This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thorpe, R. Articles by Swanson, S. J Search for Related Content PubMed PubMed Citation Articles by Thorpe, R. Articles by Swanson, S. J

MINIREVIEW

Current Methods for Detecting Antibodies against Erythropoietin and Other Recombinant Proteins

Division of Immunobiology, The National Institute for Biological Standards and Control, Hertfordshire, United Kingdom,¹ Department of Clinical Immunology, Amgen Inc., Thousand Oaks, California²

	INTRODUCTION

Top Introduction References

 
Recombinant DNA technology has revolutionized the production and use of a number of therapeutic proteins, including cytokines and cellular growth factors. These proteins have similar or identical biologic activities as their natural counterparts and are particularly useful for treating patients who are deficient in these cytokines or growth factors because of congenital abnormalities, certain viral or toxicologic pathologies, or cancer chemotherapy. However, in some instances, the use of recombinant therapeutic proteins or proteins contained in certain viral delivery vehicles has led to the production of antibodies (Abs) that can neutralize protein function (12, 22, 27, 41, 46, 73, 86, 91, 92). Therefore, the production of Abs is a potentially limiting factor for the successful clinical use of recombinant therapeutic proteins (72).

Ab responses to therapeutic proteins may impact the safety, efficacy, and pharmacokinetic and pharmacodynamic parameters of therapeutic proteins (18, 46, 54, 73, 97). For example, complexes formed between Abs and the therapeutic agents they recognize can adversely affect the pharmacokinetic or pharmacodynamic characteristics of recombinant proteins by limiting or altering the volume of distribution in vivo (55). Neutralizing Abs can bind to the portions of drug molecules involved in receptor binding or cell activation, thereby blocking the therapeutic effect of the drug (98). In addition, neutralizing Abs formed against a recombinant protein may also inhibit the activity of the corresponding endogenous factor, resulting in a failure to respond to that factor (10). Rarely, Abs may be part of an allergic response that could potentially lead to inflammation, skin reactions, or other adverse events (20, 94).

Therefore, the development and use of sensitive and specific assays to identify and measure Abs against therapeutic proteins is important to successful clinical use of these agents. In addition to measuring high levels (titers) of specific Abs, some assays possess a level of sensitivity sufficient to detect low levels of naturally occurring Abs or auto-Abs.

This article reviews the different types of Ab assays that have been used to measure and characterize Abs specific to certain recombinant therapeutic proteins, with emphasis on recombinant human erythropoietin (EPO), as well as the benefits, disadvantages, and limitations of the different assays used for screening and diagnostic purposes.

	Ab-MEDIATED PURE RED BLOOD CELL APLASIA

 
A recent example of the negative impact of Abs on recombinant protein therapy is the increased prevalence of Ab-mediated pure red blood cell aplasia (PRCA). Patients with Ab-mediated PRCA present with a rapid onset of EPO resistance, followed by severe decreases in blood hemoglobin levels and reticulocyte counts (i.e., erythroblastopenia). Ab-mediated PRCA arises in a small number of patients receiving recombinant human EPO for the treatment of anemia associated with chronic kidney disease (CKD). It is etiologically distinct from more common forms of PRCA caused by certain toxins, viral infections, malignancies, or congenital abnormalities.

Prior to 1998, there were few reports of Abs in patients treated with recombinant EPO. Patients do not generally produce anti-EPO Abs because of the immunologic similarity between recombinant and endogenous EPO and the maintenance of immunologic tolerance to self proteins (73). In recent years, the number of cases of Ab-mediated PRCA in patients with CKD has increased substantially and in most cases is associated with subcutaneous (SC) administration of epoetin alfa formulated without human serum albumin stabilizer (Eprex, Ortho Biologics LLC, Manati, P.R.) (28). Table 1 lists the currently available number of confirmed Ab-mediated PRCA cases worldwide for five currently approved recombinant erythropoietic agents.

	SENSITIVITY AND SPECIFICITY OF Ab ASSAYS

 
There is a critical need to understand the sensitivity, specificity, and utility of assays that measure anti-EPO Abs in patients with CKD-associated anemia and to reach consensus on a standard assay method. A standard assay may prove useful as an early diagnostic tool for patients developing Ab-mediated PRCA and for monitoring their disease progression and response to treatment. The terms sensitivity and specificity are used to describe the characteristics of Ab assays. Sensitivity refers to the ability of an assay to detect very small concentrations of Abs in a test sample. A highly sensitive assay is more likely to detect low levels of specific Abs in a cohort of patients and therefore has a low probability for producing false-negative findings. The referred relative sensitivities of different assays vary, according to (i) the practical limitations imposed by the types and affinities of Abs in serum samples and internal standards (58), (ii) the type of detection system employed (e.g., radiometric, colorimetric, or chemiluminescent) (66, 80), (iii) the method of analysis (e.g., capture assay, direct or indirect assay, or competitive inhibition assay) (2, 76), (iv) the presence of inhibitory substances such as immune complexes, cross-reactive antigens, or heterophilic Abs in serum samples (7, 32, 50), (v) the nature and concentration of specific target proteins (EPO, in this case) and their epitopes (5, 34, 66), and (vi) the range of Ab concentrations that each assay can measure.

Thus, the practical or perceived sensitivity of each assay is generally more conservative than the actual or real sensitivity, which often lies outside of the range of statistical confidence for calculating Ab concentrations. Therefore, the practical (lower) limit of detection is the lowest threshold at which Ab measurements can be made within the design of each assay system. In addition, assay sensitivity is affected strongly by the relative affinity of different Abs for a particular target protein or antigen. For example, high-affinity immunoglobulin G (IgG) Abs bind to antigens more tightly than the low-affinity IgM and IgG Abs that are typically produced in the early stages of Ab maturation. Naturally occurring Abs, which can interfere with assay results, tend to have low affinity and are present at low concentrations, because there is little or no active immunization (80).

Specificity refers to the ability of an assay to detect only those Abs directed against a specific target protein or ligand. The specificity of each assay is a product of the biochemical mechanics of Abs binding to the test ligand and the subsequent detection method used. Therefore, specificity is largely influenced by the nature and type of Abs in patients' samples, the presence of confounding serum factors, and the target ligands (antigens) that are used in the procedure. A highly specific assay has a very low probability of measuring false positives resulting from nonspecific binding of Abs or cross-recognition of antigenically related proteins or molecular mimics. Most assays use blocking reagents that reduce or eliminate nonspecific Ab binding.

Ideally, a useful Ab assay possesses both high sensitivity and high specificity, but in practical experience, a highly sensitive assay suitable for screening large numbers of samples may not have the level of specificity required to prevent samples from being falsely identified as having Abs present. Comparative analyses of different types of assays have often shown wide variability in their relative sensitivity and specificity for various diagnostic purposes (23, 25).

	TYPES OF Ab ASSAYS

 
A number of different assays have been used to measure Abs specific to recombinant proteins. These assays are generally grouped into two major categories: (i) binding Ab assays, which measure the binding capacity of Abs to proteins; and (ii) neutralizing Ab assays, which measure the ability of Abs to neutralize a biological effect of the protein in biological systems (bioassays). There are several types of binding Ab assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoprecipitation (RIP) assays, and the BIAcore biosensor assay. In contrast, only bioassays characterize neutralizing Abs.

ELISA ELISAs colorimetrically detect Abs bound to protein antigens either directly or indirectly. Variations of these assays can improve sensitivity and/or specificity (39, 42, 60). For example, indirect ELISAs detect primary (e.g., serum sample) Abs that bind to target proteins immobilized on plastic wells by using enzyme-linked secondary Abs, such as goat anti-human Ig Abs, and enzyme substrates that change color in the presence of enzyme-labeled Abs. The intensity of substrate color change is proportional to the amount of enzyme-linked Abs present in the sample wells and correspondingly the amount of Abs that bound previously to the plastic-immobilized protein. Properly optimized indirect ELISAs generally give low background readings and high levels of sensitivity and specificity because confounding results from nonspecific Ab binding and interfering serum factors (e.g., rheumatoid factors) are reduced or eliminated (40). Some indirect ELISAs employ the use of avidin-biotin complexes between Abs and protein antigens to increase assay sensitivity (15, 74).

Bridging ELISAs reduce background readings by using fewer amplification steps in the procedure and the requirement for two specific binding events for the target drug, which increases specificity of the assay. The bridging ELISA takes advantage of the two antigen binding sites on each Ab molecule (Ab bivalency) that allows Abs to form a bridge between (i.e., cross-link) drug immobilized on plastic wells with enzyme-labeled drug that is added in the detection step (61). In this assay, the unlabeled drug is first immobilized onto the plastic wells, and optimally diluted Abs in a test serum sample are allowed to bind, followed by the usual washing step to remove unbound Abs. Enzyme-labeled drug is then added to complete the bridge, and a colorimetric substrate is then added to visually detect the presence of specific Abs.

Schematic examples of indirect and bridging ELISAs are shown in Fig. 1, in which an enzyme-linked, anti-Ig (secondary) Ab amplifies the signal for detecting serum (primary) Abs that have bound to immobilized proteins. A version of the indirect ELISA was used by Urra and colleagues to characterize anti-EPO Abs in a patient with EPO-resistant PRCA (87).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Schematic illustrations of indirect and bridging ELISA testing procedures for measuring Abs against protein drugs (e.g., EPO). In the indirect ELISA, the protein drug is coated onto the wells of plastic assay plates prior to the addition of serum samples. Any specific Abs that are bound to the immobilized protein are subsequently detected with enzyme-linked, anti-Ig Abs (monoclonal or polyclonal) and enzyme substrate. In the bridging ELISA shown here, the unlabeled protein drug is first coated onto the wells, serum Abs are allowed to bind to the immobilized drug, and Abs are then detected with enzyme-labeled drug plus enzyme substrate. The bridging ELISA depicted here relies on the ability of bivalent Abs (IgG) to bridge the immobilized drug with enzyme-labeled drug in solution.

View larger version (40K): [in this window] [in a new window]   FIG. 2. RIP testing procedure for measuring Abs against a ligand (protein). This method uses fluid-phase binding of sample Abs in a patient's serum with a specific ligand (e.g., radiolabeled EPO [A*]) alone or in a mixture of proteins) (step 1), the subsequent binding of protein A-Sepharose or protein G-Sepharose beads that attach to ligand-Ab complexes (step 2), and the collection of beads by centrifugation (step 3). This assay method can incorporate competitive free (nonradiolabeled) ligand to test the specificity of Abs for individual proteins or peptides. Reproduced from reference 36 with permission.

View larger version (23K): [in this window] [in a new window]   FIG. 3. The principle of optical detection of Ab binding to surfaces of sensor chips in a BIAcore immunoassay device. Binding of Abs to a sensor chip is detected by the internal reflection of incident, polarized light off the chip surface. The angle of internal reflectance is dependent on the mass of Abs binding to the chip surface. Reproduced with permission from BIAcore, Inc.

A number of different bioassays have been used to quantify EPO-neutralizing Abs, including EPO-stimulated growth of erythroid precursors in plasma-clot cultures of mononuclear blood cells or bone marrow cells (13, 52) and proliferation of the EPO-dependent human erythroleukemia cell line, UT-7 (13). An example of erythroid colonies growing in culture is shown in Fig. 4. The usefulness of immortalized cell lines lies in the ability to culture these cells over extended periods of time before cell death occurs. Proliferation of such cells can be quantified by using tritiated thymidine to assess nucleic acid synthesis and tetrazolium dyes to assess metabolic activity (33, 96). In addition, genetic variants of cell lines can be produced and cultured for the purposes of biochemical or immunochemical studies. For example, a genetic variant of the UT-7 cell line transfected with the wild-type c-mpl thrombopoietin receptor gene or gene mutants was used to map the functional domains on the thrombopoietin receptor protein responsible for cellular proliferation and megakaryocyte differentiation (85). Similar work on the EPO receptor was done by Harris and colleagues using the murine myeloid cell line 32D transfected with full-length or truncated human EPO receptor (37). The UT-7 cell line and a murine erythroleukemia cell line, HCD57, were used to map the receptor binding domains of EPO (8). The HCD57 cells, which are susceptible to apoptosis during EPO withdrawal, have been used to study gene regulation of programmed cell death in erythroid cells (44). In vitro studies with the UT-7 cell line have elucidated the cytotoxic mechanism of human parvovirus B19 on erythroid cells that is induced through cellular apoprosis mediated by the nonstructural viral protein NS1 (64). Another human erythroleukemia cell line, TF-1, that expresses a truncated EPO receptor, was used to study transcription factor activation of erythroid cell growth and differentiation following EPO binding (17); retroviral transfection of this cell line with a fully functional EPO receptor restored normal transcription factor activation.

View larger version (113K): [in this window] [in a new window]   FIG. 4. Photomicrograph of "burst" human erythroid colonies forming in vitro in the presence of EPO and other growth factors. Reproduced from reference 65 with permission. Erythroid colony assays can be performed with blood or bone marrow-derived cells.

	ASSAYS USED TO MEASURE Abs AGAINST PROTEIN BIOLOGICALS

	ASSAYS FOR MEASURING Abs AGAINST RECOMBINANT EPO

Early reports of Ab-mediated PRCA and development of resistance to EPO increased interest in the potential immunogenicity of EPO and the induction of autoimmunity against endogenous EPO. Studies were carried out to address the immunogenicity of EPO in larger groups of patients with renal anemia and the occurrence of natural anti-EPO Abs in normal, healthy volunteers by different Ab assays. These studies are listed and described briefly in Table 4. These data reveal wide variance in the sensitivity of different assays, with RIP and bioassays used most often. Quantitative results with ELISA have not been described to a great extent in the literature, possibly because of poor sensitivity, specificity, or both. Generally, there appears to be excellent concordance between RIP and different bioassays for anti-EPO Abs. The BIAcore assay technology has only been used recently to measure anti-EPO Abs.

ELISA ELISAs were used to measure EPO binding Abs in two early reports of an epoetin-treated, hemodialysis patient who had become EPO resistant (67, 87): the 1997 report by Urra and colleagues also compared serum Ab titers in an Ab-positive patient to sera from 30 hemodialysis patients and 10 healthy control subjects. In these reports, titers were determined for the serum from the EPO-resistant patient with renal anemia and compared to fixed amounts of epoetin alfa and epoetin beta in an ELISA with a peroxide-labeled, anti-human secondary Ab for detection of EPO binding Abs (67, 87). A single negative control serum (1 patient) was used in the first study, while sera from the 10 hemodialysis patients and 30 normal patients served as negative controls for the second study. Test results were expressed in optical absorbance units. The PRCA serum, which neutralized EPO in a bioassay, reduced binding of the positive standard serum in the ELISA test by 42 to 75%, depending on the dilution of serum tested and which epoetin was used to coat wells. Sera from the 30 normal donors and 10 hemodialysis patients were negative for anti-EPO Abs.

An indirect ELISA was used by Weber and colleagues to demonstrate the presence of anti-EPO Abs in a patient with Ab-mediated PRCA who had received multiple recombinant EPOs (20, 94). Abs in serum samples collected during the period of EPO resistance were shown to recognize each of the three recombinant EPOs this patient received—epoetin alfa, epoetin beta, and darbepoetin alpha. Specificity of the assay was confirmed by homologous and heterologous displacement (i.e., competition) with the various recombinant proteins (20, 94).

Swanson and colleagues recently developed a bridging ELISA for detecting anti-EPO Abs (78). Sera from 8 of 13 patients with Ab-mediated PRCA, originally reported by Casadevall and colleagues (14), were analyzed by the bridging ELISA, RIP, the BIAcore 3000 immunoassay system, and a bioassay with a murine cell line transfected with the human EPO receptor (78). The sensitivity limits of these four assays for anti-EPO serum Abs were reported to be, in decreasing order, 10 ng of EPO/ml (RIP), 390 ng of EPO/ml (BIAcore), 600 ng of EPO/ml (ELISA), and 900 ng of EPO/ml (bioassay). The bridging ELISA detected anti-EPO Abs in six of the eight PRCA sera, while the other three assays successfully detected Abs in all eight sera tested. These investigators concluded that the two Ab-negative sera with the bridging ELISA represented false-negative results and, consequently, this assay was not used to quantify anti-EPO Abs.

RIP The first description of an RIP assay for measuring anti-EPO Abs was provided by Bergrem and colleagues in 1993 (6), who used a modified version of the RIP assay for serum EPO described by Egrie and colleagues in 1987 (24). This assay demonstrated the presence of circulating, anti-EPO binding Abs in an EPO-refractory patient with renal anemia at a high titer of 400 (arbitrary) radioimmunoassay units (RUs). Scatchard analysis calculated the plasma EPO-binding capacity to be 32 pmol of EPO/ml of plasma (100 IU of EPO/ml of plasma). The lower limit of detection was given as 10 RUs.

Prabhakar and colleagues used a similar RIP assay to quantitate EPO binding Abs in a hemodialysis patient who had become resistant to EPO (69). Four months after EPO treatment was discontinued, anti-EPO Abs were present at a serum titer of 1:50 (i.e., 50 IU of EPO bound per ml of serum) and decreased slowly to a titer of <1:10 by 24 months following cessation of EPO treatments. Of interest, this patient had disproportionately elevated serum EPO levels during the time when serum Abs were detected. The sensitivity limits of this RIP assay were not specified either in this study (69) or the original Egrie article (24), but estimates from the published data indicated that a titer of 1:50 or higher was meaningful (about two times above the background).

Casadevall and colleagues analyzed serum from a transfusion-dependent elderly patient with severe anemia by a modified RIP assay for anti-EPO Abs (13). Scatchard analysis with the RIP assay demonstrated a single class of high-affinity anti-EPO Abs in this patient's serum (dissociation constant, 430 ± 80 pM). An indirect ELISA demonstrated the presence of IgG Abs but not IgM Abs in this serum. The binding capacity of the patient's serum Abs was calculated to be about 2.7 IU of EPO/ml of serum. Thus, even relatively small amounts of serum Abs could apparently neutralize most of the circulating EPO in vivo. Serum Abs from this patient effectively neutralized the proliferation of EPO-dependent UT-7 cells in a bioassay.

Casadevall and colleagues later used RIP and bioassays to screen a cohort of 13 renal anemia patients who became EPO resistant during their course of treatment with one or more EPOs (14). The relative binding capacities of the patients' sera ranged from 4 to 86 IU of EPO/ml of serum. The lower limit of detection was calculated to be 200 mIU of EPO/ml of serum. All of these patients had EPO-neutralizing Abs in their serum, as determined by a bioassay. Thus, 100% concordance between the two assays was achieved with these sera.

BIAcore The BIAcore surface plasmon resonance assay system has proven useful for many biochemical and immunologic purposes, including epitope mapping, kinetic binding, Ab-ligand affinity measurements, and product purity analysis (Table 3). However, it has just recently been used to test for the presence of anti-EPO Abs in patients' sera. Results of the BIAcore analysis of 8 of 13 PRCA sera by Swanson (78) showed that this assay was equivalent to RIP and a bioassay for quantifying anti-EPO Abs (in micrograms per milliliter) in individual serum samples, thus demonstrating excellent concordance among assay methods. Ab isotype analysis by BIAcore qualitatively demonstrated the predominant presence of the IgG4 and IgG1 Ab subclasses in these patients' sera. The BIAcore assay also provided Ab dissociation data, which confirmed the specificity of Abs for epoetin alfa (78).

Bioassays In 1967, Krantz and colleagues characterized a serum factor that inhibited erythropoiesis in patients with EPO-resistant PRCA by using an in vitro culture system of autologous bone marrow cells (47-49). The inhibitor substance, shown to be serum IgG, halted new heme synthesis by cultured autologous bone marrow erythroid cells and produced Ab-dependent cellular cytotoxicity (lysis) of erythroid cells. In serum from one patient, this IgG factor bound to erythroblast nuclei (47).

In 1975, Peschle and colleagues first demonstrated the presence of neutralizing anti-EPO Abs in the serum of a renal anemia patient with PRCA with a polycythemic mouse model of erythropoiesis (68). These investigators coined the terms PRCA type A and type B to distinguish between red blood cell aplasia associated with serum inhibition of erythroid colony formation and red blood cell binding (type A) but not EPO neutralization, and serum neutralization of EPO without red blood cell binding (type B). The limit of sensitivity of the in vivo mouse assay was not determined in this study.

Casadevall and colleagues tested serum obtained from patients with confirmed Ab-mediated PRCA for the presence of EPO-neutralizing Abs (14). Using bone marrow cells from healthy donors, they showed that all PRCA serum samples effectively inhibited proliferation of bone marrow cultures in vitro, thus demonstrating 100% concordance between this bioassay and results with RIP. In this study, the EPO-dependent UT-7 cell line was used to further characterize neutralizing Abs obtained from one of the patients. Similar bioassays with autologous bone marrow cells and the UT-7 cell line were used earlier by Casadevall and colleagues to demonstrate the presence of EPO-neutralizing Abs in a patient with confirmed Ab-mediated PRCA, and the RIP assay was used to confirm the bioassay results (13).

	INTERPRETATION OF Ab TESTING RESULTS

 
Researchers have used a number of different immunologic assays to detect the presence of anti-EPO Abs in patients with Ab-mediated PRCA. Generally, Abs associated with Ab-mediated PRCA are high affinity in nature and recognize distinct epitopes on the protein portion of the EPO molecule (14). Although anti-EPO Abs can be detected by the different assay methods, there are no universal reagent standards for the various assays, and there is currently no single assay that is considered superior to the others. Furthermore, the assay most often used to measure anti-EPO Abs, the RIP assay, is used in various laboratory conditions and formats, making it difficult to directly compare results from different laboratories. A similar situation exists for ELISA, in that researchers use different reagents, incubation times, and enzyme substrates to develop the plates.

Tables 2, 3, and 4 show that there are large differences in the sensitivities and specificities of different assays. In the case of Ab-mediated PRCA, ELISA and RIP assays measure binding Abs that may or may not be correlated with neutralizing Abs, and the production of binding Abs does not always parallel the production of neutralizing Abs in amplitude or time to appearance. Data from one study indicated that RIP and EPO-neutralization assays are highly correlated (100%), but the study size was quite small (8 patients) (79).

The utility of the RIP assay for screening large numbers of patients was shown by Wu and colleagues (96), who found only three borderline Ab-positive patients and one low-titer Ab patient in a cohort of over 1,500 EPO-treated renal anemia patients. In this study, the RIP titers appeared to correspond to the severity of symptoms of PRCA; that is, there was no evident PRCA in the three borderline-positive patients and a generally mild and reversible EPO-refractory anemia in the 1 Ab-positive patient. The RIP assay can also be used to determine Ab binding affinities (14).

Results with the ELISA are less clear. In a controlled study, the ELISA detected low-affinity Abs in a relatively high percentage of EPO-treated patients (16). Since these low-affinity Abs did not correlate with any disease symptoms, the ELISA used in this study may have limited clinical utility as a screening assay for PRCA. In a later study, a bridging ELISA did not detect anti-EPO Abs in two of eight patients with confirmed Ab-mediated PRCA (78); this result may be related to the poor ability of at least some ELISA to detect lower-affinity, anti-EPO Abs.

Bioassays are essential for detecting neutralizing Abs or other factors that can functionally inhibit cytokine or growth factor activity and may be the most clinically relevant Ab assay for measuring neutralizing capability of Abs against EPO. However, neutralizing Ab assays may not be suitable for rapid screening of large patient groups, because of their lack of sensitivity and length of time required to culture erythroid cells (7 to 14 days). However, the use of continuously growing EPO-dependent cell lines will overcome this problem.

The BIAcore immunoassay has been used successfully to measure high-affinity, anti-EPO Abs in the sera of patients with Ab-mediated PRCA, and these Abs appeared to correlate well with the presence of EPO-neutralizing Abs. The ability of the BIAcore assay to readily determine Ab concentration and binding affinities may ultimately prove useful in differentiating low-affinity from high-affinity Abs and their relative contribution to the development of Ab-mediated PRCA.

Currently, there are no recommendations for which assay(s) should be used as the standard method for screening patients for anti-EPO Abs or which should be used as the definitive diagnostic method. To date, diagnosis of Ab-mediated PRCA remains dependent on results from bone marrow examination plus demonstration of anti-EPO Abs by BIAcore, RIP, or ELISA. The question of which patients should be tested remains unresolved, because there is no published case definition of Ab-mediated PRCA. An association between the presence of baseline anti-EPO Abs and increased risk for developing Ab-mediated PRCA has not been demonstrated.

Demographically, patients receiving human serum albumin-free formulations of the Eprex brand of epoetin alfa by the SC route appear to have the greatest probability of developing Ab-mediated PRCA (28). However, the relative risks for developing Ab-mediated PRCA with the use of any of the commercially available EPO preparations (e.g., epoetin beta [NeoRecormon], epoetin alfa [Epogen], or darbepoetin alfa [Aranesp]) are very low. Most published studies present Ab data on patients already diagnosed with PRCA or on patients in whom the clinical signs of PRCA are emerging. Currently, there are no longitudinal data for Ab responses before and during the course of EPO therapy in anemia patients. Therefore, a clear need exists for standardization of Ab testing in patients with developing or full-blown Ab-mediated PRCA.

Other issues that remain to be addressed include which patients should be tested for Abs and when, how often they should be tested, which assay results are the most clinically relevant to the development of Ab-mediated PRCA and its resolution, and what factors impact cost and reimbursement. Reaching consensus on these issues will facilitate effective diagnosis and treatment of patients with this serious disorder.

	FOOTNOTES

 
* Corresponding author. Mailing address: Division of Immunobiology, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Phone: 44-1707-641-000. Fax: 44-1707-646-730. E-mail: rthorpe{at}nibsc.ac.uk.

	REFERENCES

Top Introduction References

 

1. Amgen, Inc. Amgen statement on pure red cell aplasia. [Online.] www.amgen.com/clinicians/prca.html.
2. Arranz, O., J. Ara, R. Rodriguez, L. Quinto, J. Font, E. Mirapeix, and A. Darnell. 2001. Comparison of anti-PR3 capture and anti-PR3 direct ELISA for detection of antineutrophil cytoplasmic antibodies (ANCA) in long-term clinical follow-up of PR3-ANCA-associated vasculitis patients. Clin. Nephrol. 56:295-301.[Medline]
3. Baron, E. J., D. E. Weber, and L. G. Weide. 1996. Lack of agreement among two commercial enzyme-linked immunosorbent antibody assays and a conventional immunofluorescence-based method for detecting islet cell autoantibodies. Clin. Diagn. Lab. Immunol. 3:429-431.[Abstract]
4. Basser, R. L., E. O'Flaherty, M. Green, M. Edmonds, J. Nichol, D. M. Menchaca, B. Cohen, and C. G. Begley. 2002. Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor. Blood 99:2599-2602.[Abstract/Free Full Text]
5. Bendtzen, K., M. B. Hansen, C. Ross, and M. Svenson. 2000. Detection of autoantibodies to cytokines. Mol. Biotechnol. 14:251-261.[CrossRef][Medline]
6. Bergrem, H., B. G. Danielson, K. U. Eckardt, A. Kurtz, and M. Stridsberg. 1993. A case of antierythropoietin antibodies following recombinant human erythropoietin treatment, p. 265-275. In P. Sciqalla (ed.), Erythropoietin: molecular physiology and clinical application. Marcel Dekker, New York, N.Y.
7. Black, J. B., T. F. Schwarz, J. L. Patton, K. Kite-Powell, P. E. Pellett, S. Wiersbitzky, R. Bruns, C. Muller, G. Jager, and J. A. Stewart. 1996. Evaluation of immunoassays for detection of antibodies to human herpesvirus 7. Clin. Diagn. Lab. Immunol. 3:79-83.[Abstract]
8. Boissel, J. P., W. R. Lee, S. R. Presnell, F. E. Cohen, and H. F. Bunn. 1993. Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure. J. Biol. Chem. 268:15983-15993.[Abstract/Free Full Text]
9. Bommer, J., and J. Wagner. 2003. Safety of Aranesp (darbepoetin alfa) in dialysis patients with renal anaemia—results of a German mulitcentre study in 1502 patients. Nephrol. Dial. Transplant. 18(Suppl. 4):165.
10. Bonfield, T. L., M. S. Kavuru, and M. J. Thomassen. 2002. Anti-GM-CSF titer predicts response to GM-CSF therapy in pulmonary alveolar proteinosis. Clin. Immunol. 105:342-350.[CrossRef][Medline]
11. Buchalter, M. B., G. Suntharalingam, I. Jennings, C. Hart, R. J. Luddington, R. Chakraverty, S. K. Jacobson, P. L. Weissberg, and T. P. Baglin. 1992. Streptokinase resistance: when might streptokinase administration be ineffective? Br. Heart J. 68:449-453.[Abstract]
12. Casadevall, N. 2002. Antibodies against rHuEPO: native and recombinant. Nephrol. Dial. Transplant. 17(Suppl. 5):42-47.[Abstract]
13. Casadevall, N., E. Dupuy, P. Molho-Sabatier, G. Tobelem, B. Varet, and P. Mayeux. 1996. Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N. Engl. J. Med. 334:630-633.[Free Full Text]
14. Casadevall, N., J. Nataf, B. Viron, A. Kolta, J. J. Kiladjian, P. Martin-Dupont, P. Michaud, T. Papo, V. Ugo, I. Teyssandier, B. Varet, and P. Mayeux. 2002. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N. Engl. J. Med. 346:469-475.[Abstract/Free Full Text]
15. Cassano, W. F. 1989. Murine monoclonal anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining. J. Immunol. Methods 117:169-174.[CrossRef][Medline]
16. Castelli, G., A. Famularo, C. Semino, A. M. Machi, A. Ceci, G. Cannella, and G. Melioli. 2000. Detection of anti-erythropoietin antibodies in haemodialysis patients treated with recombinant human-erythropoietin. Pharmacol. Res. 41:313-318.[CrossRef][Medline]
17. Chretien, S., P. Varlet, F. Verdier, S. Gobert, J. P. Cartron, S. Gisselbrecht, P. Mayeux, and C. Lacombe. 1996. Erythropoietin-induced erythroid differentiation of the human erythroleukemia cell line TF-1 correlates with impaired STAT5 activation. EMBO J. 15:4174-4181.[Medline]
18. Conlon, K. C., W. J. Urba, J. W. Smith II, R. G. Steis, D. L. Longo, and J. W. Clark. 1990. Exacerbation of symptoms of autoimmune disease in patients receiving alpha-interferon therapy. Cancer 65:2237-2242.[CrossRef][Medline]
19. Cotes, P. M., and D. R. Bangham. 1966. The international reference preparation of erythropoietin. Bull. W. H. O. 35:751-760.[Medline]
20. Crombet, T., O. Torres, V. Rodriguez, A. Menendez, A. Stevenson, M. Ramos, F. Torres, R. Figueredo, I. Veitia, N. Iznaga, R. Perez, and A. Lage. 2001. Phase I clinical evaluation of a neutralizing monoclonal antibody against epidermal growth factor receptor in advanced brain tumor patients: preliminary study. Hybridoma 20:131-136.[CrossRef][Medline]
21. Crooks, S. R., G. A. Baxter, M. C. O'Connor, and C. T. Elliot. 1998. Immunobiosensor—an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs. Analyst 123:2755-2757.[CrossRef][Medline]
22. Dai, Y., E. M. Schwarz, D. Gu, W. W. Zhang, N. Sarvetnick, and I. M. Verma. 1995. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc. Natl. Acad. Sci. USA 92:1401-1405.[Abstract/Free Full Text]
23. Dayan, P., F. Ahmad, J. Urtecho, M. Novick, P. Dixon, D. Levine, and S. Miller. 2002. Test characteristics of the respiratory syncytial virus enzyme-linked immunoabsorbent assay in febrile infants < or = 60 days of age. Clin. Pediatr. (Philadelphia) 41:415-418.[Abstract/Free Full Text]
24. Egrie, J. C., P. M. Cotes, J. Lane, R. E. Gaines Das, and R. C. Tam. 1987. Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J. Immunol. Methods 99:235-241.[CrossRef][Medline]
25. Field, P. R., J. L. Mitchell, A. Santiago, D. J. Dickeson, S. W. Chan, D. W. Ho, A. M. Murphy, A. J. Cuzzubbo, and P. L. Devine. 2000. Comparison of a commercial enzyme-linked immunosorbent assay with immunofluorescence and complement fixation tests for detection of Coxiella burnetii (Q fever) immunoglobulin M. J. Clin. Microbiol. 38:1645-1647.[Abstract/Free Full Text]
26. Garotta, G., L. Ozmen, M. Fountoulakis, Z. Dembic, A. P. van Loon, and D. Stuber. 1990. Human interferon-gamma receptor. Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins. J. Biol. Chem. 265:6908-6915.[Abstract/Free Full Text]
27. Ge, Y., S. Powell, M. Van Roey, and J. G. McArthur. 2001. Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX. Blood 97:3733-3737.[Abstract/Free Full Text]
28. Gershon, S. K., H. Luksenburg, T. R. Cote, and M. M. Braun. 2002. Pure red-cell aplasia and recombinant erythropoietin. N. Engl. J. Med. 346:1584-1586.[Free Full Text]
29. Giovanella, L., and L. Ceriani. 2002. High-sensitivity human thyroglobulin (hTG) immunoradiometric assay in the follow-up of patients with differentiated thyroid cancer. Clin. Chem. Lab. Med. 40:480-484.[CrossRef][Medline]
30. Griffiths, P. D., and H. O. Kangro. 1984. A user's guide to the indirect solid-phase radioimmunoassay for the detection of cytomegalovirus-specific IgM antibodies. J. Virol. Methods 8:271-282.[CrossRef][Medline]
31. Grossberg, S. E., Y. Kawade, M. Kohase, H. Yokoyama, and N. Finter. 2001. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems. J. Interferon Cytokine Res. 21:729-742.
32. Gussin, H. A., K. L. Russo, and M. Teodorescu. 2000. Effect of circulating immune complexes on the binding of rheumatoid factor to histones. Ann. Rheum. Dis. 59:351-358.[Abstract/Free Full Text]
33. Hammerling, U., R. Kroon, T. Wilhelmsen, and L. Sjodin. 1996. In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity. J. Pharm. Biomed. Anal. 14:1455-1469.[CrossRef][Medline]
34. Hansen, K. 1994. Lyme neuroborreliosis: improvements of the laboratory diagnosis and a survey of epidemiological and clinical features in Denmark 1985-1990. Acta Neurol. Scand. Suppl. 151:1-44.[Medline]
35. Hansen, M. B., C. Ross, and K. Berg. 1990. A sensitive antiviral neutralization bioassay for measuring antibodies to interferons. J. Immunol. Methods. 127:241-248.[CrossRef][Medline]
36. Hansen, P. J. Analysis of proteins by immunoprecipitation. [Online.] http://www.dps.ufl.edu/hansen/protocols/imp98.prt.htm.
37. Harris, K. W., X. J. Hu, S. Schultz, M. O. Arcasoy, B. G. Forget, and N. Clare. 1998. The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells. Blood 92:1219-1224.[Abstract/Free Full Text]
38. Hellmich, B., E. Csernok, H. Schatz, W. L. Gross, and A. Schnabel. 2002. Autoantibodies against granulocyte colony-stimulating factor in Felty's syndrome and neutropenic systemic lupus erythematosus. Arthritis Rheum. 46:2384-2391.[CrossRef][Medline]
39. Hennes, U., W. Jucker, E. A. Fischer, T. Krummenacher, A. V. Palleroni, P. W. Trown, S. Linder-Ciccolunghi, and M. Rainisio. 1987. The detection of antibodies to recombinant interferon alfa-2a in human serum. J. Biol. Stand. 15:231-244.[CrossRef][Medline]
40. Hennig, C., L. Rink, U. Fagin, W. J. Jabs, and H. Kirchner. 2000. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J. Immunol. Methods 235:71-80.[CrossRef][Medline]
41. Hjelm Skog, A. L., M. Wadhwa, M. Hassan, B. Gharizadeh, C. Bird, P. Ragnhammar, R. Thorpe, and H. Mellstedt. 2001. Alteration of interleukin 2 (IL-2) pharmacokinetics and function by IL-2 antibodies induced after treatment of colorectal carcinoma patients with a combination of monoclonal antibody 17-1A, granulocyte macrophage colony-stimulating factor, and IL-2. Clin. Cancer Res. 7:1163-1170.[Abstract/Free Full Text]
42. Itri, L. M., M. Campion, R. A. Dennin, A. V. Palleroni, J. U. Gutterman, J. E. Groopman, and P. W. Trown. 1987. Incidence and clinical significance of neutralizing antibodies in patients receiving recombinant interferon alfa-2a by intramuscular injection. Cancer 59:668-674.[CrossRef][Medline]
43. Jacobs, K., C. Shoemaker, R. Rudersdorf, S. D. Neill, R. J. Kaufman, A. Mufson, J. Seehra, S. S. Jones, R. Hewick, E. F. Fritsch, et al. 1985. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313:806-810.[CrossRef][Medline]
44. Jacobs-Helber, S. M., A. Wickrema, M. J. Birrer, and S. T. Sawyer. 1998. AP1 regulation of proliferation and initiation of apoprosis in erythropoietin-dependent erythroid cells. Mol. Cell. Biol. 18:3699-3707.[Abstract/Free Full Text]
45. Johnson & Johnson. Summary of PRCA case reports. [Online.] http://www.jnj.com/news/jnj_news/1021024_095632.htm.
46. Koren, E., L. A. Zuckerman, and A. R. Mire-Sluis. 2002. Immune responses to therapeutic proteins in humans—clinical significance, assessment and prediction. Curr. Pharm. Biotechnol. 3:349-360.[CrossRef][Medline]
47. Krantz, S. B., and V. Kao. 1967. Studies on red cell aplasia. I. Demonstration of a plasma inhibitor to heme synthesis and an antibody to erythroblast nuclei. Proc. Natl. Acad. Sci. USA 58:493-500.
48. Krantz, S. B., and V. Kao. 1969. Studies on red cell aplasia. II. Report of a second patient with an antibody to erythroblast nuclei and a remission after immunosuppressive therapy. Blood 34:1-13.
49. Krantz, S. B., W. H. Moore, and S. D. Zaentz. 1973. Studies on red cell aplasia. V. Presence of erythroblast cytotoxicity in G-globulin fraction of plasma. J. Clin. Investig. 52:324-336.
50. Kuroki, M., Y. Matsumoto, F. Arakawa, M. Haruno, M. Murakami, M. Kuwahara, H. Ozaki, T. Senba, and Y. Matsuoka. 1995. Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer. J. Immunol. Methods 180:81-91.[CrossRef][Medline]
51. Lackmann, M., T. Bucci, R. J. Mann, L. A. Kravets, E. Viney, F. Smith, R. L. Moritz, W. Carter, R. J. Simpson, N. A. Nicola, K. Mackwell, E. C. Nice, A. F. Wilks, and A. W. Boyd. 1996. Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach. Proc. Natl. Acad. Sci. USA 93:2523-2527.[Abstract/Free Full Text]
52. Lacombe, C., N. Casadevall, O. Muller, and B. Varet. 1984. Erythroid progenitors in adult chronic pure red cell aplasia: relationship of in vitro erythroid colonies to therapeutic response. Blood 64:71-77.[Abstract/Free Full Text]
53. Leon, A. D., M. Tellez Araiza, J. Arellano Garcia, and E. Martinez-Cordero. 2002. Interference by rheumatoid factor activity in the detection of antiavian antibodies in pigeon breeders disease. Clin. Exp. Med. 2:59-67.[CrossRef][Medline]
54. Li, J., C. Yang, Y. Xia, A. Bertino, J. Glaspy, M. Roberts, and D. J. Kuter. 2001. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98:3241-3248.[Abstract/Free Full Text]
55. Liebe, V., M. Bruckmann, K. G. Fischer, K. K. Haase, M. Borggrefe, and G. Huhle. 2002. Biological relevance of anti-recombinant hirudin antibodies—results from in vitro and in vivo studies. Semin. Thromb. Hemost. 28:483-490.[CrossRef][Medline]
56. Lin, F. K., S. Suggs, C. H. Lin, J. K. Browne, R. Smalling, J. C. Egrie, K. K. Chen, G. M. Fox, F. Martin, Z. Stabinsky, et al. 1985. Cloning and expression of the human erythropoietin gene. Proc. Natl. Acad. Sci. USA82:7580-7584.[Abstract/Free Full Text]
57. Linardaki, G. D., K. A. Boki, A. Fertakis, and A. G. Tzioufas. 1999. Pure red cell aplasia as presentation of systemic lupus erythematosus: antibodies to erythropoietin. Scand. J. Rheumatol. 28:189-191.[CrossRef][Medline]
58. Lynch, M., B. L. Pentecost, W. A. Littler, and R. A. Stockley. 1996. The distribution of antibodies to streptokinase. Postgrad. Med. J. 72:290-292.[Abstract]
59. Meager, A., M. Wadhwa, P. Dilger, C. Bird, R. Thorpe, J. Newsom-Davis, and N. Willcox. 2003. Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis. Clin. Exp. Immunol. 132:128-136.[CrossRef][Medline]
60. Millett, B., A. M. Sullivan, M. Morimoto, and G. Parsons. 1994. A third generation immunoassay for tumor necrosis factor alpha. BioTechniques 17:1166-1171.[Medline]
61. Mimms, L., A. Goetze, S. Swanson, M. Floreani, B. Edwards, J. Macioszek, G. Okasinski, and W. Kiang. 1989. Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg. J. Virol. Methods 25:211-231.[CrossRef][Medline]
62. Mobini, R., M. Fu, G. Wallukat, Y. Magnusson, A. Hjalmarson, and J. Hoebeke. 2000. A monoclonal antibody directed against an autoimmune epitope on the human beta1-adrenergic receptor recognized in idiopathic dilated cardiomyopathy. Hybridoma 19:135-142.[CrossRef][Medline]
63. Mobini, R., A. Staudt, S. B. Felix, G. Baumann, G. Wallukat, J. Deinum, H. Svensson, A. Hjalmarson, and M. Fu. 2003. Hemodynamic improvement and removal of autoantibodies against beta(1)-adrenergic receptor by immunoadsorption therapy in dilated cardiomyopathy. J. Autoimmun. 20:345-350.[CrossRef][Medline]
64. Moffatt, S., N. Yaegashi, K. Tada, N. Tanaka, and K. Sugamura. 1998. Human parvovirus B19 nonstructural (NS1) protein induces apoprosis in erythroid lineage cells. J. Virol. 72:3018-3028.[Abstract/Free Full Text]
65. Moore, M. Developmental hematopoiesis. [Online.] http://www.mskcc.org/mskcc/html/10645.cfm.
66. Nice, E., J. Layton, L. Fabri, U. Hellman, A. Engstrom, B. Persson, and A. W. Burgess. 1993. Mapping of the antibody- and receptor-binding domains of granulocyte colony-stimulating factor using an optical biosensor. Comparison with enzyme-linked immunosorbent assay competition studies. J. Chromatogr. 646:159-168.
67. Peces, R., M. de la Torre, R. Alcazar, and J. M. Urra. 1996. Antibodies against recombinant human erythropoietin in a patient with erythropoietin-resistant anemia. N. Engl. J. Med. 335:523-524.[Free Full Text]
68. Peschle, C., A. M. Marmont, G. Marone, A. Genovese, G. F. Sasso, and M. Condorelli. 1975. Pure red cell aplasia: studies on an IgG serum inhibitor neutralizing erythropoietin. Br. J. Haematol. 30:411-417.[Medline]
69. Prabhakar, S. S., and T. Muhlfelder. 1997. Antibodies to recombinant human erythropoietin causing pure red cell aplasia. Clin. Nephrol. 47:331-335.[Medline]
70. Rasooly, A. 2001. Surface plasmon resonance analysis of staphylococcal enterotoxin B in food. J. Food Prot. 64:37-43.[Medline]
71. Ritter, G., L. S. Cohen, C. Williams, Jr., E. C. Richards, L. J. Old, and S. Welt. 2001. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33. Cancer Res. 61:6851-6859.[Abstract/Free Full Text]
72. Rosenberg, A. S. 2003. Immunogenicity of biological therapeutics: a hierarchy of concerns. Dev. Biol. (Basel) 112:15-21.
73. Schellekens, H. 2002. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin. Ther. 24:1720-1740.[CrossRef][Medline]
74. Suter, M., and J. E. Butler. 1986. The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies. Immunol. Lett. 13:313-316.
75. Svenson, M., M. B. Hansen, C. Ross, M. Diamant, K. Rieneck, H. Nielsen, and K. Bendtzen. 1998. Antibody to granulocyte-macrophage colony-stimulating factor is a dominant anti-cytokine activity in human IgG preparations. Blood 91:2054-2061.[Abstract/Free Full Text]
76. Svojanovsky, S. R., K. L. Egodage, J. Wu, M. Slavik, and G. S. Wilson. 1999. High sensitivity ELISA determination of taxol in various human biological fluids. J. Pharm. Biomed. Anal. 20:549-555.[CrossRef][Medline]
77. Swanson, S. J. 2004. Detection of baseline anti-erythropoietin antibodies in patients with chronic kidney disease, using the BIA core immunoassay. Presented at the 41st Congress of the European Renal Association and European Dialysis and Transplant Association, Lisbon, Portugal, July 17, 2003.
78. Swanson, S. J. 2003. New technologies for the detection of antibodies to therapeutic proteins, p. 127-133. In A. R. Mire-Sluis (ed.), Immunogenicity of therapeutic biological products, vol. 112. Karger, Basel, Switzerland.
79. Swanson, S. J., J. Ferbas, P. Mayeux, and N. Casadevall. 2004. Evaluation of methods to detect and characterize antibodies against recombinant human erythropoietin. Nephron. Clin Pract. 96:c88-c95.[CrossRef][Medline]
80. Swanson, S. J., S. J. Jacobs, D. Mytych, C. Shah, S. R. Indelicato, and R. W. Bordens. 1999. Applications for the new electrochemiluminescent (ECL) and biosensor technologies. Dev. Biol. Stand. 97:135-147.[Medline]
81. Swedler, W., J. Wallman, C. J. Froelich, and M. Teodorescu. 1997. Routine measurement of IgM, IgG, and IgA rheumatoid factors: high sensitivity, specificity, and predictive value for rheumatoid arthritis. J. Rheumatol. 24:1037-1044.[Medline]
82. Swissmedic. 2003. Nouvel effet indésirable grave mais rare de l’érythropoïétine recombinante. Swissmedic medical update letter. [Online.] http://www.swissmedic.ch/en/laien/overall.asp?lang=2&theme=0.00062.00004.00001&theme_id=810&news_id=2588&page=1.
83. Sytkowski, A. J., and K. A. Donahue. 1987. Immunochemical studies of human erythropoietin using site-specific anti-peptide antibodies. Identification of a functional domain. J. Biol. Chem. 262:1161-1165.[Abstract/Free Full Text]
84. Takacs, M. A., S. J. Jacobs, R. M. Bordens, and S. J. Swanson. 1999. Detection and characterization of antibodies to PEG-IFN-2b using surface plasmon resonance. J. Interferon Cytokine Res. 19:781-789.[CrossRef][Medline]
85. Takatoku, M., M. Kametaka, R. Shimizu, Y. Miura, and N. Komatsu. 1997. Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells. J. Biol. Chem. 272:7259-7263.[Abstract/Free Full Text]
86. Ullenhag, G., C. Bird, P. Ragnhammar, J. E. Frodin, K. Strigard, O. I. A., R. Thorpe, H. Mellstedt, and M. Wadhwa. 2001. Incidence of GM-CSF antibodies in cancer patients receiving GM-CSF for immunostimulation. Clin. Immunol. 99:65-74.[CrossRef][Medline]
87. Urra, J. M., M. de la Torre, R. Alcazar, and R. Peces. 1997. Rapid method for detection of anti-recombinant human erythropoietin antibodies as a new form of erythropoietin resistance. Clin. Chem. 43:848-849.[Free Full Text]
88. VanCott, T. C., L. D. Loomis, R. R. Redfield, and D. L. Birx. 1992. Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1. J. Immunol. Methods 146:163-176.[CrossRef][Medline]
89. Van Der Geld, Y. M., P. C. Limburg, and C. G. Kallenberg. 1999. Characterization of monoclonal antibodies to proteinase 3 (PR3) as candidate tools for epitope mapping of human anti-PR3 autoantibodies. Clin. Exp. Immunol. 118:487-496.[CrossRef][Medline]
90. Vyse, A. J., W. A. Knowles, B. J. Cohen, and D. W. Brown. 1997. Detection of IgG antibody to Epstein-Barr virus viral capsid antigen in saliva by antibody capture radioimmunoassay. J. Virol. Methods 63:93-101.[CrossRef][Medline]
91. Wadhwa, M., C. Bird, P. Dilger, R. Gaines-Das, and R. Thorpe. 2003. Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals. J. Immunol. Methods 278:1-17.[CrossRef][Medline]
92. Wadhwa, M., C. Bird, J. Fagerberg, R. Gaines-Das, P. Ragnhammar, H. Mellstedt, and R. Thorpe. 1996. Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy. Clin. Exp. Immunol. 104:351-358.[CrossRef][Medline]
93. Wadhwa, M., A. L. Skog, C. Bird, P. Ragnhammar, M. Lilljefors, R. Gaines-Das, H. Mellstedt, and R. Thorpe. 1999. Immunogenicity of granulocyte-macrophage colony-stimulating factor (GM-CSF) products in patients undergoing combination therapy with GM-CSF. Clin. Cancer Res. 5:1353-1361.[Abstract/Free Full Text]
94. Weber, G., J. Gross, A. Kromminga, H. H. Loew, and K. U. Eckardt. 2002. Allergic skin and systemic reactions in a patient with pure red cell aplasia and anti-erythropoietin antibodies challenged with different epoetins. J. Am. Soc. Nephrol. 13:2381-2383.[Abstract/Free Full Text]
95. Wong, R. L., D. Mytych, S. Jacobs, R. Bordens, and S. J. Swanson. 1997. Validation parameters for a novel biosensor assay which simultaneously measures serum concentrations of a humanized monoclonal antibody and detects induced antibodies. J. Immunol. Methods 209:1-15.[CrossRef][Medline]
96. Wu, G., B. Nathoo, D. Mendelssohn, S. Pandeya, and P. Tam. 2003. Antibody to erythropoietin surveillance study in 5 Ontario renal centres. Nephrol. Dial. Transplant. 18(Suppl. 4):162.
97. Zang, Y. C., D. Yang, J. Hong, M. V. Tejada-Simon, V. M. Rivera, and J. Z. Zhang. 2000. Immunoregulation and blocking antibodies induced by inter-feron beta treatment in MS. Neurology 55:397-404.[Abstract/Free Full Text]
98. Zhong, D., E. L. Saenko, M. Shima, M. Felch, and D. Scandella. 1998. Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood 92:136-142.[Abstract/Free Full Text]
99. Zhu, G., B. Yang, and R. N. Jennings. 2000. Quantitation of basic fibroblast growth factor by immunoassay using BIAcore 2000. J. Pharm. Biomed. Anal. 24:281-290.[CrossRef][Medline]

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thorpe, R. Articles by Swanson, S. J Search for Related Content PubMed PubMed Citation Articles by Thorpe, R. Articles by Swanson, S. J