Rapid method for detection of anti-recombinant human erythropoietin antibodies as a new form of erythropoietin resistance.
Because of anemia, with hemoglobin of 65 g/L, a patient with end-stage renal disease in chronic hemodialysis required treatment with rHuEPO. rHuEPO-alpha, 20 U/kg body weight three times weekly (Eprex, Cilag, Switzerland), was scheduled, with good initial hematological response. In the following 12 months, hemoglobin started to fall gradually despite continued rHuEPO treatment at dosage of 30 U/kg body weight thrice weekly. The reduction in hemoglobin led us to consider rHuEPO resistance and to investigate the possible causes. rHuEPO-alpha was changed to rHuEPO-beta (Erantin, Boehringer Mannheim, Germany), at 35 U/kg subcutaneous thrice weekly, without a hematological response. As no other cause of rHuEPO resistance was found, serum was screened by ELISA for anti-rHuEPO antibodies.
An ELISA for detection of antibodies was developed. Ninety-six-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with rHuEPO-alpha or beta at 10 mg/L in PBS pH 7.4, and then incubated overnight at 4[degrees]C. The plates were emptied and washed five times with PBS containing 0.5 mL/L Tween 20. Subsequently, the plates were postcoated with PBS containing 30 g/L bovine serum albumin (BSA) for 4 h at room temperature. The contents of the wells were flicked out and 100 [micro]L of different serum dilutions (1:50 to 1:800) was added to the wells and incubated for 1 h at room temperature. Plates were then washed five times as described previously. Subsequently, 100 [micro]L of horseradish peroxidase-conjugated goat anti-human IgG or IgM (Sanofi, Chasca, MN) was added to the wells and incubated at room temperature for 1 h. After washing, 100 [micro]L of freshly prepared peroxidase substrate solution (0.2 g/L tetramethylbenzidine in a citrate buffer containing 0.01% [H.sub.2][O.sub.2]) were added to each well. After 30 min the reactions were stopped by adding 100 [micro]L of 1.25 mol/L sulfuric acid. The absorbance was measured with a microplate reader (Whittaker EIA 400 FW; SLT Labinstruments, Graz, Austria) at 450 nm against a reference blank of 620 nm. Sera from 30 healthy blood donors and sera from 10 hemodialysis patients receiving rHuEPO treatment were used as controls. All control samples were <0.2 absorbance (no antibodies found). All experiments were performed in triplicate. Patient sera at 1:50 dilution was assayed four times on wells coated with BSA (without EPO). Observed absorbance was negligible (0.017-0.065).
The ELISA revealed the presence of IgG but not IgM anti-rHuEPO antibodies directed to both rHuEPOs (Fig. 1). Note that when the serum at different dilutions (1:50 to 1:800) was assayed, the absorbance decreased progressively, and with the dilution 1:800, the absorbance was similar to that of the controls. Ten serum samples from patients receiving treatment with rHuEPO were negative. rHuEPO-alpha is formulated in a solution containing human serum albumin (molar ratio 1:14), whereas rHuEPO-beta is formulated as pure lyophilized product. Although antibodies of patient samples bound to both rHuEPOs, results with rHuEPO-alpha-coated wells disclosed lower absorbance.
[FIGURE 1 OMITTED]
To evaluate the specificity of the ELISA, patient serum samples at 1:50, 1:100, and 1:400 dilutions were preincubated with 1.5 mg/L (3 x [10.SUP.5] U/L) of rHuEPO-beta for 30 min before assay. There was a significant reduction in absorbance, 42%, 46%, and 75%, respectively, as compared with samples without previous incubation with rHuEPO. The biologic activity of rHuEPO could be inhibited by the patient serum. The patient serum sample inhibited an EPO-dependent growth assay when it was preincubated with rHuEPO .
Despite a large number of patients having been treated with rHuEPO in the last several years, the recombinant hormone has not yet been reported to be antigenic. Its protein moiety of 165 amino acids is identical to that of human urinary EPO and corresponds to predictions from the human EPO gene , except for a terminal arginine residue that is lacking in both recombinant and native EPO isolated from human urine . This protein component could, at least theoretically, give rise to an immunological response. Extensive glycosylation of the EPO protein, however, results in some heterogeneity of EPO molecules. Differences have been found in the carbohydrate moieties of native urinary EPO among individuals , and some differences have also been observed between rHuEPO produced by CHO cells and human urinary EPO . A comparison of the two different preparations of rHuEPO, produced by mouse C 127 fibroblasts or by CHO cells, suggests that glycosylation patterns differ, depending on the cell type used for EPO synthesis . This may have an impact on the antigenicity of different preparations of the recombinant hormone. However, both glycoforms of rHuEPO were recognized by the antibodies of the patient.
In conclusion, although rHuEPO is weakly immunogenic and even though the production of rHuEPO antibodies seems to be extremely rare, it must be considered as a potential risk. We have developed a simple, sensitive, and specific ELISA to detect antibodies against rHuEPO.
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Jose Miguel Urra (1), (a), Miguel de la Torre (2), Roberto Alcazar (2) and Ramon Peces (2)
(1) Services of Immunol. and
(2) Nephrol., Hosp. Alarcos, 13002 Ciudad Real, Spain;
(a) author for correspondence: Lab. Hosp. Alarcos, 13002 Ciudad Real, Spain, fax 3426-210298
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|Title Annotation:||Technical Briefs|
|Author:||Urra, Jose Miguel; de la Torre, Miguel; Alcazar, Roberto; Peces, Ramon|
|Date:||May 1, 1997|
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