TNT detection using llama antibodies and a two-step competitive fluid array immunoassay

Llamas possess unique subclasses of antibodies that lack light chains, and thus are made by the pairing of two heavy chains. IgG was purified from two llamas which had been immunized with trinitrobenzene-keyhole limpet hemocyanin. Conventional IgG1 and heavy chain IgG2 and IgG3 subclasses were fractionated using affinity chromatography. The effectiveness of heavy chain antibodies for the detection of trinitrotoluene (TNT) using a competitive fluid array immunoassay was evaluated and compared to both the llama IgG1 as well as a murine monoclonal anti-TNT antibody. It was found that heavy chain antibody bound TNT with selectivity similar to conventional antibodies, yet the heavy chain antibodies possessed greater thermal stability. The titer of the heavy chain antibodies however was found to be 10-fold lower than the IgG1; thus analytical assays were best demonstrated using the llama IgG1 conventional antibody. The TNT competitive immunoassay on the Luminex fluid analyzer had a dynamic range from ∼ 100 ng/mL to 10 μg/mL. Utilizing the same two-step competitive assay format the dynamic range of the monoclonal antibody was found to have a broad range (1 ng/mL to 1 μg/mL). This method was demonstrated on TNT contaminated soil extracts using both the llama IgG1 and the mouse monoclonal validating the utility of method for analysis of field samples.