RE: Affimer LFD and anti-viral paper4 Jun 2020 14:48
After thousands of posts here this is a gem. It shows that the university of leads have used affimers in LFT successfully to identify CCHFV by binding to the spike protein and providing a read out on a membrane all proven with ELISA tests....
“Lateral flow assay for detection of CCHFV NP
Functionalization of beads.
For the test line, anti-CCHFV NP IgGs were conjugated to 300 nm red latex particles (Ikerlat) and for the control line, BSA-biotin was conjugated to 260 nm blue latex particles (Ikerlat). Beads were washed in MES 10 mM pH 6 and their size was measured by dynamic light scattering (DLS) using a Zetasizer Nano S system (Malvern). Beads were activated using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) and added to the protein of interest. After incubation for 2 h, 500 mM imidazole was added, and beads were washed with 0.1% Tween-20. Blue and red functionalized beads were mixed, diluted in tris-HCl 25 mM, pH 9.5 and dispensed onto a rayon conjugate pad 25-mm (Operon) using a Matrix 1600 dispensing module (Kinematic Automation). Conjugate pad was incubated 30 min in a MMUFE 500 oven (Memmert) at 45°C.
Membrane dispensation.
For the test line, Affimer-NP was diluted in tris-HCl 20 mM pH 8.5, containing 5.0% sucrose and 0.095% sodium azide. Anti-biotin IgG for the control line was diluted (1 mg/mL) in the same buffer (pH 7.5). Both reagents were dispensed in two parallel lines on nitrocellulose membrane at 1 µL/cm. Dispensed membranes were dried for 5 min at 45°C.
LFA assembly.
A master card was assembled on a 72-mm backing card (Kenosha) as follows: HiFlow Plus Nitrocellulose Membrane (HF120) (Millipore), the conjugate pad (Operon) and a 25-mm absorbent pad (Ahlstrom) were pasted and covered with a 65-mm transparent adhesive film (Lohmann). The master card was cut to 4.2-mm width strips using a Matrix 2360 machine (Kinematic Automation)....”
Trek
Sign In
Follow the stocks
