Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces, milk, and colostrum

Abstract

The aim of this study was to develop and evaluate a method for detecting Mycobacterium avium ssp. paratuberculosis (MAP) bacteria in bovine fecal, milk, and colostrum samples using immunomagnetic beads (IMB) and a rhodamine hydrazone immunosensor. Immunomagnetic beads were prepared by using purified antibodies from hyperimmunized sera that were coupled to Fe nanoparticles with diethylene triamine pentaacetic acid (DTPA) or ethyl (dimethyl aminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) as linkers. Rhodamine hydrazone particles were synthesized and coupled to IgY anti-MAP antibodies using DTPA or EDC-NHS linkers. Separation efficiency of the IMB was tested on bovine fecal, milk, and colostrum samples experimentally contaminated with MAP. The studied methods were evaluated on their ability to detect MAP and separate bacteria in complex mediums. The ELISA results indicated 95% efficacy in antibody coupling to IMB, with the DTPA-IMB method being more efficient than the EDC-NHS-IMB method. By using the DTPA-IMB method, MAP bacteria were successfully recovered from fecal, milk, and colostrum samples. The DTPA-IMB method used in combination with the rhodamine hydrazone immunosensor had a limit of detection equal to 30 and 30,000 MAP cells/mL using chromogenic and fluorescent properties, respectively. Combining the DTPA-IMB separation method with the rhodamine hydrazone immunosensor provides a fast, sensitive, and cost-beneficial method for detecting MAP in bovine feces, milk, and colostrum.

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody

“…Their main characteristics include: (i) fast separation speed, high efficiency, and good repeatability; (ii) simple operation without the need for expensive equipment; (iii) minimal impact on the biological characteristics and functions of the isolated cells; (iv) specific separation achieved by coupling specific antibodies and probes [22]. Magnetic bead-based separation has been widely used in clinical diagnosis, isolation and detection of various tumor cells, bacteria, and other microorganisms due to its high efficiency, rapidity, simplicity, non-toxicity, low cost, high separation purity, and retention of cell activity [23][24][25]. In our study, we utilized biotin-avidin coupling between antibodies and magnetic beads, resulting in strong covalent bonds [26,27].…”

Detection of bacteria causing mastitis in cows with immunoelectric tool

“…According to Khosravi et al (2021), 14 Fe nanoparticles were produced by the heat solvent method. Then, DTPA 200 mg was dissolved in 1 mL of DMSO; the volume of the mixture was adjusted to 5 mL using 0.1 M Tris–HCL pH 7.2; 10 μL of Triton X 100 was added to the reaction, and the pH of this mixture was adjusted to 9.6.…”

Recent Progress in Photoresponsive Biomaterials

“…The degree of quenching was used as an internal indicator to determine the amount of drug released. Khosravi et al [60] reported the fabrication of immunomagnetic nanoparticles using a rho-damine hydrazone immunosensor (see Figure 20) for separating Mycobacterium avium spp. paratuberculosis (MAP) bacteria from bovine feces, milk, and colostrum.…”