Horseradish peroxidase hrp conjugated anti m13 antibody

After three rounds of panning, the phage clones were screened by an enzyme-linked immunosorbent assay (ELISA). The 96-well ELISA plate (Costar, Corning, NY, USA) was coated with 40 nM TeNT-Hc, and also coated with 130 nM Yersinia pestis capsular F1, 54 nM Yersinia pestis capsular V protein (F1 and V, purified in our lab) and 91 nM interferon-omega (IFN-ω, purified in our lab) individually as negative antigens.50 μL of phage particles at a concentration of approximately 8 μM in 3% Marvel™ PBS (pH 7.4) were added, and the binding of the phages to the immobilized antigen was performed at 37 °C for 2 h. The unbound phages were removed by washing extensively with PBST. The plate wells were detected by horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE Healthcare), followed by the addition of peroxidase substrate, o-phenylenediamine dihydrochloride (OPD). 2 M H2SO4 was used to stop the reaction, and the results were monitored at OD492/630 with a microplate reader (Multiskin MK3, Shanghai Labsystems, Shanghai, China). The particular clones that were bound to TeNT-Hc were selected, and the corresponding phage plasmids were sequenced.

Maxisorp 96-well microtiter plates were coated overnight with 5 µg/mL streptavidin (50 µL/well) in NaHCO₃ buffer (50 mM, pH 9.6), then washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) for one hour. Peptides (2 µg/mL) were then loaded on the plate for 30 min. Rabbit sera, individual phage clones or purified antibodies were added to the plates for a one-hour incubation. The plates were then washed with PBST and incubated with the detecting antibody: alkaline phosphatase (AP)-conjugated goat anti rabbit for rabbit serum (Sigma-A8025), horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE healthcare, Little Chalfont, UK) for phage clones and AP-conjugated anti-human IgG (Sigma-A3187) for scFv-Fc antibodies. Detection of HRP conjugates was carried out with 3,3′,5,5′-tetramethybenzidine (TMB/E, Millipore, Billerica, MA, USA). Detection of AP-conjugated antibodies was carried out with SIGMAFAST p-nitrophenyl phosphate tablets (Sigma-N2770).

The phage displayed peptide clones with different amino acid sequences were subjected to ELISA. Briefly, ELISA plates were coated with the G₁ protein at a concentration of 10 μg/well diluted in 0.1 mol/L NaHCO₃ (pH 8.6) overnight at 4°C. Meanwhile, the purified pET32a(+) vector control protein coated with the same concentration was used as control group. All ELISA plates were blocked with 200 μL blocking buffer (5% BSA) for 2 h at room temperature. Ten-fold serial dilutions of the selected phages were added to both coated plates starting with 10¹² phages in the first well. Plates were incubated for 2 h at room temperature and then washed 6 times with TBST (TBS containing 0.5% Tween-20). Bound phage was then detected with 200 μL 1:1000 diluted horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE Healthcare, 27–9421-01, USA) in blocking buffer. Finally, the peroxidase activity was rapidly detected with substrate solution TMB upon addition of a sulfuric acid stop solution. The absorbance of the reaction was determined at 450 nm with an ELISA microplate reader (Shanghai Utrao Medical Instrument Co., Ltd., Shanghai, China).