This large glycoprotein is defined by the carbohydrate epitope Ca19-9 located on MUC-1, and is referred to as sialylated lewis A (Lea). It is expressed by epithelial tumors of the gastrointestinal (GI) tract including the stomach, small and large intestines, colon, pancreas, and cervix. Ca19-9 mucin circulates in the blood and accumulates in ascites or pleural fluid of patients with GI cancer. Using a RIA kit specific for the Ca19- 9 epitope (Centocor/Fujirebio), 1 Unit of Ca19-9 corresponds to 0.59 ng/mL of the 116NS-19-9 antibody reactive determinants. This corresponds to ~1.7 U/ng of total protein or 1 KU equals 0.59 ug of Ca19-9 antigen. Other antibodies have been produced that recognize different determinants on the same molecule, including Ca50, Span-1, Dupan-2, and SSEA-1, which are measured in terms of each specific antibodies reactive epitope units [23, 24].

Purity Ratios
Bioprocessing controls the consistency of the Ca 19-9 purification by establishing limits on the purity ratio (U/ml/OD280nm). The purity ratio for the Ca 19-9 Ag purified from natural product and cell culture supernate material must be greater than 1.0 x 10 5U/m./ODnm. The purity of the natural sourced material can vary greatly due to patient variability but it must always have a purity ratio greater than 100,000 (1.0 x 10 5). The cell culture conditioned media is collected and chromatographed. Because we prepare the natural product and cell culture supernatant differently prior to purification, we also obtain differences in relative purity ratios.

ELISA
Through the use of our antigens for several development projects involving testing GI Ag to develop GI MoAb HP (4250-8215 and 4250-8235) and testing against NS-1116 (Centocor), optimal coating concentrations are as follows:

Immunization
Based on our experience in generating monoclonal antibodies against the GI Ag produced at Bioprocessing, Inc., we have extensive experience with using the HP Ag for immunizations. For monoclonal antibodies, we normally immunize and boost with the following:

PAGE
We employ PAGE analysis to determine Ca19-9 purity for both natural product and cell culture supernatant. Loading 1-2 KU (0.5-1 μg) followed by staining with GelCode Blue (Thermo Fisher Scientific Inc., Rockford, IL). It is difficult to detect Ca19-9 based on the aggregates that it forms when in a concentrated solution. Western blot analysis of high pure Ca19-9 demonstrates the high degree of glycosylation present on the MUC-1 antigen (Figure 1).

Figure 1. Western Blot of Pure Ca19-9 Antigen Purified Ca19-9 (500 U of catalogue #4250-2000) was subjected to western blotting with GI MoAb HP, Clone 4C11 (catalogue # 4250-8215), a monoclonal antibody that is specific for Ca19-9 epitope on MUC-1. Results demonstrate the purity in addition to the degree of glycosylation, as seen by the smearing pattern on the blot.

Gastrointestinal Tumor Marker (Ca19-9) Brief

Literature Cited
1. Ho, J.J., et al., Expression of CA19-9, DU-PAN-2, and SPan-1 antigens on two types of normal salivary mucins. Oncol Res, 1993. 5(9): p. 347-56. 2. Lan, M.S., et al., Co-expression of human cancer-associated epitopes on mucin molecules. Int J Cancer, 1987. 39(1): p. 68-72. 3. Haga, Y., et al., Partial purification and characterization of CA19-9 antigen from the ascitic fluid of a patient with pancreatic cancer. Clin Biochem, 1989. 22(5): p. 363-8. 4. Ho, J.J. and Y.S. Kim, Serological pancreatic tumor markers and the MUC1 apomucin. Pancreas, 1994. 9(6): p. 674-91.

Structure and Function
The mucin family of glycoproteins is classified by the presence of tandem repeat structures rich in serines, threonines, and prolines that are extensively modified by Oglycosylation. The human MUC family consists of 20 members, of which are classified into subcategories based on whether they are secreted or membrane bound [1]. Secreted mucins (MUC-2, 3, 5AC, 5B, and 6) form a physical gel barrier that protects epithelial cells lining the respiratory and gastrointestinal tracts and ductal surfaces of specialized organs such as the pancreas, kidney, and liver. Membrane bound mucins (MUC-1, 3, 4, 12, 13, 16, and 17) also contribute to the formation of a protective mucous gel through ectodomains of O-glycosylated tandem repeats that extend from the apical surface of the cell. Membrane bound mucins typically contain a sea urchin sperm protein, enterokinase and agrin (SEA) domain that resides between the glycosylated ectodomain and the transmembrane domain (Figure 1). Autoproteolysis of the MUC-1 SEA domain results in the formation of a stable non-covalent dimer, consisting of the N-terminal ectodomain and a C-terminal transmembrane subunit [2- 4].

Figure 1. MUC-1 Overview The mucin family of glycoproteins is characterized by their tandem repeat domain, which undergoes extensive Olinked glycosylation following translation (italicized in RED). MUC- 1 is cleaved by autoproteolysis and forms a stable non-covalent dimer, consisting of a small cytoplasmic tail and a large N-terminal ectodomain. Following insertion into the plasma membrane, the N-terminal ectodomain is shed into circulating blood, while the C-terminal tail returns to the cytoplasm where it is phosphorylated, thus activating signal transduction pathways. At Bioprocessing, Inc., we purify carbohydrate antigens that are presented on the surface of the MUC-1 molecule. These epitopes are considered Tumor Antigens based on their increased frequency detected in cancer patients circulating fluids.

MUC-1 localizes to the apical border of normal epithelial cells [5]. Transformation and a loss of polarity in epithelial cells results in MUC-1 expression over the entire surface of the cell, as seen in Figure 2 [5].

Figure 2. Immunohistochemistry Immunohisochemistry was performed on Breast Tumor cells. Cells were washed and resuspended in 1X PBS followed by fixation in 4% paraformaldehyde. Fixed cells were permeabilized followed by staining with our MUC-1 specific monoclonal antibody (Br MoAb HP, Clone 6A4, catalogue #2250-8215) that is specific for the MUC-1 antigen Ca15-3.

Furthermore, overexpression of MUC-1 causes transformation [6] and resistance to stress induced apoptosis in normal epithelial cells [7-12]. The MUC-1 N-terminal ectodomain (MUC1-N) contains variable numbers of highly glycosylated 20 amino acid tandem repeats, that have a high density of O-linked oligosaccharide residues (Ser/Thr), and is shed into circulating blood. The shed form of MUC-1 has been reported to have a molecular weight of 330-400 kD [13-15]. There have been three variant isoforms of MUC-1 identified, MUC-1X, Y, or Z, that contain variable amounts of carbohydrate content ranging from 50-80% [16, 17]. MUC-1 isolated from normal epithelium contains less carbohydrate by having shorter chains of galactose N-acetyl glucosamine and N-acetyl galactosamine than in tumor epithelium. Carbohydrates are added to MUC-1 by core 2 O-glycans of sialyl lewis x structure (NeuAc2- 3Galβ1-4 (Fuc1-3) GlcNAc-R (SLe x )) through post-translational modifications. In tumor cell lines and serum collected from patients with cancer, the sialylated lewis A moiety can be part of the core structure of MUC-1, which is detected by using a monoclonal antibody for sialylated lewis A (Ca19-9) [18-20]. Additionally, MUC-1 has been shown to be autoproteolytically cleaved from a 110-residue SEA transmembrane domain on tumor cell surfaces, which is also part of the Ca125 molecule expressed in ovarian cancer [4, 21, 22].

Purity Ratios
At Bioprocessing, Inc., we isolate breast tumor antigen (Ca15-3) from natural product and cell culture supernatant collected from human breast adenocarcinoma cells, obtaining average ratios of 2.5 X 10 4 Units/OD unit using optical density (OD) at 280 nm. Ca15-3 immunoblots at ~400 kD using a Ca27.29 monoclonal antibody (Figure 4). Additionally, we have observed that Br antigen forms aggregates that migrate at >400 kD when placed over a size exclusion column.

Currently, a conversion factor for the MUC-1 cancer epitope that will convert units/mL to mg/mL does not exist. This is primarily due to the high degree of heterogeneity that exists among the MUC-1 cancer antigen species. The MUC-1 epitope is recognized by B27.29 and DF3 monoclonal antibodies located within the 20 residue tandem repeat domain (SAPDTRPA) [23]. Within this epitope-binding site, there are 2 sites for O-linked glycosylation in addition to a variably number of tandem repeats within MUC-1, contributing to the inability to determine an exact unit to mass ratio. Despite the difficulty in determining a mass for Br Ag produced at Bioprocessing, Inc., we have optimized the use of this antigen in several assays.

ELISA
The following are optimal coating concentrations for ELISA assays. These were developed internally during several projects involving testing Br Ag against B27.29:

Immunization
Based on our experience in generating monoclonal antibodies against the Br Ag produced at Bioprocessing, Inc., we have extensive experience using the HP Ag for immunizations. For the production of monoclonal antibodies, we normally immunize and boost mice with the following concentrations:

PAGE
We employ PAGE analysis to determine Ca15-3 purity for both natural product and cell culture supernatant. We load 250-500 U of Br Ag on a 4-20% PAGE gel run under reducing conditions followed by staining with GelCode Blue (Thermo Fisher Scientific Inc., Rockford, IL) (Figure 3).

Figure 3. PAGE Analysis of Pure Br Antigen In order to determine the purity of Ca15-3 antigen that is purified using size exclusion chromatography, we subject 200 U (Br Ag HP CC, catalogue #2250-2000) to PAGE analysis. Samples are run on a 4-20% PAGE gel under reducing conditions and stained with Pierce glycoprotein stain. Results demonstrate the relative purity of Br Antigen purified at Bioprocessing, Inc.. Ca15-3 antigen is a glycoprotein that does not stainwith GelCode Blue, a coomassie stain.

Immunoblot
Immunoblotting for purified Ca15-3 (250 U) using Ca27.29 under reducing conditions on a 4-20% gradient PAGE gel (Bio-Rad, Hercules, CA) displays a double band at ~200 - 300 kD (Figure 4). As seen in Figure 4, a faint double band appears as a result of post-translational modifications of MUC-1, which is recognized by Ca27.29.

Figure 4. Western Blot of Pure Br Antigen Purified samples of Br Antigen (250 U of Br Ag HP CC, catalogue #2250-2000) were subjected to western blotting with Ca27.29, a monoclonal antibody that is specific for Ca15-3 epitope on MUC-1. Results demonstrate the purity in addition to the degree of glycosylation, as seen by the linear staining pattern on the blot.

Breast Tumor Antigen (Ca15-3) Brief

Literature Cited
1. Taylor-Papadimitriou, J., Report on the first international workshop on carcinomaassociated mucins. Int J Cancer, 1991. 49(1): p. 1-5. 2. Levitin, F., et al., The MUC1 SEA module is a self-cleaving domain. J Biol Chem, 2005. 280(39): p. 33374-86. 3. Ligtenberg, M.J., et al., Cell-associated episialin is a complex containing two proteins derived from a common precursor. J Biol Chem, 1992. 267(9): p. 6171-7. 4. Macao, B., et al., Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat Struct Mol Biol, 2006. 13(1): p. 71-6. 5. Kufe, D., et al., Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma, 1984. 3(3): p. 223-32. 6. Li, Y., et al., Human DF3/MUC1 carcinoma-associated protein functions as an oncogene. Oncogene, 2003. 22(38): p. 6107-10. 7. Raina, D., et al., MUC1 oncoprotein blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage. EMBO J, 2006. 25(16): p. 3774-83. 8. Ren, J., et al., Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents. Cancer Cell, 2004. 5(2): p. 163-75. 9. Wei, X., H. Xu, and D. Kufe, Human MUC1 oncoprotein regulates p53-responsive gene transcription in the genotoxic stress response. Cancer Cell, 2005. 7(2): p. 167-78. Figure 3. Western Blot of Pure Br Antigen Purified samples of Br Antigen (250 U of Br Ag HP CC, catalogue #2250-2000) were subjected to western blotting with Ca27.29, a monoclonal antibody that is specific for Ca15-3 epitope on MUC-1. Results demonstrate the purity in addition to the degree of glycosylation, as seen by the linear staining pattern on the blot. BA-19-00-03 Rev 01 Effective Date: 9/30/2010 7 10. Yin, L., L. Huang, and D. Kufe, MUC1 oncoprotein activates the FOXO3a transcription factor in a survival response to oxidative stress. J Biol Chem, 2004. 279(44): p. 45721-7. 11. Yin, L., S. Kharbanda, and D. Kufe, Mucin 1 oncoprotein blocks hypoxia-inducible factor 1alpha activation in a survival response to hypoxia. J Biol Chem, 2007. 282(1): p. 257-66. 12. Yin, L., et al., Human MUC1 carcinoma antigen regulates intracellular oxidant levels and the apoptotic response to oxidative stress. J Biol Chem, 2003. 278(37): p. 35458- 64. 13. Minkiewicz-Radziejewska, I., A. Gindzienski, and Z. Namiot, Isolation of MUC 1 antigen from human gastric juice. Rocz Akad Med Bialymst, 2001. 46: p. 69-76. 14. Sekine, H., T. Ohno, and D.W. Kufe, Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas. J Immunol, 1985. 135(5): p. 3610-5. 15. Strous, G.J. and J. Dekker, Mucin-type glycoproteins. Crit Rev Biochem Mol Biol, 1992. 27(1-2): p. 57-92. 16. Irimura, T., et al., Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity. J Biochem, 1999. 126(6): p. 975-85. 17. Oosterkamp, H.M., et al., Comparison of MUC-1 mucin expression in epithelial and non-epithelial cancer cell lines and demonstration of a new short variant form (MUC- 1/Z). Int J Cancer, 1997. 72(1): p. 87-94. 18. Ho, J.J., B. Siddiki, and Y.S. Kim, Association of sialyl-Lewis(a) and sialyl-Lewis(x) with MUC-1 apomucin ina pancreatic cancer cell line. Cancer Res, 1995. 55(16): p. 3659-63. 19. Sikut, R., et al., Distinct sub-populations of carcinoma-associated MUC1 mucins as detected by the monoclonal antibody 9H8 and antibodies against the sialyl-Lewis a and sialyl-Lewis x epitopes in the circulation of breast-cancer patients. Int J Cancer, 1996. 66(5): p. 617-23. 20. Zhang, K., et al., Secreted MUC1 mucins lacking their cytoplasmic part and carrying sialyl-Lewis a and x epitopes from a tumor cell line and sera of colon carcinoma patients can inhibit HL-60 leukocyte adhesion to E-selectin-expressing endothelial cells. J Cell Biochem, 1996. 60(4): p. 538-49. 21. Engelmann, K., et al., Transmembrane and secreted MUC1 probes show traffickingdependent changes in O-glycan core profiles. Glycobiology, 2005. 15(11): p. 1111-24. 22. Maeda, T., et al., Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16). J Biol Chem, 2004. 279(13): p. 13174-82. 23. Reddish, M.A., et al., Epitope Mapping of Mab B27.29 Within the Peptide Core of The Malignant Breast Carcinoma-Associated Mucin Antigen Coded for by the Human MUC-1 Gene. Journal of Tumor Marker Oncology, 1992. 7(1).

The AFP glycoprotein has been used to monitor carcinomas of hepatic and germ cell origins for more than two decades. However, it is also elevated in benign diseases of the liver and germ cells. Hepatic and germ cell carcinomas exhibit different Concanavalin A (Con A) molecular affinity variant AFP patterns compared to AFP patterns in cord serum. (1) AFP antigen, in fact, has three distinct isoforms as defined by differential binding to the lectins ConA or to Lens culinaris (LCA). The three isoforms are designated L1 (Con A binding), L2 ( LCA binding) and L3(strong LCA binding). In patients with testicular tumors AFP-L2 can not be separated from AFP-3. (2) The L3 form or AFP-LCA reacting form has an additional 1-6 fucose residue attached at the reducing end of N-acetylglucosamine. (3,4) The high affinity to LCA is based on this modification.

The percentage of AFP-L3 to total AFP is higher in hepatic cancers as compared to non cancer hepatic diseases (5,6) Wako Pure Chemicals Industries reports a specificity of greater than 92% using their AFP-L3% assay to diagnose HHC compared to 75% for a total AFP assay.(7)

Bioprocessing, Inc. cell culture derived AFP products, both part and highly pure , contain 65-70% L-3 isoform while our cord serum derived AFP contains only 3% of the L3 form. ( as measured by FDA approved Wako Pure Chemical Industries AFP-L3% assay (8)

Diagnostic manufacturers who make AFP assays should be aware that the cord serum derived AFP used most commonly in these assays does not represent the same carbohydrate isoform ratio as compared to the serum of cancer patients. In addition, cord serum derived AFP is usually pool tested for infectious diseases which adds to the risk of using cord serum derived AFP in assays or controls.

Alpha-fetoprotein Antigen Brief

References:
1. Smith CJ et.al., Concanavalin-A-affinity molecular heterogeneity of human hepatoma AFP and cord-serum AFP, Ann NY Acad Sci 1983; 417: 69-74 2. Li,D, et al, AFP-L3: a new generation of tumor marker for hepatocellular carcinoma, Clin Chem Acta 2001; Nov; 313 (1-2):15-9 3. Naitoh A, et al., Highly enhanced fucosylation of serum glycoprotein in patients with hepatocellular carcinoma, J. Gastotroenterol. Hepatol 1999 May; 14 (5): 436-45 4. Mita Y, et. al., Plasma fucosyltransferase activity in patients with hepatocellular carcinoma with special reference to correlation with fucosylated species of AFP, J Hepatol 2000 Jun; 32(6): 946-54 5. Sato Y et. al. Early recognition of hepaocellular carcinoma based on altered profiles of AFP, N Engl J Med 1993 Jun 24;328(25):1802-6 6. Taketa K et. al. A collaborative study for the evaluation of lectin-reactive alpha fetoproteins in early detection of hepatocellular carcinoma, Cancer Res 1993 Nov15;53(22):5419-23 7. Wako LBA AFP-L3 technical bulletin 2006 8. Bioprocessing, Inc. internal data 2006

Safe and Environmentally responsible Ferritin antigen production

Cadmium Sulfate is routinely used to precipitate Ferritin as part of a purification procedure in the diagnostic industry. Large volumes of buffered salts containing Cd are required on average in the 2 parts per million or greater range. All cadmium waste must be removed and disposed of by licensed hazardous waste haulers. These wastes contribute to the already large amounts of hazardous waste in landfills throughout the U.S. and Canada.

"In surface water and groundwater, cadmium can exist as the hydrated ion, or as ionic complexes with other inorganic or organic substances. While soluble forms may migrate in water, cadmium is relatively nonmobile in insoluble complexes or adsorbed to sediments. Similarly, cadmium in soil may exist in soluble form in soil water, or in insoluble complexes with inorganic and organic soil constituents. Cadmium in soil tends to be more available when the soil pH is low. Cadmium is taken up and retained by aquatic and terrestrial plants and is concentrated in the liver and kidney of animals that eat the plants." (Elinder 1985). From HHS:ATSDR , Toxicology profile for Cd .

Working with Cadmium in the laboratory poses a risk to laboratory personnel in terms of accidental exposure through splashes in eyes or mouth and dermal routes. Even with proper handling and protective clothing this risk is high. In addition OSHA requires PEL value reporting and air quality monitoring in workplaces using Cadmium. Companies which use Cd in their Ferritin processing create serious risks to personnel and the environment.

We produce Ferritin Antigen using a cadmium free method.

It involves more steps, product losses and expenses to produce and is therefore more expensive per mg than Ferritin produced using Cadmium. Since Ferritin antigen is a relatively inexpensive biological reagent raw material we feel that the increased price is wholly justifiable for our clients as well as ourselves.

Tumor Marker Antigen Mucin Biosynthetic Pathways Brief

References:
1. Marcos N, et. al. Polypeptide GalNac-transferases, ST6GalNAc-transferase I, and ST3 Gal-transferase I expression in Gastric Carcinoma Cell lines, J Histochem & Cytochem; 2003 51(6): 761-71 2. Magnani J, the discovery, biology and drug development of sialyl Le a and sialyl Le X, Arch Biochem Biophys ;2004; 426: 122-31 3. Whitehouse D et. al. The relative activities of the C2GnT1 and ST#Gal-1 gglycosyltransferases determine Oglycan structure and expression of a tumor-associated epitope on MUC-1, J Biol Chem 2001 276(14):11007-15 4. Dziadek S, et.al. Synthesis and structural model of an alpha(2,6)-sialyl-t-glycosylated MUC-1 eicosapeptide under physiological conditions, Chem 2006; 12(19):4981-93

Structure and Function
Gold and Freedman discovered Carcinoembryonic Antigen (CEA) in 1965 when they identified an antigen present at high concentrations in fetal colon and colon adenocarcinoma, but low in healthy colon (60-fold less) [1-3]. The gene encoding CEA has been classified as a member of the immunoglobin supergene family, which includes genes coding for adhesion molecules and major histocompatibility antigens [6]. The human CEA gene family consists of 29 genes, 18 of which are expressed with 7 in the CEA subgroup and 11 belonging to the pregnancy-specific glycoprotein subgroup [6]. The protein core of CEA contains an N-terminal domain of 108 amino acids homologous to the immunoglobin variable domain (IgV-like) and six domains homologous to the immunoglobin constant domain of the C-2 set (IgC2-like). [6, 7] CEA is attached to the cell membrane by a glycosyl phosphatidylinositol anchor (GPI) and is released as a soluble form by phospholipase C or phospholipase D [6]. Liver CEA is a glycoprotein that is ~60% carbohydrate and has a molecular mass of 180-200 kD [8]. CEA carbohydrate content is very heterogeneous, primarily composed of mannose, galactose, N-acetylglucosamine, fucose, and sialic acid [8]. Post-translational modifications, in particular glycosylation, of CEA, alter the already complex and differential antigenic profile of this molecule.

CEA function as an adhesive protein is neither calcium nor temperature dependent and may be homophilic (CEA to CEA) or heterophilic (CEA to non-CEA) [6, 7, 9]. Adhesion is facilitated by membrane localization on tissue specific cells expressing CEA.

The role of CEA has been well established as a circulating serum tumor marker for several cancer types, in particular colorectal cancer. CEA may also be elevated in gastric, pancreatic, breast, and lung cancers and also in non-neoplastic malignancies such as cirrhosis, ulcerative colitis, pancreatitis and smoking.

Purified CEA Specifications
Our natural high pure (HP) CEA antigen (Ag) (Product Code 3000-1000) is purified from human fluids. Final specifications for CEA Ag HP purified from pleural fluids is determined by PAGE analysis (³95% purity), Roche Elecsys CEA Assay, and purity ratio (determined by CEA mg/mL / OD280nm, ³95% purity). A final concentration of natural CEA Ag HP is between 1.0 and 5.0 mg/mL.

Figure 2 CEA PAGE and Western Blot A. CEA Ag CC and Nat are subjected to PAGE analysis and staining with a protein and glycoprotein stains. B. Western blot analysis was performed and CEA Ag CC HP (3000-2100) was blotted with a monoclonal antibody specific for CEA (Product Code 3000-8220, CEA mAb clone 5D9). The degree of glycosylation is evident from the glycoprotein stain and the extensive band seen on the PAGE and Western Blot respectively.

We also manufacture a cell culture (CC) CEA antigen, which is produced by a colon adenocarcinoma cell line. As with natural CEA, CEA Ag CC HP (Product Code 3000-2100) is purified by using multiple chromatography steps including affinity. Purity for CEA Ag CC HP is done using PAGE analysis (³95% purity by Coomassie Stain) and Roche Elecsys CEA Assay. A final concentration of CEA Ag CC HP is between 1.0 and 5.0 mg/mL.

CEA Ag, CC, PP, (Product Code 3000-2300), is purified using affinity chromatography. A final concentration of CEA Ag CC PP (Product Code 3000-2300) is between 1.0 and 5.0 mg/mL

Tumor Marker Antigen Mucin Biosynthetic Pathways Brief

References:
1. Gold, P. and S.O. Freedman, Specific carcinoembryonic antigens of the human digestive system. J Exp Med, 1965. 122(3): p. 467-81. 2. Gold, P. and S.O. Freedman, Demonstration of Tumor-Specific Antigens in Human Colonic Carcinomata by Immunological Tolerance and Absorption Techniques. J Exp Med, 1965. 121: p. 439-62. 3. Boucher, D., et al., Studies on the control of gene expression of the carcinoembryonic antigen family in human tissue. Cancer Res, 1989. 49(4): p. 847-52. 4. Hefta, S.A., et al., Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site. Proc Natl Acad Sci U S A, 1988. 85(13): p. 4648-52. 5. Zimmermann, W., et al., Isolation and characterization of cDNA clones encoding the human carcinoembryonic antigen reveal a highly conserved repeating structure. Proc Natl Acad Sci U S A, 1987. 84(9): p. 2960-4. 6. Thompson, J.A., F. Grunert, and W. Zimmermann, Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Lab Anal, 1991. 5(5): p. 344-66. 7. Hammarstrom, S., The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol, 1999. 9(2): p. 67-81. 8. Thomas, P., et al., The structure, metabolism and function of the carcinoembryonic antigen gene family. Biochim Biophys Acta, 1990. 1032(2-3): p. 177-89. 9. Jessup, J.M. and P. Thomas, Carcinoembryonic antigen: function in metastasis by human colorectal carcinoma. Cancer Metastasis Rev, 1989. 8(3): p. 263-80.