detection limit of the strip was 106 CFU/ml with a result that could be observed within 10 min. The IFS could detect eight strains of Ac and display no cross-reactions with 30 other pathogenic strains. The detection results would not be affected by impurities in plant or unknown microorganisms in natural field samples and were consistent with PCR results, indicating that the IFS has high accuracy. This is the first report of using only one unlabeled McAb to develop a direct-type immunofluorescence strip for the rapid detection of Ac. The IFS reduced detection time and simplified operation procedures compared with the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. In addition, this simple and inexpensive method will play a significant role in monitoring plant pathogens on field detection.

Dual-Affinity Ratiometric Quenching (DARQ) Assay for the Quantification of Therapeutic Antibodies in CHO-S Cell Culture Fluids

“…To date, a myriad of assays have been developed for antibody detection and measurement, including lateral flow assays, , enzyme-linked immunosorbent assay (ELISA), , and immunoaffinity chromatography. , These, however, differ widely in terms of duration (from minutes to hours), performance (sensitivity, limit of detection, and dynamic range), and cost. Likewise, the biorecognition moieties utilized to target the antibody analyte include protein-based affinity tags such as antigens, secondary antibodies, and antibody-binding receptors (e.g., Protein A, Protein G, and Fc receptors FcγRs), as well as synthetic affinity tags such as aptamers and peptides. − Finally, detection modalities vary significantly ranging from optical (e.g., UV/vis, fluorescence, and surface plasmon resonance) , to electrochemical (e.g., impedance and amperometry) and acoustic (e.g., photoacoustic and quartz crystal microbalance) . Fluorescence holds a preeminent place among detection modalities, owing to its high sensitivity, flexibility, and availability of fluorescence spectrophotometers. , The generation of a fluorescence signal by the affinity tags can be accomplished either by chemical conjugation, i.e., by labeling them with synthetic fluorophores, or enzymatically by fusing them with enzymes (e.g., horseradish peroxidase or luciferase) that convert substrates into fluorescent products. , Of major interest are combinations of fluorophores and labeling strategies that can engage in phenomena such as static or dynamic quenching. − Such combinations of fluorophores and labeling strategies enable continuous monitoring of the titer of analytes as their concentration evolves with time and provides information regarding the biomolecular structure of the analyte–tag complex …”

“…In this work, we adopted Trastuzumab (brand name: Herceptin) and Adalimumab (brand name: Humira) as model κI-mAbs, owing to their demonstrated therapeutic value in fighting cancer and autoimmune diseases. , Accordingly, we employed tumor necrosis factor-α (TNF-α) and human epidermal growth factor receptor 2 (Her2) as model antigen (AG) molecules. The fluorophores used in this work, namely, fluorescein (λ ex /λ em : 494/512 nm) and rhodamine (λ ex /λ em : 552/575 nm), were chosen for their expected absorbance–emission spectral overlap and extensive use in fluorescent labeling applications. ,, Specifically, the antigens were labeled with fluorescein ( Fl Her2 or Fl TNF-α), whereas PrL was labeled with rhodamine ( Rh PrL). Within the affinity complex, the mAb acts as a molecular scaffold that maintains 2 Rh PrL and 2 fluorescein-labeled antigen molecules ( Fl Her2 or Fl TNF-α) in a predefined orientation and distance (∼3.10 nm between the centers of mass of PrL and TNF-α and ∼4.98 nm between the centers of mass of PrL and Her2), as illustrated in Figure .…”

Development of a fluorescent immunoassay strip for the rapid quantitative detection of cadmium in rice

“…First, the immunogen Cd-ITCBE-BSA 5 and goat anti-mouse mAb were coated on different positions of the NC membrane to generate the T and C lines, respectively. The test strip was assembled as previously reported and cut to the appropriate size (Zeng, Zhai, Xie, & Liu, 2018). The prepared test strips were sealed and stored in a dry environment at 37°C.…”

New Functional Tracer—Two-Dimensional Nanosheet-Based Immunochromatographic Assay for Salmonella enteritidis Detection

“…5 In this sandwich format, the binding affinity or interaction strength between a specific antigen and antibody can intrinsically determine the detection performance. 6 Unfortunately, the practical applications of ICTs are often limited by some uncontrollable factors of antibodies: (1) complicated cross-linking procedures between antibodies and nanomaterials are still required for probe preparation; 7 (2) the binding ability of the antibody is likely to be affected during the labeling process; and (3) the capacity of resistance to rough chemical conditions of the labeled antibody is a key issue that must to be considered because the antibody often needs to face unsatisfactory circumstances, such as organic extracts or complex sample matrices, when detecting targets. 8 With the above issues taken into account, the preparation of advanced label-free materials, which can be directly used as efficient adsorbents to bacteria, is advantageous and urgently needed.…”