Detection of chemicals by a reporter immunoassay: application to fluoride.

This report describes a concept in which an immunoassay is used indirectly to quantify a nonantigenic very low molecular weight compound participating in a chemical reaction with a haptenic reporter. The detection limit of each reagent is, therefore, governed only by the affinity of the antibodies toward the reporter. Fluoride was used as a model, and silylated estradiol was used as a reporter. Upon silylation with N-O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-O-bis(dimethylterbutylsilyl) trifluoroacetamide (MTBSTFA), estradiol is no longer recognized by antibodies specific to estradiol. After reaction with hydrofluoric acid (HF) or fluoride salts (KF, CsF, NaF), its immunoreactivity is restored, and native estradiol is formed and is detected by immunoassay. The level of synthesized estradiol is dependent on the concentration of fluoride. A fluoride detection limit of 0.3 microg/L (15 nM) is obtained. Potential interference with other acids has been eliminated by choosing the silyl group (trimethylsilyl vs tert-butyldimethylsilyl) and by selecting optimal reaction conditions for the desilylation. The method has been applied to the detection of fluoride salts in natural waters (range 0.28-9.0 mg/L) and in an atmosphere artificially contaminated with HF between 8 and 160 microg/m(3) in the parts-per-billion range. This indirect immunoassay combines simplicity and high sensitivity and, therefore, can be used in field monitoring. Finally, the extension of the concept to other chemicals is discussed.

Use of DNA immunization to produce polyclonal antibodies against the native human neurokinin-1 receptor for substance P.

Antibodies against the native form of the human NK1 receptor (hNK1R) for the neuropeptide substance P (SP), an important immunoregulator, are difficult to produce using classical immunization techniques. We show here that mice immunized with a plasmid harboring hNK1R cDNA developed antibodies recognizing extracellular epitopes of native hNK1R expressed on CHO cell membranes, as shown by FACS and immunofluorescence analysis, some antibodies being specifically directed against the second extracellular loop (E2) of the receptor. This original strategy, DNA immunization, thus efficiently generated new immunological tools to further analyse the role of SP in the regulation of immune cell functions.