Detection of isoforms of recombinant human erythropoietin by various plant lectins after isoelectric focusing

Abstract

A screening method to determine the binding behavior of lectins toward recombinant human erythropoietin (rHuEPO) was developed. Twenty-three different lectins were tested for this purpose. rHuEPO isoforms were separated by isoelectric focusing using the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) accredited method for the direct detection of the prohibited doping substance erythropoietin (EPO). For the visualization of the rHuEPO isoforms lectins were used instead of antibodies. Optimization of the screening protocol enabled the detection of a maximum number of rHuEPO isoforms. By means of this protocol information about the binding properties of a lectin toward each individual rHuEPO isoform was accessible. All evaluated lectins showed significant differences in their binding behavior. The most intense response was obtained with WGA, DSL, PHA-E, LEL, PSA, and LCA. While WGA, DSL, PHA-E, and LEL were able to bind all isoforms detected by the standard antibody, LCA and PSA demonstrated a clear preference for rHuEPO isoforms located in the more basic region of the electropherogram. Further lectins tested were ConA, succWGA, PHA-L, RCA, SNA, MAA, STL, ECL, GSL-II, SJA, SBA, UEA-I, Jacalin, PNA, DBA, GSL-I, and VVA. Compared to the lectins mentioned above, they showed reduced sensitivity. Endogenous and recombinant EPO only differ in the composition of their N- and O-glycan moieties. As lectins possess the unique ability to recognize subtle differences in glycan substructures, they represent an interesting approach for their structural characterization. Furthermore, they might be useful for affinity enrichment/purification of rHuEPO in doping control.

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

“…Structural characterization of rHuEPO has been usually performed by peptide mapping after enzymatic digestion [2,[8][9][10] or glycan analysis after chemical or enzymatic release [11][12][13]. At the intact protein level, rHuEPO has been mainly analysed by CZE [14][15][16][17], IEF [18][19][20], 2-DE [21], CIEF [22] and MALDI-TOF-MS [23]. Nevertheless, the structural information provided by these methods is limited.…”

The Glycosylation and Pharmacokinetics of CTLA4Ig Produced in Rice Cells

“…28) As shown in Fig. 1B, apo-human transferrin had the highest value followed by CHO-EPO and crhCTLA4Ig.…”

Lectin‐binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes)

“…Con A binds to the common sugar a-D-mannose and has been used in numerous experimental studies. The specificities of LCA and PSA are similar but they possess a narrower specificity than Con A (Nagano et al, 2005) and show a preference for branched a-D-mannoses (Kawai et al, 2002). Similar to the lectins of group I, the extracellular material in the lobular lumen was also strongly stained as well as the basement membrane in both species (Table 1).…”

“…These lectins consist of four subunits and are known to recognize complex type N-glycans, but the detailed mechanisms of molecular recognition are poorly understood (Kaneda et al, 2002). While the ''E''-subunit of Phaseolus vulgaris agglutinin is directed against the erythrocyte cell surface, the ''L'' subunit is able to agglutinate lymphocytes, but in many cases, the specificities of PHAE and PHAL overlap (Nagano et al, 2005). In our studies both lectins strongly stain the basement membrane (and presumably other components of the extracellular matrix in the stroma) as well as all analysed types of germ cells.…”