Here, we present a novel signal amplification strategy in LFIA, which adopts three amplification steps: (a) biotin-streptavidin amplification; (b) polylysine amplification; (c) fluorescence dye signal amplification. The biotin-streptavidin system (BSAS) has been widely applied in immunohistochemistry and immunoassay for its high specificity and strong affinity [45,46]. Streptavidin selleck chem Crenolanib (SA) contains four binding sites with an extraordinarily high affinity for biotin.In this paper, we explored the use of this novel signal amplification conjugate as label for direct electronic signal measurement in Inhibitors,Modulators,Libraries LFIA. This efficient way to increase the sensitivity was achieved by amplification of the signals, which were generated from the fluorescence dye-antibody conjugate with a high fluorescence dye-to-antibody ratio.
When FLPL-BSAS-mAb1 conjugate is bound to one antigen, tens or hundreds of fluorescence dye molecules would bind to a single antigen, consequently leading to signal amplification. In this assay, the resulting conjugates achieved a detection limit Inhibitors,Modulators,Libraries 100-fold lower than that of the magnetic beads-based ELISA [13] and gold-based LFIA [5]. The influence of some important parameters Inhibitors,Modulators,Libraries such as the type of nitrocellulose (NC) membrane, the structure of FLPL-BSAS-mAb1 conjugates and detection time of the present method were investigated in detail. Furthermore, the analytical performance of FLPL-BSAS-mAb1-based LFIA was further evaluated and its precision was also discussed.2.?Materials and Methods2.1. Reagents and MaterialsA nitrocellulose (NC) membrane, absorbent pad, sample pad, conjugate pad, and backing cards were purchased from Millipore (Bendford, MA, USA).
Purified Cry1Ab protein, rabbit polyclonal antibody against Cry1Ab (pAb2), mouse monoclonal antibody against Cry1Ab (mAb1) and Bt Cry1Ab/1Ac/1F ELISA Kit were obtained from Abraxis LLC (Warminster, PA, USA), while Atto 647N (��absmax = 644 nm, ��emmax = 669 nm), polylysine (30�C70 KD), bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-N��-ethylcarbodiimide Inhibitors,Modulators,Libraries hydrochloride (EDC), N-hydroxysuccinimide (NHS), streptavidin (SA), biotin and dimethyl sulfoxide (DMSO) were from Sigma (St. Louis, MO, USA). Other Cry proteins (Cry1C, Cry2A, Cry3A) were from Agdia Inc. (ElKart, IN, USA). Goat anti-rabbit IgG (GAR, >95%), rabbit IgG (RIgG, >95%) were obtained from Longji (Hangzhou, China).
Dialysis tubing (20 KD) was from Spectrum Labs (Rancho Dominguez, CA, USA). All other analytical purified reagents Brefeldin_A were purchased domestically without further treatment or purification.2.2. ApparatusAn XYZ Biostrip selleck products Dispenser and CM 4000 Cutter were purchased from Bio-Dot (Irvine, CA, USA). A portable fluorescence strip reader ESE-Quant FLUO was purchased from Invitrogen (Carlsbad, CA, USA). The ultracentrifuge is from Heraeus Biofuge Stratos (Sollentum, Germany). The SepectraMax M5 multi-mode microplate reader was from Molecular Devices (Sunnyvale, CA, USA).2.3. Preparation of FLPL-BSAS-mAb1 Conjugates2.3.1.