Combination of ELISA and dried blood spot technique for the quantification of large molecules using exenatide as a model.

INTRODUCTION: To explore the feasibility of coupling dried blood spot (DBS) technique with ELISA for the quantification of large molecules, exenatide was used as a model. A method for the quantification of exenatide in human blood was developed and evaluated. METHODS: Exenatide standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. The extraction conditions were optimized by comparing different extraction solutions, with/without protease inhibitors, and various incubation times. A competitive ELISA assay was used for quantification of exenatide from DBS samples. RESULTS: The assay range of exenatide standards in blood was 100-5000 pg/mL. The intra-assay precision (%CV) was from 1.2% to 16.3%, and the accuracy (%Recovery) was from 87.5% to 117.0%. The inter assay precision (%CV) was from 1.7% to 14.3%, and the accuracy was from 95.0% to 115.5%. All the above assay parameters met acceptance criteria. Furthermore, the storage stability of exenatide on DBS cards was tested at ambient temperature as well as at 4°C and -70°C, and it was found that change of storage temperature did not affect the stability of exenatide significantly. DISCUSSION: Our results demonstrated a successful coupling of DBS technique with ELISA for quantification of exenatide in human blood, and the DBS-ELISA combination has a great potential to be further applied for the quantification of other large molecule drugs or biomarkers.