Goat anti mouse iga hrp

Serum and intestinal-mucosal antibody responses were determined by ELISA. High binding ELISA trays (Greiner) were coated overnight at 4°C with 1 μg/ml of OVA. Plates were washed 3 times and then blocked with 1% BSA for 1 h to minimize unspecific binding. Samples and a known sample used as a standard were included in each plates and titrated by 3-fold serial dilutions. Plates for IgG analysis were incubated for 90 min at room temperature and those for IgA determination for 4 h at 37°C. All plates were washed twice with 0.05% (v/v) Tween 20 in PBS and once with PBS. HRP-conjugated goat-anti-mouse IgG was added to the plates with serum samples and goat-anti-mouse IgA-HRP (Southern Biotech) to the plates with fecal, or small intestine extracts. The plates were incubated at 4°C overnight and after twice washing then developed with OPD for 20 min at which time the enzyme reaction was stopped with H₂SO₄ and absorbance values analyzed at 490 nm. Endpoint titers were determined as the extrapolated sample dilution giving an absorbance value of 0.4 above the no-sample background.

Serum IgG or IgA reactivity toward CBL or HhL was analyzed by immunoblot analysis. For screening multiple sera from Ctr and DC-LMP1/CD40 animals, the Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad, Cat: 1704017) was used. CBL (30 µg per lane or 600 µg for Mini-PROTEAN II Multiscreen Apparatus) or HhL (20 µg per lane or 200 µg for Mini-PROTEAN II Multiscreen Apparatus) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Sera of mice were used as primary antibodies. Differences in serum antibody concentrations between Ctr and DC-LMP1/CD40 mice were adjusted by using 2.5 µg ml⁻¹ serum IgG or 1 µg ml⁻¹ serum IgA for all samples. In some experiments, mouse IgG1 anti-human heat shock protein 60 (aHSP60) antibody (clone LK-2, Enzo, Cat: ADI-SPA-807-E) was additionally used as the primary antibody (1:10,000 in PBS/1% nonfat dried milk). HRP-conjugated secondary antibodies were used as follows: goat anti-mouse IgG-HRP (SouthernBiotech, Cat: 1030–05; 1:10,000) or goat anti-mouse IgA-HRP (SouthernBiotech, Cat: 1040–05; 1:10,000). Western Lightning Plus-ECL Detection Reagent (PerkinElmer, Cat: NEL103E001EA) and X-ray films (Amersham, Cat: 45–001-504) were used for protein detection.

Total IgA levels were measured in gastric wash samples using the Invitrogen IgA mouse uncoated ELISA kit (88-05450-22, Thermo Fisher Scientific) following the manufacturer’s instructions. Dilutions of gastric wash (1:5, 1:25, and 1:125) were prepared in assay buffer (1× PBS, 0.05% Tween 20, and 0.5% BSA) for each sample. Total IgA concentration was analyzed relative to a second-order polynomial standard curve.
To determine H. pylori-specific Ab, 96-well plates were coated with 100 μl of 10 μg/ml H. pylori PMSS1 lysate overnight at 4°C. After washing with wash buffer (00-0400-59, Thermo Fisher Scientific), wells were blocked with blocking buffer (1× PBS, 1% Tween 20, and 10% BSA) for 2 h at room temperature before washing again. Samples were added at 1:5, 1:25, and 1:125 dilution in 1× PBS before incubating overnight at 4°C. After washing, 100 μl/well goat anti-mouse IgA-HRP (1040-05, SouthernBiotech, Birmingham, AL) diluted 1:6000 was added and incubated for 1 h before washing again. Color was developed by incubation with tetramethylbenzidine substrate (88-50450-88, Thermo Fisher Scientific) and then stopped after 15 min with the addition of 1 M H₂SO₄. The plate was then immediately read at 450 nm (BioTek ELx808). Data were reported as fold change above the OD reading of gastric wash samples from H. pylori–negative mice.