Chromatographic enzyme immunoassay for T-2 toxin.

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(gamma-maleimidobutyryl)oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1 microgram of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel.

Repetitive hit-and-run fluoroimmunoassay for T-2 toxin.

A monoclonal antibody for T-2 toxin is converted to a Fab'-fluorescein derivative. The latter is specifically complexed onto a T-2 agarose gel. Fifteen successive doses of T-2 ranging from 1 to 50 ng are then repetitively and linearly detected using a column packed with a small volume (0.2 ml) of this gel without recharging with Fab'-fluorescein. For these assays the effluent from the column is monitored with a spectrofluorometer.

Single-step, quantitative derivatization of amino, carboxyl, and hydroxyl groups in iodothyronine amino acids with ethanolic pivalic anhydride containing 4-dimethylaminopyridine.

Reaction of thyroxine with ethanol and pivalic anhydride in the presence of 4-dimethylaminopyridine quantitatively forms N,O-dipivalyl thyroxine ethyl ester. Other iodothyronines react similarly and the procedure is moisture insensitive. Apparently this reaction is successful, in contrast to similar procedures reported for the derivatization of alpha-amino acids, because it overcomes the problem in other procedures of irreversible side reactions arising from an oxazolone intermediate.

Electrophore-labeling and alkylation of standards of nucleic acid pyrimidine bases for analysis by gas chromatography with electron-capture detection.

The pyrimidine bases cytosine, uracil and thymine, along with some analogues, are electrophore-labeled either with pentafluorobenzoyl chloride (PFBC), pentafluorophenylsulfonyl chloride (PPSC), or heptafluorobutyric anhydride. Subsequent alkylation is most successful for PFB-cytosine, PPS-uracil, and PPS-thymine. These same alkylated compounds also have the highest aqueous stability and respond most strongly by gas chromatography-electron-capture detection. One of these derivatives, determined to be N4-PFB-1,3-dimethylcytosine by authentic synthesis, and its 5-methyl analogue, can be detected with good precision down to the 100-fg level. Poor reproducibility is encountered at the 10-fg level.

Evidence for surface effects in an electron-capture detector.

We have found surface effects in a conventional electron-capture detector that are significantly reduced in an experimental, more inert version of this detector, both by Varian. These surface effects generate unique patterns of solute response for both pesticides-herbicides, and derivatized cytosine strong electrophores. They also cause a minimum followed by a more pronounced maximum in the response factor with increasing solute concentration, demonstrated with lindane. Certain speculations are presented to account for these observations.