Hrp conjugated goat anti mouse igg

ELISA plates were coated overnight at 4 °C with 100 μL of inactivated SVV-LNSY01-2017 (1 μg/mL) diluted in bicarbonate coating buffer (1.59 g/L Na₂CO3 and 2.93 g/L NaHCO3, pH = 9.6). The wells of the plate were washed 3 times with PBS and then blocked with 5.0% bovine serum albumin (BSA) in PBS (PBSA) for 2 h at 37 °C. The wells were drained and incubated with 100 μL of two-fold serially diluted mAbs (from 1:100 to 1:12,800) for 1 h at 37 °C. The wells were then washed three times with PBS containing 0.05% Tween-20 (PBST) and then incubated with 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000, Boster, Wuhan, China) for 1 h at 37 °C. After 3 times washing with PBST, the reactions were developed with 50 μL/well substrate A (0.1 M citrate/phosphate buffer [pH 5.0]) and 50 μL/well solution B (0.04% o-phenylenediamine; 0.14% H₂O₂) for 10 min at room temperature and then terminated with 50 μL/well of 2 M H₂SO₄. The optical densities (OD) at 630 nm were measured using a microplate reader.

The binding activities of rLP78 to hFn or hPlg were determined by ELISA and western blotting. For ELISA analysis, microtiter ELISA plates were coated with 100 μL of hFn or hPlg (Sigma-Aldrich, Burlington, MA, United States) at 5 μg/mL, and incubated overnight at 4°C. After blocking with 5% skim milk, 100 μL of rLP78 at different concentrations (ranging from 1.56 μg/mL to 100 μg/mL) was added and incubated for 2 h at 37°C. After being washed with PBST, the plates were treated with 100 μL of anti-His-tag monoclonal antibody (1:1,000 dilution) (Boster, Hubei, China), followed by 100 μL of HRP-conjugated goat anti-mouse IgG (1:10,000 dilution) (Boster, Hubei, China). Finally, the optical density (OD) values of the solutions were measured at 450 nm.
For western blotting, rLP78 was separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking with 10% skim milk, the membrane was incubated with 5 μg/mL hFn or hPlg, followed by incubation with rabbit anti-fibronectin antibody or rabbit anti-plasminogen antibody (1:1,000 dilution) (Boster, Hubei, China) as the primary antibody, and HRP-conjugated goat anti-rabbit IgG (1:10,000 dilution) (Boster, Hubei, China) as the secondary antibody.

Serum anti-HBs levels were assessed using corresponding radioimmunoassay (RIA) kits (Beijing North Institute of Biological Technology, China) according to the manufacturer's instructions. OVA-specific antibodies were detected by ELISA. In brief, stripwell flat bottom polystyrene plates (Corstar, NY, USA) were coated with 1 µg/µl OVA (Sigma) overnight at 4°C, and then the membranes were blocked by a 5% BSA solution in a 200 µl volume. After being washed, plates were filled with 100 µl mice sera at appropriate dilution ratios for 1 hour followed by incubation with an HRP-conjugated goat anti-mouse IgG (Boster, Wuhan, China). Antibody levels were visualized by TMB substrate (eBioscience, San Diego) and collected as OD450 values.

Whole-cell ELISA for M2e-specific antibodies was performed in 96 well plates using a previously published procedure with some modifications [29 (link)]. In brief, Madin-Darby Canine Kidney (MDCK) cells were grown in 96-well culture plates in minimum essential medium (MEM) complete medium containing 10% fetal bovine serum (FBS) at 37°C until the cells were almost confluent. The cells were then incubated with 10⁶ EID₅₀ of H1N1 viruses (100 μL) in PBS or with medium alone (for uninfected controls). After incubating for 2 h at 37°C, 200 μL of complete medium was added to each well and plates were incubated at 37°C for 16 h. The plates were then washed with PBS and fixed with 10% formalin at RT for 10 min. Then, the cells were washed three times with PBS and blocked with 200 μL/well PBS/3% BSA for 2 h at 37°C. Serial dilutions of tested sera were added to the wells and incubated for 1–2 h at 37°C. After incubation, the cells were washed and incubated with HRP-conjugated goat anti-mouse IgG (Boster, Wuhan, China) for 1 h at 37°C, followed by TMB Horseradish Peroxidase Color Development Solution for ELISA (Beyotine Biotechnology, Shanghai, China) for 10 min at 37°C. The reaction was stopped with the addition of 50 μL of 2 mol/L H₂SO₄ and the OD₄₅₀ was measured on a microplate spectrophotometer. Data reflect the mean change in OD (infected–uninfected) of triplicate wells per sample.

To extract total protein from harvested CRC cells, Pierce IP lysis buffer (Thermo Scientific, #87788) was employed. The protein concentration was determined via the BCA method (Thermo Scientific, #23227) before adding protein buffer and boiling the samples for 10 min. Next, electrophoresis was performed using 10% SDS-PAGE gels (Solarbio, #P1200-50T), and the proteins were transferred to a PVDF membrane (Thermo Scientific, #88518). The membrane was then sealed with 5% skimmed milk at room temperature for 1 h, followed by overnight incubation at 4 °C with the appropriate primary antibodies: EFTUD2 (Proteintech, #67855-1-Ig, 1:2000), c-MYC (Proteintech, #10828-1-AP, 1:4000), Flag (Abcam, #ab236777, 1:500) and β-Actin (Proteintech, #81115-1-RR, 1:5000). Subsequently, the membrane was washed 3 times with 1× TBST (Thermo Scientific, #28360) for 10 min each wash, and then incubated with the appropriate secondary antibody, including HRP-conjugated goat anti-rabbit IgG (Proteintech, #PR30011, 1:5000) and HRP-conjugated goat anti-mouse IgG (BOSTER, #BA1050, 1:5000), for 1 h at room temperature. Finally, the luminescence signal was visualized using ECL (Solarbio, #PE0020), and the data were analyzed using ImageJ software.

Protein homogenates from the cells were extracted as previously described [5 (link)]. Briefly, the cells were lysed for 20 min on ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 ×g for 20 min at 4°C to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scientific). Then, equal amounts of protein were separated on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Membranes were probed overnight at 4°C with primary antibodies. The bands were visualized using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:15000, Boster) or goat anti-rabbit IgG (1:20000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA). As an internal standard, all membranes stripped with primary antibodies were reprobed with anti-GAPDH antibody (Invitrogen). Changes in protein expression were determined after normalizing the band intensity of each lane to that of GAPDH. Signal was visualized using Konica SRX 101A developer (Konica Minolta Medical Imaging, Wayne, NJ, USA) and the Quantity One software (Bio-Rad, Mississauga, ON, Canada) was used for densitometrical analysis.

The cell membrane proteins of PK-15 and NCI-H292 cells were prepared by a commercial Membrane and Cytosol Protein Extraction Kit according to the manufacturers’ instructions (Tiangen Biotech, China). The ability of rMhr-DnaK to bind cell membrane proteins was quantitatively determined by a microtiter plate adhesion assay (MPAA) (Xiong et al., 2016 (link)). In brief, a 96-well ELISA plate was coated with 100 μL cell membrane proteins (10 μg/mL) overnight at 4°C. After blocking with 5% BSA, the plate was incubated for 2 h at 37°C with 100 μL of rMhr-DnaK solution at different concentrations (ranging from 1.56 to 100 μg/mL) or PBS. Unbound proteins were removed by washing with PBS Tween (PBST), and the adherence was evaluated by adding 100 μL of mouse anti-Trx-tag monoclonal antibody (1:1,000 dilution; Sangon, China) followed by 100 μL of HRP-conjugated goat anti-mouse IgG (1:10,000 dilution; Boster, China). After washing, the substrate containing 3,3′,5,5′-tetramethylbenzidine and H₂O₂-urea was added, and the plates were incubated at 37°C for 15 min. Then, 2 M H₂SO₄ was added to stop the reaction, and the optical density (OD) of the solution was measured at 450 nm. For the adherence inhibition assay, 50 μg/mL of rMhr-DnaK was mixed with anti-DnaK or preimmune serum at various dilutions (1:10, 1:25, and 1:50) and added into the microtiter plate.