Biology Reference
In-Depth Information
Methods for the detection of anti-EPO antibodies
Detection of anti-EPO antibodies can be achieved using biological or bio-
chemical tests. Biological tests detect only antibodies able to neutralize the
activity of EPO. Normal erythroid progenitors or EPO-sensitive cell lines are
cultured in the presence of serum to be tested and inhibition of cell prolifera-
tion is recorded [8]. Typical results using the UT-7 cell line are shown in
Figure 1. The sensitivity of these tests is rather good, and antibodies able to
neutralize less than 500 mU EPO/mL serum are easily detectable. Their speci-
ficity can be tested by increasing EPO concentrations that must relieve prolif-
eration inhibition. Some EPO-sensitive cell lines, such as UT-7, are also sen-
sitive to other growth factors like GM-CSF [9] that can be used to control the
specificity of the inhibition. Anti-EPOR antibodies could also inhibit cell pro-
liferation with the same characteristics, although such antibodies have not
been reported to date. Other inhibitory molecules present in the serum to be
tested could also inhibit cell proliferation. Although they can be easily distin-
guished from anti-EPO antibodies, they could strongly decrease the efficiency
of these tests. This method presents other limitations: it requires a good expert-
ise in the use of these growth factor-sensitive cell lines, requires several days
(up to 14 days when using normal erythroid progenitors), and only neutraliz-
ing antibodies can be detected. For these reasons, biological tests are used to
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Epo (U/ml)
Figure 1. Biologic assay for the evidence of anti-EPO neutralizing antibodies. EPO-dependent UT-7
cells were incubated for 48 hours with increasing concentrations of EPO in the presence of 10% con-
trol serum (squares) or 10% serum from patient with pure red cell aplasia due to anti-EPO antibodies
(circles). Incubation was done in 96 well plates (total volume 100 µL, 5,000 cells seeded per well).
The cells were labeled with 1 µCi 3 H-thymidine during the last four hours of incubation.
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