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1.  Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia 
Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin.
PMCID: PMC3535764  PMID: 23114696
2.  Immunogenicity of panitumumab in combination chemotherapy clinical trials 
Panitumumab is a fully human antibody against the epidermal growth factor receptor that is indicated for the treatment of metastatic colorectal cancer (mCRC) after disease progression on standard chemotherapy. The purpose of this analysis was to examine the immunogenicity of panitumumab and to evaluate the effect of anti-panitumumab antibodies on pharmacokinetic and safety profiles in patients with mCRC receiving panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapies.
Three validated assays (two screening immunoassays and a neutralizing antibody bioassay) were used to detect the presence of anti-panitumumab antibodies in serum samples collected from patients enrolled in four panitumumab combination chemotherapy clinical trials. The impact of anti-panitumumab antibodies on pharmacokinetic and safety profiles was analyzed using population pharmacokinetic analysis and descriptive statistics, respectively.
Of 1124 patients treated with panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapy with postbaseline samples available for testing, 20 (1.8%) patients developed binding antibodies and 2 (0.2%) developed neutralizing antibodies. The incidence of anti-panitumumab antibodies was similar in patients with tumors expressing wild-type or mutant KRAS and in patients receiving oxaliplatin- or irinotecan-based chemotherapies. No evidence of an altered pharmacokinetic or safety profile was found in patients who tested positive for anti-panitumumab antibodies.
The immunogenicity of panitumumab in the combination chemotherapy setting was infrequent and similar to the immunogenicity observed in the monotherapy setting. Panitumumab immunogenicity did not appear to alter pharmacokinetic or safety profiles. This low rate of immunogenicity may be attributed to the fully human nature of panitumumab.
Trial registration NCT00339183 (study 20050181), NCT00411450 (study 20060277), NCT00332163 (study 20050184), and NCT00364013 (study 20050203).
PMCID: PMC3231982  PMID: 22070868
3.  A Step-wise Approach for Transfer of Immunogenicity Assays during Clinical Drug Development 
The AAPS Journal  2009;11(3):526-534.
We designed a three-step statistical approach to transfer bioanalytical assays (ELISA and Biacore) which evaluates the (1) average equivalence between the two labs (2) concordance in individual sample results between the two labs, and (3) long-term stability of assay performance. Each experimental design evaluated the contribution of four critical variables to the overall variability. Two lots of each variable were examined in a controlled experiment. The variables tested for ELISA were analyst, plate washer, biotinylated-therapeutic protein, and streptavidin–horseradish peroxidase; and for Biacore were analyst, instrument, chip lot, and conjugation chemistry reagent lots. Equivalence in the mean signal to noise (S/N) or mean relative units (RU) between the two labs was established through statistical evaluation of the assay performance characteristics across multiple assay variables. Concordance between the two labs in the individual sample results was subsequently verified both quantitatively and qualitatively. The long-term maintenance of assay stability was monitored by performance testing of a predefined set of samples which were prepared in sufficient quantities to last several years. The process of method validation for biomarker testing in clinical trials is to analyze the variability of the assay performance. However, different factors contribute to this variability and need to be evaluated when the method is transferred to another site/lab. Lack of understanding the critical variables can potentially result in unexpected problems and delays. The three-step statistical approach of assay transfer provides a robust process for transferring complex biological assays.
PMCID: PMC2758123  PMID: 19626442
biacore; bioanalytical; concordance; ELISA; method transfer; robustness

Results 1-3 (3)