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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

Effect of crossing over antibodies between assays to determine which antibody–antigen interaction accounts for the metal-ion-dependent effects seen in the measurement of CA IX. Results are shown for four matched pairs of EDTA plasma and serum samples assayed using either the R&D assay, the Siemens assay or each of these but with the capture antibodies swapped between the assays. Absorbances were measured at 450 nm in both cases but background subtraction at 540 nm carried out in the case of the R&D systems assay. CA, carbonic anhydrase
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ACB-10-240F4: Effect of crossing over antibodies between assays to determine which antibody–antigen interaction accounts for the metal-ion-dependent effects seen in the measurement of CA IX. Results are shown for four matched pairs of EDTA plasma and serum samples assayed using either the R&D assay, the Siemens assay or each of these but with the capture antibodies swapped between the assays. Absorbances were measured at 450 nm in both cases but background subtraction at 540 nm carried out in the case of the R&D systems assay. CA, carbonic anhydrase

Mentions: When capture antibodies were exchanged between assays, it was very apparent that introduction of the Siemens capture antibody into the R&D assay maintained the absence of difference in absorbance values in plasma versus serum. However, pairing the R&D capture antibody with the Siemens detection antibody resulted in marked differences in absorbance values for the two fluid types as seen in the Siemens assay (Figure 4). This clearly indicates that the Siemens detection antibody i.e. the M75 clone29 is responsible for the differences seen in CA IX in plasma and serum and together with the previous results is consistent with the recognition by this antibody of an epitope which is metal-ion dependent.


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)

Effect of crossing over antibodies between assays to determine which antibody–antigen interaction accounts for the metal-ion-dependent effects seen in the measurement of CA IX. Results are shown for four matched pairs of EDTA plasma and serum samples assayed using either the R&D assay, the Siemens assay or each of these but with the capture antibodies swapped between the assays. Absorbances were measured at 450 nm in both cases but background subtraction at 540 nm carried out in the case of the R&D systems assay. CA, carbonic anhydrase
© Copyright Policy - open-access
Related In: Results  -  Collection

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

ACB-10-240F4: Effect of crossing over antibodies between assays to determine which antibody–antigen interaction accounts for the metal-ion-dependent effects seen in the measurement of CA IX. Results are shown for four matched pairs of EDTA plasma and serum samples assayed using either the R&D assay, the Siemens assay or each of these but with the capture antibodies swapped between the assays. Absorbances were measured at 450 nm in both cases but background subtraction at 540 nm carried out in the case of the R&D systems assay. CA, carbonic anhydrase
Mentions: When capture antibodies were exchanged between assays, it was very apparent that introduction of the Siemens capture antibody into the R&D assay maintained the absence of difference in absorbance values in plasma versus serum. However, pairing the R&D capture antibody with the Siemens detection antibody resulted in marked differences in absorbance values for the two fluid types as seen in the Siemens assay (Figure 4). This clearly indicates that the Siemens detection antibody i.e. the M75 clone29 is responsible for the differences seen in CA IX in plasma and serum and together with the previous results is consistent with the recognition by this antibody of an epitope which is metal-ion dependent.

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