Colloidal gold-based immunochromatographic strip assay for the rapid detection of diminazene in milk.

Diminazene (DIM) is widely used to treat trypanosomiasis in livestock, but its residues in animal products can cause food safety problems. Thus, a rapid colloidal gold-based immunochromatographic strip assay was established to analyse DIM residue in milk samples. A highly sensitive and specific monoclonal antibody (mAb) against DIM was produced with a new synthetic immunogen by the active ester method. The titre of the prepared antibody was up to 1:1.0 × 106, the affinity constant was 2.2 × 108 L mol-1 and the half-maximal inhibitory concentration (IC50) was 0.4 ng mL-1 based on enzyme-linked immunosorbent assay (ELISA). Then, the colloidal gold-labelled mAb probe was successfully prepared and used for preparing the immunochromatographic strip. The strip showed high sensitivity and specificity, the IC50 for DIM was 5.2 ng mL-1, the limit of detection was 1.2 ng mL-1 and the linear range of detection was 1.8-15.4 ng mL-1. The average recoveries ranged from 89.5% to 91.7%, and coefficients of variation were 6.6-7.2%. The immunochromatographic strip assay can be employed in the rapid detection of DIM residue in milk samples.

Preparation of polyclonal antibodies to diminazene and its detection in animal blood plasma.

This work was undertaken to examine the possibility of preparing polyclonal antibodies to diminazene by using colloidal gold nanoparticles as an antigen carrier and adjuvant. The antibodies prepared by us were used for immunodot determination of diminazene in calf-blood plasma. The immunochemical results agreed with those obtained by high-performance liquid chromatographic estimation of diminazene content in animal biological fluids.

Generation of monoclonal antibodies to the anti-trypanosomal drug isometamidium.

Mice were immunized with either an isometamidium-human serum albumin (HSA) conjugate or an isometamidium-porcine thyroglobulin conjugate (PTG). Thereafter, monoclonal antibodies (MAbs) IL-A 1001, IL-A 1002, IL-A 1003, 5F7.B7, and 5F7.C9 were generated and selected on the basis that they recognized conjugated and unconjugated isometamidium, but lacked cross-reactivity with the carrier molecules. All five MAbs were of the IgG1 isotype. Each of the five MAbs was assessed in a competitive ELISA for isometamidium; in each case, the minimum level of detection was approximately 10 ng/ml. Each MAb exhibited approximately 0.1% cross-reactivity with the anti-trypanosomal compound diminazene. However, based on their cross-reactivity with the anti-trypanosomal compound homidium, the MAbs could be divided into two groups; IL-A 1001, IL-A 1002, and IL-A 1003, produced using an isometamidium-HSA conjugate as an immunogen, exhibited low levels of cross-reactivity (approximately 0.1%). In contrast, 5F7.B7 and 5F7.C9, produced using an isometamidium-PTG conjugate as an immunogen, exhibited high levels of cross-reactivity.