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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

Representative examples of standard curves for the (a) Siemens (corrected for pre-dilution) and (b) R&D assays. (c) and (d) Relationship between concentrations of CA IX in 15 matched pairs of EDTA plasma or serum, respectively, using these two assays. The dotted line in each case is the line of equivalence. CA, carbonic anhydrase
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ACB-10-240F1: Representative examples of standard curves for the (a) Siemens (corrected for pre-dilution) and (b) R&D assays. (c) and (d) Relationship between concentrations of CA IX in 15 matched pairs of EDTA plasma or serum, respectively, using these two assays. The dotted line in each case is the line of equivalence. CA, carbonic anhydrase

Mentions: Typical standard curves are shown (Figures 1a and b). Analysis of rCA IX on both assays showed a ratio of Siemens:R&D values obtained of 2.8. In support of this indicating differences in standardization, analysis of five Siemens standards (35–750 pg/mL) on the R&D assay showed concentrations ranging from 12.0 to 236.1 pg/mL, respectively, with an average ratio of Siemens:R&D values of 3.13 (SD 0.17). Conversely, a reciprocal analysis analysing three R&D standards (62.5–250 pg/mL assigned concentrations) on the Siemens assay showed concentrations ranging from 154.7 to 626.5 pg/mL, respectively, with an average ratio of Siemens:R&D values of 2.51 (SD 0.03). These support the difference seen with the purified recombinant protein with slight differences presumably due to differing backgrounds with the different standards across the two assays. However, subsequent comparison of 15 plasma samples across both assays showed no such simple relationship between assay results with Siemens:R&D ratios varying from 1.95 to 17.3. Similarly, comparison of the 15 matched serum samples across both assays showed ratios varying from 0 to 7.1 (Figures 1c and d). These results support a difference in standardization between assays but also indicate that with clinical samples there are other potential factors affecting the results.


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)

Representative examples of standard curves for the (a) Siemens (corrected for pre-dilution) and (b) R&D assays. (c) and (d) Relationship between concentrations of CA IX in 15 matched pairs of EDTA plasma or serum, respectively, using these two assays. The dotted line in each case is the line of equivalence. CA, carbonic anhydrase
© Copyright Policy - open-access
Related In: Results  -  Collection

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

ACB-10-240F1: Representative examples of standard curves for the (a) Siemens (corrected for pre-dilution) and (b) R&D assays. (c) and (d) Relationship between concentrations of CA IX in 15 matched pairs of EDTA plasma or serum, respectively, using these two assays. The dotted line in each case is the line of equivalence. CA, carbonic anhydrase
Mentions: Typical standard curves are shown (Figures 1a and b). Analysis of rCA IX on both assays showed a ratio of Siemens:R&D values obtained of 2.8. In support of this indicating differences in standardization, analysis of five Siemens standards (35–750 pg/mL) on the R&D assay showed concentrations ranging from 12.0 to 236.1 pg/mL, respectively, with an average ratio of Siemens:R&D values of 3.13 (SD 0.17). Conversely, a reciprocal analysis analysing three R&D standards (62.5–250 pg/mL assigned concentrations) on the Siemens assay showed concentrations ranging from 154.7 to 626.5 pg/mL, respectively, with an average ratio of Siemens:R&D values of 2.51 (SD 0.03). These support the difference seen with the purified recombinant protein with slight differences presumably due to differing backgrounds with the different standards across the two assays. However, subsequent comparison of 15 plasma samples across both assays showed no such simple relationship between assay results with Siemens:R&D ratios varying from 1.95 to 17.3. Similarly, comparison of the 15 matched serum samples across both assays showed ratios varying from 0 to 7.1 (Figures 1c and d). These results support a difference in standardization between assays but also indicate that with clinical samples there are other potential factors affecting the results.

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