Development of an ELISA for the detection and determination of contaminating proteins in recombinant DNA derived human erythropoietin.
An enzyme linked immunosorbent assay (ELISA) has been developed for the quantitative determination of the most probable contaminating proteins (MCP) of recombinant human Erythropoietin produced in the mouse fibroblast cell line C-127. The developed ELISA is a double polyclonal sandwich type immunoassay, which allows a quantitation of the MCPs in the range of parts per million. The polyclonal antibody, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference MCPs (a collection of the most probable protein contaminants) by immunoblot analysis and immunoabsorption of radiolabeled MCPs. Affinity purification of this antibody preparation using the immobilized MCPs resulted in an assay with higher signal-to-noise ratio. The assay was demonstrated to be very specific for the MCPs obtained from the rhu EPO purification process. Since the purification of each recombinant DNA derived protein expressed in mammalian cells requires its own unique process, no generic assay for contaminating proteins can be developed. There are only a few common criteria for the development of such multi-antigen ELISA, which will be discussed in this paper.
Pauly
J U
JU
Research Laboratories, Behringwerke AG, Marburg, W.-Germany.
Siebold
B
B
Schulz
R
R
List
W
W
Lüben
G
G
Seiler
F R
FR
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification
D004274:DNA, Recombinant; D011506:Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin