Horseradish peroxidase hrp conjugated goat anti mouse igg

The concentration of recombinant proteins (rPoMSP4) was determined through the Bradford method using bovine serum albumin (BSA) as standard (Bradford protein assay kit, Solarbio). Purified proteins were analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Beyotime Biotech) to assess the expression level and immunoreactivity. The separated proteins from SDS-PAGE were electrophorectically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon) and blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk at 4 °C. The membranes were probed with anti-His antibody (ABclonal) at 1:5000 dilution along with primary antibody dilution buffer (Meilunbio) overnight at 4 °C. Membranes were washed three times with 0.1% TBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for 90 min. Finally, the membranes were analysed with a ChemiDoc MP imaging system (Bio-Rad).

To map the linear epitope, a GST-tagged fragment of the B. melitensis EF-Tu gene was designed and expressed in E. coli BL21(DE3). Three overlapping fragments of the gene were amplified using specific primers (Supplementary Table 1) and cloned into vector pGEX-6p-1, generating recombinant plasmids pGEX-EF-Tu-1-1, pGEX-EF-Tu-1-2, and pGEX-EF-Tu-1-3. The recombinant proteins were expressed and purified as described above and then analyzed by western blotting with BD₆ as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) as secondary antibody.
Based on the results of the western blot, two fragments (EF-Tu-2-1 and EF-Tu-2-2) of the positive region were amplified (Supplementary Table 1), cloned, and expressed as described above to determine the position of the epitope. Recombinant proteins were detected by western blotting using BD₆ as described above. A further five fragments (EF-Tu-3-1, EF-Tu-3-2, EF-Tu-3-3, EF-Tu-3-4, and EF-Tu-3-5) were then amplified (Supplementary Table 1), expressed, and examined in the same way. Lastly, four fragments (EF-Tu-4-1, EF-Tu-4-2, EF-Tu-4-3, and EF-Tu-4-4) were designed by reducing the number of amino acids one by one on each side until the accurate position of the epitope was determined (Supplementary Table 1). The epitope was referred to as EF.

H. pylori cells were harvested and washed with phosphate-buffered saline (PBS, pH 7.4). The pellets were subjected to ultrasonication. After centrifugation at 16,000 g for 5 min at 4°C, cell debris were removed and the cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the SDS-PAGE gel were transferred onto a 0.45 μm Immobilon-P PVDF membrane (Millipore, MA, USA). After blocked with skim milk, the membrane was incubated with monoclonal mouse antibody (anti-CagA) (Santa Cruz Biotechnology, CA, USA) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech, Beijing, China) successively. Finally, proteins on the membrane were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) according to the instructions.