Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Division of Hematology, Center for Biologics Evaluation and Research, United States Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA.
Biologicals (Impact Factor: 1.21). 11/2007; 35(4):285-95. DOI: 10.1016/j.biologicals.2006.11.002
Source: PubMed


Human alpha-1-proteinase inhibitor1Also known as antitrypsin.1 (α1-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. α1-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) α1-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-α1-PI), proposed as alternatives to pd-α1-PI products, have been under intensive investigation. For accurate determination of α1-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for α1-PI quantification and potency measurements. As a part of our multi-step research focused on α1-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-α1-PI and r-α1-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, r2 = 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 α1-PI ELISA indicates that this assay can be used for monitoring concentration levels of α1-PI in multi-component biological matrices, based on the following: (a) quantification of r-α1-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of α1-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized α1-PI; (d) quantification of α1-PI in human serum; and (e) comparative quantification of α1-PI in commercially available products.

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Available from: Elena Karnaukhova, Dec 20, 2014
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    • "Fig. 4 compares r-α1-PI from A. niger with standard pd-α1-PI, enzymatically deglycosylated pd-α1-PI (de-pd-α1-PI), and with r-α1-PI from E. coli. Unlike r-α1-PI in the soluble protein fraction from E. coli (see SDS-PAGE in [21]), the raw supernatant from A. niger has a relatively simple protein composition. "
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    ABSTRACT: Human alpha1-proteinase inhibitor (alpha1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alpha1-PI (r-alpha1-PI) could provide an attractive alternative. Although r-alpha1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. We have explored the possibility of expressing the gene for human alpha1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha1-PI was similar to that of the pd-alpha1-PI. In vitro stability of the r-alpha1-PI from A. niger was tested in comparison with pd-alpha1-PI reference and non-glycosylated human r-alpha1-PI from E. coli. We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.
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