An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines

Abstract

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

Keywords: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); ABTS; ADP; ADP-Ribosyltransferase enzymatic unit; Adenosine diphosphate; BSA; D; DTT; ELISA; EU; Enzyme-ELISA; Enzyme-linked immunosorbent assay; F; FHA; Fims2/3; H(2)O(2); HIST; HPLC; HRP; Haemophilus influenzae type b; Hib; His; IPV; LPC; Macro domain; Mono ADP-Ribosyltransferase; Monoclonal antibody; NAAD; NAD; OD; OVA; PBS; PRN; PTd; PTx; Pertussis toxin; Pertussis toxoid; Pertussis vaccines; SHD; T; TBS; Tris buffer saline; bovine serum albumin; diphtheria toxoid; dithiothreitol; filamentous haemagglutinin; fimbriae type 2 and type 3; formaldehyde; high performance liquid chromatography; histamine sensitisation test; histidine; horseradish peroxidase; hydrogen peroxide; inactivated poliovirus vaccine; lysophosphatidylcholine; mAb; nicotinamide adenine dinucleotide; nicotinic acid adenine dinucleotide; optical density; ovalbumin; pertactin; phosphate buffered saline; single human dose; tetanus toxoid.