Development of a chemiluminescent imaging assay for the detection of anti-erythropoietin antibody in human sera.
Measuring low amounts of anti-erythropoietin antibodies (anti-EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high-throughput chemiluminescent (CL) imaging assay was developed for the detection of anti-EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12-13.91 ng/mL. The limit of detection (LOD, 3sigma) for the CL-imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme-linked immunosorbent assay (ELISA), the LOD of the CL-imaging assay is 50-fold lower. The recoveries of anti-EPO Abs in the fortified serum were in the range 87.1-116.9% using the present method, which highlighted the validity of the CL-imaging assay system to accurately determine the anti-EPO Abs in serum samples. CL-imaging assay was used to evaluate the presence of anti-EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti-EPO Abs.
Wang
Wenjun
W
Department of Chemistry, Key Laboratory for Atomic and Molecular Nanosciences of the Education Ministry, Tsinghua University, 100084 Beijing, People's Republic of China.
Lu
Yanyan
Y
Zhang
Sichun
S
Wang
Shidong
S
Cao
Po
P
Tian
Yaping
Y
Zhang
Xinrong
X
D000906:Antibodies / Q000097:blood* / Q000276:immunology*; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000276:immunology*; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods*; D011994:Recombinant Proteins; D013997:Time Factors
D000906:Antibodies; D011994:Recombinant Proteins; D004921:Erythropoietin