Morphine-6-glucuronide assay using a competitive ELISA assay

Morphine 6–glucuronide (M6G), a metabolite of morphine, is considerably more potent analgesic than morphine itself. M6G in serum is particularly difficult to measure, due to low lipophilicity of the molecule and low serum concentrations after usual parenteral doses of morphine.

A competitive ELISA assay was developed to measure the concentration of M6G in serum and buffer samples. This competitive ELISA method effectively measured the degree to which samples containing an unknown amount of antigen (M6G) compete with a fixed concentration of an anti body (R29).

The method was shown to be sensitive and reproducible with low intra-assay and inter-assay variation, low matrix effect and low cross reactivity. The method was simple and easy to use, and several samples (10 to 35 samples on each plate) could be analyzed simultaneously.

The major advantage of the ELISA method over HPLC was the use of a very small volume (as little as 20 μl) for the assay. This advantage is important in the case of measuring M6G when it is not feasible to obtain a large volume of sample, for example in samples from newborn babies and children.