Peroxidase conjugated goat anti mouse igg

Total protein extraction was performed as previously described [6 (link)]. Anti-phospho-ERK (E-4, sc-7383), total ERK (K-23, sc-94) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-claudin-10 (38–8400) from Invitrogen (Invitrogen Corporation, CA), anti-β actin (Sigma, St Louis, MO) or anti-GAPDH (AB9485) both from Abcam® (Abcam®, Cambridge, USA) were used as primary antibodies. Peroxidase-conjugated goat anti-mouse IgG (Sigma, St Louis, MO) and anti-rabbit (BioRad, Hercules, CA, USA) were used as secondary antibodies. The reactions were developed using a chemiluminescence ECL kit (Amersham Pharmacia, Uppsala, Sweden). Bands were visualized using UVItec (UVItec Limited, Cambridge, UK) and analyses were performed using Uviband 1D analysis software (UVItec Limited, Cambridge, UK).

Purified recombinant TTCF was coated at 10 µg/ml, 100 µl/well onto high-binding 96-wells ELISA microtiter plates (Greiner, UK) in Na₂CO₃ buffer, pH 9.6 overnight at 4 °C. Plates were washed three times with phosphate buffered saline (PBS) and then blocked for 1 hour in PBS-Tween 0.05% (PBS-T) containing 1% Casein. Plates were washed 3x with PBS-T. Anti-TTCF mouse monoclonal antibody (clone 10G5^{20 (link)}) at a concentration of 1 µg/ml was added to act as a positive control and allow normalisation across ELISA plates. Fifty times pre-diluted samples (in PBS-T) and 10G5 were serially two-fold diluted before transfer to the ELISA plates and incubated for 1 hour. Plates were then washed 3 times. Peroxidase conjugated goat-anti-mouse IgG (Sigma UK), at 1:10.000 dilution in PBS-T was then added for 1 hour. Plates were washed 4x and freshly prepared tetra-methyl-benzidine (TMB) substrate solution (0.1 M Sodium Acetate pH 6.0, 10 µg/ml TMB, 0.015% H₂O₂) added, followed by 10% H₂SO₄ to stop the reaction. Absorbances were read at 450 nm and corrected by subtraction of the reference read at 650 nm and normalised to the positive control (10G5 monoclonal antibody) absorption. Statistical analysis was performed using SPSS v13 (IBM, USA), one-way ANOVA with post-hoc Tukey HSD test.

Antigen-capture ELISA was performed as described [12 (link)] to determine the antibody titers of anti-VLP sera. In brief, 50 μL of diluted fish sera (1:100) were coated in a well of 96-well microtiter plate at 4 °C overnight. The plate was conducted to room temperature and washed three times with 200 μL PBS. After blocking with 5% BSA in PBS for 1 h and washing three times with 1% PBST (all wash steps below indicated three times of 200 μL 1% PBST), 1 ng VLP was added to each well and incubated for 1 h. After wash, the plate was incubated with in-house produced mouse anti-VLP antiserum at a dilution of 1:1000 in PBST for 1 h. The plate was washed and peroxidase-conjugated goat anti-mouse IgG (Sigma) at a dilution of 1:3000 was added and incubated for 1 h. Following thorough washes (five times), 100 μL of OPD substrate (Sigma) was added and the color development was conducted at room temperature. The reaction was stopped by the addition of 4 M sulfuric acid and the absorbance at 492 nm was determined. Statistical differences of titers from different groups were assessed by paired Student’s t tests. Numerical results are presented as mean ± standard deviation with 95% confidence intervals and p < 0.05 was considered statistically significant.

Enzyme-linked immunosorbent assay (ELISA) was used to measure the total IgG antibodies in serum samples. ELISA plates were coated with 10⁸ CFU of whole-cell V. cholerae as an antigen and incubated at 4°C overnight. Nonbinding sites were blocked with 2% BSA at 37°C for 2 h. Plates were washed three times with PBS-0.05% Tween 20 (PBST), and 100 μl of sera in 1/100 through 1/12800 dilutions was added to each well and incubated at 37°C for 2 h. After washing steps, 100 μl of peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich) diluted in PBST (1 : 10000) was added to each well. Plates were incubated for additional 1.5 h at 37°C. After washing 3 times, 100 μl of tetramethylbenzidine (TMB) was added to wells and incubated in the darkness for 30 min. The reaction was stopped by 1 M H₂SO₄, and the absorbance values were read by using an ELISA reader (Labsystems, model no. 352) at 450 nm [22 (link)]. An IgA antibody in saliva and serum samples was evaluated in the same procedure with goat anti-mouse IgA in 1/10000 dilution (SIGMA Aldrich) as the secondary antibody. Serum and saliva dilutions for IgA antibody evaluation was 1/100 through 1/12800 dilutions for serum and 1/25-1/800 dilutions for saliva.

Peripheral blood neutrophils from healthy donors were lysed in lysis buffer (1 % NP-40 in 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, and 1 mM PMSF). After centrifugation to remove cell debris, the supernatant was subjected to 8 % SDS-PAGE under non-reducing condition with appropriate molecular weight markers. After electrophoretic transfer of the proteins to nitrocellulose membrane, the membrane was blocked with 5 % skim milk in Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.6) containing 0.05 % Tween-20. The membrane was incubated with AP11, followed by peroxidase-conjugated goat anti-mouse IgG (Sigma, Saint Louis, 1:5000 dilution in blocking solution). Immunoreactive proteins were visualized using the ECL chemoluminescence detection system (Amersham Pharmaciam, Sweden).

The protein expression of Cx36 and Cx43 in the hippocampus was determined by Western blot technique, which has previously been explained in detail[6 (link)]. In brief, the total protein concentration of the homogenized hippocampi was determined by the Bradford assay. Equal amounts of protein from each sample (5 μg per lane for α-tubulin, 10 μg per lane for Cx36, and 25 μg per lane for Cx43) were transferred to a polyvinylidene difluoride membrane (Roche, Germany) by electroblotting. The membrane was blocked in TBST buffer (100 mM Tris base, 150 mM NaCl, and 0.2% w/v Tween 20) and then incubated with the following primary antibodies: mouse monoclonal anti-Cx36 (1:2000 dilution; Zymed, USA), anti-Cx43 (1:100,000 dilution; Upstate, USA), and mouse monoclonal anti-α-tubulin (1:200,000 dilution; Invitrogen, USA). After washing, the membrane was incubated with peroxidase conjugated goat anti-mouse IgG (1:50,000, 1:200,000, and 1:2,000,000 dilutions for Cx36, Cx43, and α-tubulin, respectively; Sigma-Aldrich, Germany). The membrane was then washed with TBST buffer and reacted with electrochemiluminescence advance Western blotting detection reagents (Pharmacia Amersham, UK). Bands were visualized on X-ray film and quantified by densitometry. The relative levels of Cx36 and Cx43 proteins were expressed as ratios (Cx43/α-tubulin×100, Cx36/α-tubulin×100).