Immunological tests for tuberculosis.

Diphtheria toxin (DT) is often used in the construction of immunotoxins. One potential problem using DT-based immunotoxins is the pre-existing anti-DT antibodies present in human blood due to vaccination. The present study examined the effect of human serum with pre-existing anti-DT antibodies on the toxicity of UCHT1-CRM9, an immunotoxin directed against CD3 molecules on T-lymphocytes. Sera with detectable anti-DT antibodies at 1:100 or greater dilutions inhibited the immunotoxin toxicity. Experiments with radio-labeled UCHT1-CRM9 indicate that anti-DT antibodies partially block its binding to the cell surface as well as inhibit the translocation from the endosome to the cytosol. The inhibitory effect could be adsorbed using a full-length DT mutant or B-subfragment. A C-terminal truncation mutant could not adsorb the inhibitory effect, suggesting that the last 150 amino acids contain the epitope(s) recognized by the inhibitory antibodies. Therefore, an anti-CD3 single-chain immunotoxin, sFv-DT390, was made with a truncated DT. The IC50 of sFv-DT390 was 4.8 x 10(-11) M, 1/16 the potency of the divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicity was only slightly affected by the anti-DT antibodies in human sera.

Antibodies, Bacterial↗

PubMed1995

FemA of Staphylococcus aureus: isolation and immunodetection.

FemA, a cytoplasmic protein necessary for the expression of methicillin resistance in Staphylococcus aureus and also involved in the biosynthesis of staphylococcal cell walls, was detected and quantified in several S. aureus strains under different growth conditions by Western immunoblot. Two types of antigens were used for the production of polyclonal antibodies against FemA: (i) a synthetic peptide comprising 14 amino acids of its C-terminal sequence; and (ii) FemA isolated by preparative gel electrophoresis and electroelution from an overproducing staphylococcal strain. Immunodetection revealed that all investigated strains, either methicillin-resistant or susceptible, expressed FemA during the exponential growth phase in varying amounts. In the stationary phase, the FemA content was diminished. Strains in which femA was inactivated by insertion of Tn551 into the control region of the femAB operon still expressed about 10% of the protein compared to their parent strains. Tn551 insertion in the middle of the femB gene did not affect the FemA expression. In 40 methicillin-susceptible and 6 resistant clinical isolates of S. aureus, the FemA content or its affinity to the antibodies was reduced compared to laboratory parent strains. In susceptible strains, an additional protein of higher molecular weight, present in large quantities, was also able to bind the FemA antibodies. Such a protein was also present in methicillin-resistant isolates, although it was not as pronounced as in the susceptible strains.

Antibodies, Bacterial↗

PubMed1995

Antibodies to lipopolysaccharide.

Lipopolysaccharides (LPS) are indispensable structural components of the Gram-negative bacterial outer membrane and are major determinants of virulence in pathogenic species. In the infected host LPS is better known as endotoxin where it acts as a potent stimulator of the inflammatory response. This article reviews the methods for the production and measurement of anti-LPS antibodies, and then describes the uses to which these methods have been employed. Antibodies to LPS (either monoclonal or polyclonal) may be used directly as immunotherapeutic agents for the treatment of Gram-negative sepsis or endotoxaemia, or as probes for the diagnosis and epidemiological investigation of Gram-negative bacterial infections. Antibodies are useful tools for investigation of the chemical structure of LPS, its expression on bacteria and to study the role of LPS in pathogenic mechanisms. The detection and quantitation of anti-LPS antibodies has formed the basis of classical and more recent serological studies of major bacterial infections.

Antibodies, Bacterial↗