4.3. Antibodies

CagA immunogold reactivity of bacteria and epithelial cells was tested on resin sections by using the following primary Abs: (1) rabbit polyclonal HPP-5003-9 or mouse monoclonal HPM-5001-5 Abs (Austral Biologicals, San Ramon, CA, USA) raised against a highly purified recombinant CagA antigen; (2) rabbit polyclonal sc-25766 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against a recombinant CagA fragment (aminoacids 1–300); (3) rabbit polyclonal Ab raised against recombinant CagA [35], kindly given by Dr. A. Covacci (Siena, Italy). In addition, possible CagA colocalization with proteasome (both 19S and 20S molecular species) and polyubiquitinated proteins, two well-established markers of PaCSs [20,22], were tested by using: (1) rabbit polyclonal 539166 Ab (Calbiochem, La Jolla, CA, USA) raised against 19S proteasome S2 subunit; (2) rabbit polyclonal BML-PW8155 Ab (Enzo Life Sciences, Farmingdale, NY, USA) raised against 20S proteasome α/β subunits; (3) mouse monoclonal FK1 Ab (BML-PW8805; Enzo Life Sciences) raised against polyubiquitinated proteins. As secondary Abs, anti-rabbit or anti-mouse immunoglobulins labelled with 5 to 20 nm colloidal gold particles (British Bio Cell, Cardiff, UK, and Aurion, Wageningen, The Netherlands) were used [17].

Tests to evaluate the specificity of immunogold labelling were carried out using antibodies absorbed with excess antigen and omitting or substituting the specific antibodies in the first layer of the immunogold procedure. Positive and negative controls were obtained by parallel investigation of H. pylori cultures, epithelial cell cultures, and H. pylori-positive or -negative gastric mucosa specimens as in previous studies [16,17,20]. In particular, anti-CagA Abs used were tested by parallel TEM investigation on well-characterized bacterial cultures either CagA-producing (H. pylori strains 60190, ATCC 49503, and CCUG 17874, from Culture Collection University of Göteborg, Sweden) or not producing CagA (H. pylori strain 60190:M22, the isogenic mutant of the 60190 strain in which the cagA gene was disrupted by insertional mutagenesis; kindly provided by Dr. T.L. Cover, Nashville, TN, USA), fixed and embedded as done for H. pylori-colonized biopsies. We found that the rabbit polyclonal HPP-5003-9 Ab from Austral Biologicals gave the best results in term of sensitivity, specificity and reproducibility. All the pictures here shown were obtained with such an Ab.