title: Combination of ELISA and dried blood spot technique for the quantification of large molecules using exenatide as a model.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Alliance Pharma, Inc., 17 Lee Blvd. Malvern, PA 19355, USA.
authors.forename: Yong-Qing
authors.initials: YQ
authors.lastname: Lin
authors.forename: Risha
authors.initials: R
authors.lastname: Khetarpal
authors.forename: Yilu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hengchang
authors.initials: H
authors.lastname: Song
authors.forename: Shawn S
authors.initials: SS
authors.lastname: Li
mesh_terms: D059788:Dried Blood Spot Testing / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000077270:Exenatide; D005240:Feasibility Studies; D006801:Humans; D007004:Hypoglycemic Agents / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D011786:Quality Control; D013048:Specimen Handling; D013696:Temperature; D014688:Venoms / Q000032:analysis*
chemical_list: D007004:Hypoglycemic Agents; D010455:Peptides; D014688:Venoms; D000077270:Exenatide