Abstract
A multiple antigen ELISA forE. coli proteins (ECPs) that may be present in purified recombinant human interferon-γ (rIFN-γ) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-γ at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-γ, 0.2 mg/mL of rIFN-γ was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls ⩽4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-γ (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-γ was obtained (r=0.999). Moreover, linearity of dilution was obtained for ECPs in “in-process” samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-γ. Therefore, these lots contained ;1.3 ppm ECPs.
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Abbreviations
- ECP(s):
-
E. coli protein(s)
- ELISA:
-
enzyme linked immunosorbent assay
- rIFN-γ :
-
recombinant human gamma interferon
- rGH:
-
recombinant human growth hormone
- BSA:
-
bovine serum albumin
- PBS:
-
phosphate buffered saline
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Chen, A.B., Championsmith, A.A., Blanchard, J. et al. Quantitation ofE. coli protein impurities in recombinant human interferon-γ. Appl Biochem Biotechnol 36, 137–152 (1992). https://doi.org/10.1007/BF02929693
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DOI: https://doi.org/10.1007/BF02929693