TABLE 4.

Assays used to measure anti-EPO Abs in single-arm or controlled studies of patients with Ab-mediated PRCA^a

No. of serum samples, PRCA or dialysis	Assay method(s) (SL)	Results
40 (plus 40 control sera) (16)	Indirect ELISA (EPO-coated wells) (SL, 1:10 serum dilution against 80 ng of EPO/ml)	Controls, negative; dialysis, 67% (27/40) positive with low-affinity Abs
13 (EPO-resistant PRCA) (14)	RIP (protein G) (SL, 200 mU of EPO/ml of serum); bioassay for neutralizing Abs with normal bone marrow cells (SL, ≥50 IU EPO/ml of serum)	100% concordance between RIP and neutralizing Ab assays (13/13); RIP Ab positive (range: 3-86 IU epoetin alfa bound/ml); all sera had neutralizing Abs against epoetin alfa
8 (EPO-resistant PRCA) (78)	RIP (protein A) (SL, 10 ng of Abs/ml; ELISA (bridging assay) (SL, 780 ng of Abs/ml); BIAcore 3000 (SL, 390 ng of Abs/ml); bioassay for neutralizing Abs using EPO-transfected mouse cell line (SL, 900 ng of Abs/ml)	Excellent concordance in Ab positivity (100%) for all eight patients using RIP, BIAcore, and bioassay, good concordance with Ab concentrations; two sera tested Ab-negative by ELISA but were positive by all other assays; BIAcore assay determined the predominant Ab isotypes
1,531 CKD patients from 5 Canadian centers treated with epoetin alfa (96)	RIP (protein A) (SL, ≥0.9% of total radioactive EPO bound by a 1:20 serum dilution; 0.4%-0.9% EPO bound = borderline positive); bioassay for neutralizing Abs (UT-7 cell line) (SL, ≥20% neutralization in first 2 serum dilutions)	Only 4 patients had anti-EPO Abs, three borderline positive at indicated SL; no neutralizing Abs detected
1,502 patients from multicenter European darbepoetin alfa trial (9)	RIP (protein A) (SL, 10 ng of Abs/ml)	No patients reported with clinical signs of Ab-mediated PRCA

^aSL, sensitivity limit of assay. Numbers in parentheses are reference numbers.