Research Use Only

MN/CAIX ELISA

Intended Use
The MN/CAIX ELISA is an enzyme-linked immunoassay for the quantitation of MN protein, also known as CA IX, in human serum and plasma. FOR RESEARCH USE ONLY. 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 to primarily 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 associated with earlier stage disease. In bladder, gallbladder and liver cancer, more MN staining was seen in non-invasive 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 showed 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]. 90% 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 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 pre-concentrated 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]. 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 significant 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 significant positive association with HER-2/neu overexpression [22].

Assay Principle
The MN/CAIX ELISA is a sandwich type immunoassay 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 that are provided in the kit allow accurate, quantitative determinations of MN/CAIX in suitable samples. Using a microtiter plate reader, one can measure the absorbance of the colored product in the Standards and sample wells simultaneously. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of MN/CAIX in a sample. Samples may be assigned a quantitative value of MN/CAIX in picograms per mL (pg/mL).

Selected References

1. Opavsky R, Pastorekova S, Zelnik V, Gibadulinova A, Stanbridge E, Zavada J, Kettmann R, Pastorek J. Human MN/CA9 Gene, a Novel Member of the carbonic Anhydrase Family: Structure and Exon to Protein Domain Relationships. Genomics 1996, 33:480-487.
2. Wykoff C, Beasley N, Watson P, Turner K, Pastorek J, Sibtain A, Wilson G, Turley H, Talks K, Maxwell P, Pugh C, Ratcliffe P, Harris A. Hypoxia-inducible Expression of Tumor-associated Carbonic Anhydrases. Cancer Res. 2000, 60:7075-7083.
3. Svastova E, Hulikova A, Rafajovoa M, Zat’ovicova M, Gibadulinova A, Casini A, Cecchi A, Scozzafava A, Supuran C, Pastorek J, Pastorekova S. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEBS Letters 2004, 439-445.
4. Ivanov S, Liao S, Ivanova A, Danilkovitch-Miakova A, Tarasova N, Proescholdt M, Merrill M, Proescholdt M, Oldfield E, Lee J, Zavada J, Waheed A, Sly W, Lerman K, Stanbridge E. Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer. Am. J. of Pathology 2001, 158(3):905
5. Leppiliampi M, Saarnio J, Karttunen T, Kivela J, Pastorekova S, Pastorek J, Waheed A, Sly W, Parrikila S. Carbonic anhydrase isozymes IX and XII in gastric tumors. World. J. Gasrto. 2003, 9(7):1398-1403.
6. Juhasz M, Chen J, Leneckel U, Kellner U, Kasper H-U, Tulassay Z, Pastorekova S, Malfertheiner P, Ebert M. Expression of carbonic anhydrase IX in human pancreatic cancer. Aliment. Pharmacol. Ther. 2003, 18:837-846.
7. Hussain S, Palmer D, Danesan R, Hiller L, Murray, P, Pastorek J, Young L, James N. Carbonic anhydrase IX, a marker of hypoxia: correlation with clinical outcome in transitional cell of the bladder.
8. Saarnio J, Parkkila S, Parkkila A, Pastorekova S, Haukipuro K, Pastorek J, Juvonen T, Karttunen T. transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours. J. Hepatol. 2002, 35(5):643-649.
9. 9. Wykoff C, Beasley N, Watson P, Campo L, Chia S, English R, Pastorek J, Sly W, Ratcliffe P, Harris A. 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):1011.
10. Saarnio J, Parkkila S, Parkkila A, Haukipuro K, Pastorekova S, Pastorek J, Kairakuoma M, Karttunen T. 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), 279.
11. Liao S, Brewer C, Zavada J, Pastorek J, Pastorekova S, Manetta A, Berman M, DiSaia P, Stanbridge E. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am. J. Pathology 1994, 145(3):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):1108.
13. Vermylen P, Roufosse C, Burhy A, Verhest A, Bosschaerts T, Pastorekova S, Ninane V, Sculier J. Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in on-small cell lung carcinoma. Eur. Respir. J. 1999, 14:806-811.
14. Liao S, Aurelio O, Jan K, Zavada J, Stanbridge E. Identification of the MN/CA IX protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res. 1997, 57(14):2827-2831.
15. Murakami Y, Kanda K, Tsuji M, Kanayama H, Kagawa S. MN/CA9 gene expression as a potential biomarker in renal cell carcinoma. BJU Internat. 1999, 83:743-747.
16. Bui M, Seligson D, Han K, Pantuck A, Dorey F, Huang Y, Horvath S, Leibovich C, Chopra S, Liao S, Stanbridge E, Lerman M, Palotie A, Figlin R, Belldegrun A. Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy. Clin. Ca. Res. 2003, 9:802-811.
17. Grabmaier K, Vissers J, De Weijert M, Oosterwijk-Wakka J, Van Bokhoven A, Brakenhoff R, Noessner E, Mulders P, Merkx, G, Figdor C, Adema G, Oosterwijk E. Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250. Int. J. Cancer 2000, 85:865-870.
18. Zavada J, Zavadova Z, Zat’ovicova M, Hyrsl L, Kawaciuk I. Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients. Brit. J. Canc. 2003, 89:1067-1071.
19. Chia S, Wykoff C, Watson P, Han C, Leek R, Pastorek J, Gatter K, Ratcliffe P, Harris A. Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma. J. of Clin. Onco. 2002, 19(16):3660-3668.
20. Loncaster J, Harris A, Davidson S, Logue J, Hunter R, Wykoff C, Pastorek J, Ratcliffe P, Stratford I, West C. 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, 61:6394-6399.
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, 91:1301-1307.
22. Bartosova M, Parkkila S, Pohlodek K, Karttunen T, Galbevy S, Mucha V, Harris A, Pastorek J, Pastorekova S. 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, 197:314-321.