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Measuring carbonic anhydrase IX as a hypoxia biomarker: differences in concentrations in serum and plasma using a commercial enzyme-linked immunosorbent assay due to influences of metal ions.

Wind TC, Messenger MP, Thompson D, Selby PJ, Banks RE - Ann. Clin. Biochem. (2011)

Bottom Line: This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay.These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid.This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

ABSTRACT

Background: There is increasing interest in measuring the soluble forms of carbonic anhydrase IX (CA IX) in blood as a marker of hypoxia for prognostic purposes or for predictive use in therapeutic trials in various cancers. Following our initial observations of marked differences in the measured concentrations of CA IX in EDTA plasma versus serum, we sought to investigate these further in order to determine their effects on results in published studies and to ensure accurate measurement in future studies.

Methods: Serum and EDTA plasma samples from healthy controls and patients with renal cancer were used in the validation of two commercially available enzyme-linked immunosorbent assays (ELISAs) for CA IX with examination of recovery, parallelism and specificity and comparison of paired plasma and serum.

Results: Successful validation of one of the ELISAs was not achieved with particular problems with parallelism and marked differences in measured CA IX concentrations between EDTA plasma and serum. This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay. The other commercially available ELISA examined was successfully validated and showed no difference in CA IX between EDTA plasma and serum.

Conclusions: These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid. This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

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Related in: MedlinePlus

Assessment of parallelism in the (a) Siemens and (b) R&D ELISAs for EDTA plasma and serum samples (open symbols = serum; closed symbols = plasma) by comparing the back-calculated concentrations against serial doubling dilutions of the samples. The arrow in each case indicates the normal working dilution used in the assay. ELISA, enzyme-linked immunosorbent assay
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ACB-10-240F2: Assessment of parallelism in the (a) Siemens and (b) R&D ELISAs for EDTA plasma and serum samples (open symbols = serum; closed symbols = plasma) by comparing the back-calculated concentrations against serial doubling dilutions of the samples. The arrow in each case indicates the normal working dilution used in the assay. ELISA, enzyme-linked immunosorbent assay

Mentions: Intra-and inter-assay precision was acceptable for both assays at <10% with the exception of the lowest concentration controls on the Siemens assay, which exhibited CVs of 14.5% and 18.6%, respectively (Table 1). Generally for both assays the SD of the duplicates for any sample was <5% of the mean result. The criteria for assessing parallelism are described above in the Data analysis section. Using these criteria, the three samples initially tested on the Siemens assay failed to dilute out in parallel (Figure 2a). When one of these plus a further two plasma samples were tested in the presence of excess EDTA, this was still the case with one of the three passing. For the R&D assay, one sample had extremely low concentrations of CA IX and was unsuitable but the remaining two serum and three EDTA plasma samples all passed on parallelism, three of which were among the samples analysed on the Siemens assay (Figure 2b).


Measuring carbonic anhydrase IX as a hypoxia biomarker: differences in concentrations in serum and plasma using a commercial enzyme-linked immunosorbent assay due to influences of metal ions.

Wind TC, Messenger MP, Thompson D, Selby PJ, Banks RE - Ann. Clin. Biochem. (2011)

Assessment of parallelism in the (a) Siemens and (b) R&D ELISAs for EDTA plasma and serum samples (open symbols = serum; closed symbols = plasma) by comparing the back-calculated concentrations against serial doubling dilutions of the samples. The arrow in each case indicates the normal working dilution used in the assay. ELISA, enzyme-linked immunosorbent assay
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104817&req=5

ACB-10-240F2: Assessment of parallelism in the (a) Siemens and (b) R&D ELISAs for EDTA plasma and serum samples (open symbols = serum; closed symbols = plasma) by comparing the back-calculated concentrations against serial doubling dilutions of the samples. The arrow in each case indicates the normal working dilution used in the assay. ELISA, enzyme-linked immunosorbent assay
Mentions: Intra-and inter-assay precision was acceptable for both assays at <10% with the exception of the lowest concentration controls on the Siemens assay, which exhibited CVs of 14.5% and 18.6%, respectively (Table 1). Generally for both assays the SD of the duplicates for any sample was <5% of the mean result. The criteria for assessing parallelism are described above in the Data analysis section. Using these criteria, the three samples initially tested on the Siemens assay failed to dilute out in parallel (Figure 2a). When one of these plus a further two plasma samples were tested in the presence of excess EDTA, this was still the case with one of the three passing. For the R&D assay, one sample had extremely low concentrations of CA IX and was unsuitable but the remaining two serum and three EDTA plasma samples all passed on parallelism, three of which were among the samples analysed on the Siemens assay (Figure 2b).

Bottom Line: This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay.These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid.This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

ABSTRACT

Background: There is increasing interest in measuring the soluble forms of carbonic anhydrase IX (CA IX) in blood as a marker of hypoxia for prognostic purposes or for predictive use in therapeutic trials in various cancers. Following our initial observations of marked differences in the measured concentrations of CA IX in EDTA plasma versus serum, we sought to investigate these further in order to determine their effects on results in published studies and to ensure accurate measurement in future studies.

Methods: Serum and EDTA plasma samples from healthy controls and patients with renal cancer were used in the validation of two commercially available enzyme-linked immunosorbent assays (ELISAs) for CA IX with examination of recovery, parallelism and specificity and comparison of paired plasma and serum.

Results: Successful validation of one of the ELISAs was not achieved with particular problems with parallelism and marked differences in measured CA IX concentrations between EDTA plasma and serum. This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay. The other commercially available ELISA examined was successfully validated and showed no difference in CA IX between EDTA plasma and serum.

Conclusions: These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid. This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

Show MeSH
Related in: MedlinePlus