title: Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures.
abstract: Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.
authors.affiliation: Division of Hematology, Center for Biologics Evaluation and Research, United States Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA. elena.karnaukhova@fda.hhs.gov
authors.forename: Elena
authors.initials: E
authors.lastname: Karnaukhova
authors.forename: Basil
authors.initials: B
authors.lastname: Golding
authors.forename: Yakir
authors.initials: Y
authors.lastname: Ophir
mesh_terms: D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism; D001704:Biopolymers / Q000032:analysis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006358:Hot Temperature; D006801:Humans; D011786:Quality Control; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000592:standards; D012015:Reference Standards; D000515:alpha 1-Antitrypsin / Q000032:analysis* / Q000235:genetics / Q000592:standards
chemical_list: D001704:Biopolymers; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin