Evaluation of a Monoclonal Antibody-Based Test for Detection of Helicobacter pylori -Specific Antigen in Stool Samples from Mice

Abstract

A test using monoclonal antibodies for detection of antigen in stool samples was compared with culture and histology for noninfected (n ‫؍‬ 25), Helicobacter pylori-infected (n ‫؍‬ 25), and Helicobacter felis-infected (n ‫؍‬ 6) mice. Sensitivity and specificity were 96%. The monoclonal antibody-based test is therefore a noninvasive technique that is able to diagnose H. pylori infection in mice.Mice infected with Helicobacter pylori are a convenient animal model for investigations (5, 6); however, noninvasive techniques to assess infection status are limited. The "gold standard" technique for indicating gastric colonization by H. pylori is considered to be a culture of gastric tissue. This requires the animal to be sacrificed, as does any histological analysis, therefore preventing the same animal to be monitored before, during, and after treatment. A noninvasive technique for detection of H. pylori is a test using monoclonal antibodies for detection of antigen in stool samples from mice. The aim of the present study, therefore, was to assess the sensitivity and specificity of the H. pylori-specific antigen test. All experiments were approved by the animal ethics committee of the Women's and Children's Hospital, North Adelaide, South Australia.Female C57BL/6 mice (age, Ͼ7 weeks) were divided into three groups, noninfected (n ϭ 25), H. pylori-infected (n ϭ 25), and Helicobacter felis-infected (n ϭ 6), and housed separately. Inoculations with H. pylori (Sydney strain 1) (6) and H. felis (5, 6) were performed via gavages of 100-l suspensions (10 9 CFU/ml). Following an infection time of 4 weeks and by using age-matched noninfected controls, mouse fecal pellets were collected from each group. Mice were then sacrificed by CO 2 asphyxiation and cervical dislocation, after which the stomachs were excised for histological examination and bacterial culture. Paraffin-embedded sections were stained with hematoxylin and eosin for histology and with a modified May-Grünwald-Giemsa stain to assess bacterial colonization (4). Gastritis was assessed in the body and the antrum by using a modified Sydney grading system for gastritis (6). The severity of gastritis and bacterial colonization density were assessed blindly by an impartial observer.The remaining tissue was homogenized, and serial 10-fold dilutions were performed, with 200 l of each dilution for all mice plated out in duplicate on Helicobacter-selective agar. In addition, 200 l of each dilution of the homogenized stomachs of the H. felis-infected group were plated out in duplicate on Campylobacter-selective agar. Plates were incubated in a 10% CO 2 incubator set at 95% humidity at 37°C for 5 to 7 days.Fecal samples were stored at Ϫ20°C until analyzed. A monoclonal antibody-based enzyme-linked immunosorbent assay (FemtoLab H. pylori Cnx; Connex, Martinsried, Germany) was used to detect H. pylori-specific antigen in stool samples by a modified method. Briefly, the size of the stool sample was reduced (mean weight, 0.047 g; range, 0.015 to 0.200 g), and the stop solution fo...

Lysozyme as a recognition element for monitoring of bacterial population

“…The specific and direct detection of bacteria using monoclonal antibody and aptamer as recognition receptors immobilized covalently onto functionalized interface of biosensor or biochip are widespread developed. However, the application of aptamer [12]based elements suffers from the limitation of discrimination between viable and dead pathogen, while for monoclonal antibody [3,6,13,14]-based recognition elements, the limitation are expensive cost, susceptibility to natural/human environmental stresses and cross-binding to other pathogen, which may cause several positive results [15][16][17]. Compared to monoclonal antibody and aptamer, bacteriophages are not uncommon in natural environment, relative highly specific to pathogen and thus harmless to human health, much less complicated technique to prepare than antibodies or aptamer, and show a far longer shelf life at room temperature [18].…”