The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.

Amino Acid Sequence↗

PubMedDate not supplied

A competitive inhibition enzyme immunoassay for detection and quantification of organophosphorus compounds.

A sensitive and specific method for detection and quantification of methyl phosphonic acid, p-aminophenyl 1,2,2-trimethyl-propyl diester (MATP) as a model substance for organophosphorus compounds is described. Different procedures for coupling the haptenic group for immunization, purification and immobilization allowed the detection of hapten-specific antibodies. The competitive inhibition enzyme immunoassay (CIEIA), using purified chicken and rabbit IgG-antibodies, was able to detect MATP-concentrations as low as 10(-10) mol/l. Based upon our results, we postulate that the CIEIA represents a good alternative to the customary diagnosis of organophosphate intoxications, measuring blood cholinesterase activity.

Animals↗