Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

Development of a new indirect ELISA method for detection of anti-tuberculosis antibodies in human serum

Tuberculosis is a crucial health problem. Establishing a rapid, reliable and still inexpensive diagnostic method for tuberculosis seems to be substantial in developing countries where TB has very high incidence rate. An Indirect Enzyme-linked immunosorbent Assay [ELISA] was established to detect serum antibodies against Mycobacterium tuberculosis. Three kinds of antigens were used to prepare the solid phase for antibody assay including: purified protein derivative [PPD], M. tuberculosis Bacilli, and Mycobacterium bovis Bacillus Calmette Guerin [BCG]. Sera of two main following groups were investigated in this study: sera samples from smear-positive, culture-positive and Tuberculin Skin Test-positive TB patients and sera samples from smear-negative, culture negative and TST-negative healthy individuals. Among the antigens used, BCG produced higher sensitivity and specificity in the assay. With PPD as the solid phase, higher sensitivity, but lower specificity was observed in comparison with BCG. Both, low response and noise [non-specific binding] were observed with TB bacilli as the solid phase in the assay. Using BCG solid phase system in this method resulted in higher sensitivity in comparison to single antigen solid phase systems. In addition, we were able to circumvent the problem of non-specific bindings in more popular multi-antigenic solid systems such as PPD. By using this new indirect ELISA, a rapid, reliable and still inexpensive diagnosis of tuberculosis might be possible. Although, further investigations are required to our result

Characterization of recombinant antibodies developed for capturing enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli O157:H7, an emerging cause of food-borne disease with the occurrence of an estimated 20,000 illnesses and 250 deaths each year in the United States, has now been reported from several countries worldwide. Infections with this bacteria, which follows the ingestion of contaminated food by humans, causes bloody diarrhea, hemolytic uremic syndrome (HUS), and renal disease, that can have serious health implications. The source of food contamination is usually associated with animals, mainly cattle. Many cattle become infected early in life when they are exposed to an environment that is contaminated by other animals shedding the organisms in their feces. Detection of E. coli O157:H7 in feces or contaminated food samples requires tests with high sensitivity, which is increased by the use of monoclonal antibodies. However, the production of concentrated monoclonal antibodies in ascites raises animal welfare concerns, and can be expensive. In this study, single chain of variable fragment (scFv) molecules were developed from hybridoma clones that produce immunoglobulins specific for the LPS and flagella antigen of E. coli O157:H7 using phage display technology. The reactivity of the soluble scFv for their respective antigens was preserved in ELISA and by partial inhibition of bacterial agglutination with polyclonal antiserum. Furthermore, the scFv were able to capture E. coli O157:H7 bacteria demonstrating their potential use in diagnostic assays.