An ELISA specific for murine erythropoietin

Abstract

Summary. Murine recombinant erythropoietin (EPO) was purified from an EPO-producing cell line and used for the production of polyclonal monospecific anti-murine EPO antibodies in rabbits. The anti-mouse EPO antibodies were purified by two affinity chromatography procedures. In order to obtain the most sensitive ELISA, different antibody combinations were tested in the ELISA sandwich assay. The best combination was achieved with an anti-human EPO antibody as coating and the biotinylated anti-murine EPO antibody as detecting antibody. With this sandwich-ELISA a sensitive standard curve in the range of 0·6-30 mU/ ml could be established. The assay provides a sensitive and reliable measure of murine EPO in serum and cell culture supernatants ranging from normal to highly elevated EPO levels.Keywords: murine erythropoietin, purification, polyclonal antibodies, sandwich-ELISA, specificity.Erythropoietin (EPO) is a glycoprotein growth factor responsible for the proliferation and differentiation of early and late erythropoietic progenitor cells. When recombinant human EPO became available, several immunological assays for measuring EPO in human serum were established (Jelkmann, 1992). In our own laboratory we developed a sensitive and reliable sandwich ELISA for human EPO (Noé et al, 1992). However, this human ELISA was not sufficiently sensitive to efficiently measure mouse EPO in serum or culture supernatants from murine-derived cells. In this report we describe a sandwich ELISA specific for mouse EPO, using polyclonal antibodies produced against murine recombinant EPO. MATERIALS AND METHODS Purification of murine EPO (m-EPO).The source for the purification of m-EPO was the cell line Ne-3, kindly provided by Dr W. Ostertag (Heinrich-Pette-Institut, Universität Hamburg, Germany). This mouse fibroblast cell line contained the entire coding sequence of the mouse EPO gene. The cells were first cultivated in CG medium (Vitromex, Vilshofen, Germany) with 10% fetal bovine serum (FBS, Boehringer Mannheim, Germany), the concentration of which was gradually reduced in order to obtain serum-free conditions. After cultivation in CG medium in the absence of FBS for 14 d, the cells were centrifuged and the supernatant was mixed with 1/20 of the volume of 20 × phosphatebuffered saline (PBS), containing 2% Tween 80, 2% sodium azide (NaN 3 ) and stored at ¹20ЊC until processed.The following three chromatographic steps were performed using a FPLC (Pharmacia, Freiburg, Germany). The first step was chromatography of the supernatant over an agarose wheat germ lectin (Pharmacia) column equilibrated with PBS, containing 0·1% NaN 3 , 0·1% Tween 80. The column was washed with the equilibration buffer and the bound protein was eluted with 0·3 M N-acetylglucosamine (Serva, Heidelberg, Germany) in equilibration buffer. The eluted fractions were measured at 280 nm. In addition, EPO present in the eluted fractions was also measured with an EPO ELISA (Noé et al, 1992) modified for recombinant m-EPO (Boehringer Mannheim, Germany). The fractions wit...

A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase

“…Parallelism tests between sample and standard curves were performed for all samples (26). Intra-assay variations were determined by replicates of the same sample in the same plate and inter-assay variation by the same sample analyzed at different days (27).…”

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Protein Levels of Escherichia coli Thioredoxins and Glutaredoxins and Their Relation to Null Mutants, Growth Phase, and Function

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A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway

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