CA IX ELISA
CA IX ELISA
For the quantitative detection of the circulating form of the CA IX (Carbonic Anhydrase IX) protein in human serum and plasma
Item # 06490025
CA IX ELISA Controls
(3 1.0 ml vials: High, Mid, Low Levels)
Item # 06490033
Use
The Oncogene Science CA IX ELISA is an enzyme-linked immunoassay used for the quantitative detection of the circulating form of the CA IX (Carbonic Anhydrase IX) protein in human serum and plasma.
- This product is for research use only and not for use in diagnostic procedures
Background
The transmembrane protein MN, also known as CA IX, is a member of the large family of enzymes known as carbonic anhydrases, which share the ability to catalyze the reversible hydration of carbon dioxide to carbonic acid, leading to a decrease in pH [1]. Up-regulation of CA9 gene expression occurs under hypoxic conditions, and is believed to be involved in sensing and maintaining the acidic environment of hypoxic cells, particularly in hypoxic regions within tumors [2,3].
Normal expression of MN protein is restricted primarily to gastric, intestinal, and liver mucosa [4,5]. Using IHC and RT-PCR, significant levels of MN protein have been detected in a variety of tumors, including those of the kidney, cervix, lung, bladder, colon, breast, liver, gall bladder, and pancreas [6]. In many of these malignancies, the presence of MN-positive cells is reportedly associated with earlier stage disease. In bladder, gallbladder, and liver cancer, more MN staining was seen in noninvasive and dysplastic lesions than in later-stage, invasive tumors [7,8]. In breast cancer, 50% of DCIS (ductal carcinoma in situ) tumors versus 29% of invasive tumors had MN staining [9].
In colorectal cancer, MN was seen in 76% of polyps but in only 64% of carcinomas. Within the carcinomas, staining intensity was higher in the Dukes A/B than in C/D tumors. Staining levels were also higher in well-differentiated tumors [10]. In cervical neoplasms, greater than 90% stained positive for MN [11]. In atypical cervical Pap smears, those that upon biopsy had neoplastic disease had atypical cells in the Pap smear that stained positive for MN, while benign lesions indicated no staining [12]. Conversely, in non-small-cell lung cancer, 80% of tumors stained positive for MN, whereas all normal and dysplastic lesions were negative [13].
Many studies have analyzed MN levels in renal cell carcinoma (RCC). Greater than 75% of RCC samples stain positive for MN by IHC or RT-PCR [14,15,16,17]. Ninety percent of all RCC are of the clear-cell type, and of these, greater than 90% are positive for MN [15,16,17]. Most studies were performed using tissue from the primary tumors; however, a comparison of MN levels between matched primary and metastatic lesions has been performed [16]. In 11/15 patients, MN staining was greater in the primary lesion than in the metastatic tumor. One group also reported no MN staining in non-renal clear-cell tumors [14], suggesting MN may be a good biomarker for renal clear-cell adenocarcinoma. Investigation into using soluble MN as a biomarker by one group was initiated by testing whether the MN protein could be detected in the serum or urine of RCC patients [18]. By western blot, they were able to detect MN in preconcentrated serum and urine at ~ 35 pg/mL in Controls and 20–3600 pg/mL in RCC.
Several groups have reported an association between MN levels in tumors and patient outcome. In bladder and renal cell cancer, higher levels of MN predicted a better patient outcome [7,15,16] in various studies. However, in invasive breast cancer, patients whose tumors had higher levels of MN had a higher relapse rate, worse overall survival, and MN level was an independent predictor for overall survival (hazard ratio = 2.61) [19]. In cervical cancer, MN level is a prognostic factor for disease-specific and metastasis-free survival, with higher levels indicating a worse prognosis [20]. In lung cancer, patients whose tumors expressed EGFr alone had a better prognosis than patients whose tumors expressed EGFr with MN and/or MMP-9 [21]. In breast cancer, using RT-PCR, MN levels had a positive association with HER-2/neu overexpression [22]. Clinical Research, using the CA IX ELISA, will help to better define the role of CA IX in cancer.
Some research has referenced potential diagnostic uses of our research use only biomarkers. However, these studies often are not consistent and the data is not yet conclusive. We offer this information and references as a service to clinical researchers that wish to know the state of the scientific literature to help guide their clinical research.
Principle of the Test
The CA IX ELISA is a sandwich-type test that uses a mouse monoclonal Capture Antibody and a biotinylated mouse monoclonal antibody as detector. The Capture Antibody has been immobilized on the interior surface of the microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the wells to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then exposed to the biotinylated Detector Antibody. A streptavidin-HRP Conjugate is then added. Addition of Substrate to the wells allows the catalysis of a chromogen into a colored product, the intensity of which is proportional to the amount of MN protein, also known as CA IX, that is bound to the plate.
Standards are provided in the kit that allow accurate, quantitative determinations of MN/CA IX in suitable samples. Using a microtiter plate reader, one can measure simultaneously the absorbance of the colored product in the Standards and sample wells. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of MN/CA IX in a sample. Samples may be assigned a quantitative value of MN/CA IX in picograms per mL (pg/mL) of serum and plasma.
Patents
Purchase of this kit licenses its use under the following U.S. patents: 5,387,676; 5,981,711; and 6,004,535
Selected References
1. Opavsky R, Pastorekova S, Zelnik V, et al. Human MN/CA9 gene, a novel member of the carbonic anhydrase
family: Structure and exon to protein domain relationships. Genomics 1996; 33: p. 480–487.
2. Wykoff C, Beasley N, Watson P, et al. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res 2000; 60: p. 7075–7083.
3. Svastova E, Hulikova A, Rafajovoa M, et al. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEBS Letters 2004; p. 439–445.
4. Ivanov S, Liao S, Ivanova A, et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J of Pathology 2001; 158(3): p. 905.
5. Leppiliampi M, Saarnio J, Karttunen T, et al. Carbonic anhydrase isozymes IX and XII in gastric tumors. World J Gastro 2003; 9(7): p. 1398–1403.
6. Juhasz M, Chen J, Leneckel U, et al. Expression of carbonic anhydrase IX in human pancreatic cancer. Aliment Pharmacol Ther 2003; 18: p. 837–846.
7. Hussain S, Palmer D, Ganesan R, et al. Carbonic anhydrase IX, a marker of hypoxia: Correlation with clinical outcome in transitional cell of the bladder. Oncol Rep 2004 May; 11(5): p. 1005–1010.
8. Saarnio J, Parkkila S, Parkkila A, et al. Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours. J Hepatol 2002; 35(5): p. 643–649.
9. Wykoff C, Beasley N, Watson P, et al. Expression of the hypoxia-inducible and tumor-associated carbonic anhydrases in ductal carcinoma in situ of the breast. Am J of Pathology 2001; 158(3): p. 1011.
10. Saarnio J, Parkkila S, Parkkila A, et al. Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J of Pathology 1998; 153(1): p. 279.
11. Liao S, Brewer C, Zavada J, et al. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am J of Pathology 1994; 145(3): p. 598–609.
12. Liao S, Stanbridge E. Expression of MN/CA IX protein in Papanicolaou smears containing atypical glandular cells of undetermined significance is a diagnostic biomarker of cervical dysplasia and neoplasia. Cancer 2000; 88(5): p. 1108.
13. Vermylen P, Roufosse C, Burhy A, et al. Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in non-small-cell lung carcinoma. Eur Respir J 1999; 14: p. 806–811.
14. Liao S, Aurelio O, Jan K, et al. Identification of the MN/CA IX protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res 1997; 57(14): p. 2827–2831.
15. Murakami Y, Kanda K, Tsuji M, et al. MN/CA9 gene expression as a potential biomarker in renal cell carcinoma. BJU Internat 1999; 83: p. 743–747.
16. Bui M, Seligson D, Han K, et al. Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: Implications for prognosis and therapy. Clin Ca Res 2003; 9: p. 802–811.
17. Grabmaier K, Vissers J, De Weijert M, et al. Molecular cloning and immunogenicity of renal cell carcinomaassociated antigen G250. Int J Cancer 2000; 85: p. 865–870.
18. Zavada J, Zavadova Z, Zat’ovicova M, et al. Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients. Brit J Canc 2003; 89: p. 1067–1071.
19. Chia S, Wykoff C, Watson P, et al. Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma. J of Clin Onco 2002; 19(16): p. 3660–3668.
20. Loncaster J, Harris A, Davidson S, et al. Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: Correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res 2001; 61: p. 6394–6399.
21. Swinson D, Cox G, O’Byrne K. Coexpression of epidermal growth factor receptor with related factors is associated with a poor prognosis in non-small cell lung cancer. Brit J Cancer 2004; 91: p. 1301–1307.
22. Bartosova M, Parkkila S, Pohlodek K, et al. Expression of carbonic anhydrase IX in breast cancer is associated with malignant tissues and is related to overexpression of c-erbB2. J of Pathology 2002; 197: p. 314–321.
