RESUMEN
The tumour-associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known as small intestinal mucin antigen (SIMA). This epitope is detected in tissue from a high proportion of patients with colorectal cancer, and elevated levels occur in serum from a significant number of such patients, highlighting the potential clinical utility of MAb 4D3. In the present study, insight into the composition and structure of the carbohydrate epitope recognised by MAb 4D3 was gained following characterisation of 2 glycopeptides that co-purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycoprotein alpha-1-anti-chymotrypsin. This glycoprotein was subsequently deglycosylated to yield 5 forms corresponding to alpha-1-anti-chymotrypsin substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was reactive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated alpha-1-anti-chymotrypsin. MAb 4D3 also reacted to different extents with ovine, bovine or porcine submaxillary mucins, each of which has a different amount of the O-linked sialylated disaccharide known as sialosyl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxillary mucin, in which almost all of the carbohydrate chains are sialosyl Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 epitope involves the sialylated N-acetyl galactosamine disaccharide as well as an additional monosaccharide present on a neighbouring carbohydrate chain. Although the preferred epitope recognised by MAb 4D3 involves this sialylated disaccharide, the specificity of MAb 4D3 was different from that of other MAbs with a reported specificity for sialosyl Tn.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Epítopos/análisis , Secuencia de Aminoácidos , Antígenos de Neoplasias/aislamiento & purificación , Antígenos de Neoplasias/metabolismo , Secuencia de Carbohidratos , Epítopos/aislamiento & purificación , Epítopos/metabolismo , Glicopéptidos/análisis , Glicopéptidos/aislamiento & purificación , Humanos , Mucosa Intestinal/química , Datos de Secuencia Molecular , Peso Molecular , Mucinas/análisis , Mucinas/metabolismo , Papaína/metabolismo , Papaína/farmacología , Glándula Submandibular/química , alfa 1-Antiquimotripsina/metabolismo , alfa 1-Antiquimotripsina/farmacologíaRESUMEN
We have developed a sensitive ELISA using MAb 4D3 for the detection of a novel epitope on Small Intestinal Mucin Antigen (SIMA) and report here that SIMA is present in the serum of patients with colorectal cancer. SIMA has been shown to occur in tissue from a high proportion of patients with colorectal cancer. SIMA derived from serum was similar to tissue-derived SIMA: both eluted in the void volume of a Superose 6 column indicating a molecular weight above 5,000 kDa and they exhibited similar buoyant densities on CsCl gradients. The ELISA was most reliable after pre-treatment of serum with 0.4 M perchloric acid to remove interfering substances. The upper limit for SIMA in normal serum was set as the mean plus 2 standard deviations determined from a group of 97 healthy control subjects. In a sample of 113 patients with colorectal cancer, SIMA serum levels were elevated in 15% of patients with Dukes' Stage A, 38% with Stage B, 32% with Stage C and 75% with Stage D colorectal cancer. SIMA serum levels were compared with those of the widely used tumor marker, carcinoembryonic antigen (CEA). The SIMA assay detected a significant number of sera that were not detected by the test for CEA. We propose that SIMA will prove to be a valuable serological tumor marker, in combination with CEA and other tumor markers, for the detection of colorectal cancer.
Asunto(s)
Adenocarcinoma/inmunología , Anticuerpos Monoclonales , Antígenos de Neoplasias/sangre , Neoplasias Colorrectales/inmunología , Glicoproteínas/sangre , Biomarcadores de Tumor/sangre , Antígeno Carcinoembrionario/sangre , Ensayo de Inmunoadsorción Enzimática , Enfermedades Gastrointestinales/inmunología , Glicoproteínas/inmunología , Humanos , Inflamación/inmunología , Intestino Delgado/química , Monosacáridos/análisis , Mucinas , Estadificación de NeoplasiasRESUMEN
This study has identified the expression in normal and neoplastic gastrointestinal (GI) tract of epitopes on the colonic mucin LIMA (large intestinal mucin antigen), which are unique markers of normal colonic differentiation. Six anti-LIMA monoclonal antibodies (MAbs) (22D4, 9B5, 2C3, 23B2, 46A2, and 10B3) were studied immunohistochemically in normal GI tract, colorectal adenomas, and colorectal and gastric cancers. All MAbs showed specificities consistent with distinct epitopes, five of which were neuraminidase-resistant and four periodate-sensitive. Each reacted with mucin in 60-100 per cent normal colons--MAbs 10B3 and 23B2 also with small intestinal mucin--but none with gastric mucin. Five MAbs showed crypt and regional gradients in normal colon, MAbs, 22D4, 9B5, and 2C3 showing a hierarchy of reactivities in the crypt. Individual adenomas showed decreasing goblet cell (GC) LIMA expression with increasing size. However, 30 per cent of familial adenomatous polyposis (FAP) patients had generalized background losses of 9B5 and 2C3 GC reactivity, retaining 22D4, whilst 44 per cent of non-FAP patients lost 22D4 GC reactivity, regaining 9B5 and 2C3--evidence for polymorphism of mucin expression. All colorectal cancers expressed LIMA epitopes (frequently weaker than normal), and three MAbs (22D4, 9B5, and 2C3) showed deeper than normal staining in adjacent crypts. Eighty-five per cent of gastric cancers also expressed LIMA epitopes.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Epítopos/análisis , Neoplasias Gastrointestinales/inmunología , Mucinas/inmunología , Adenoma/inmunología , Poliposis Adenomatosa del Colon/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Biomarcadores/análisis , Diferenciación Celular , Colon/inmunología , Sistema Digestivo/inmunología , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/metabolismoRESUMEN
We describe the production, immunochemical and immunohistochemical characterization of monoclonal antibodies (MAbs) raised against the oncofetal small intestinal mucin antigen (SIMA). Four MAbs, reacting with distinct neuraminidase-sensitive SIMA epitopes, were shown to define a novel differentiation-associated relationship of SIMA epitopes within the normal small intestinal villus. Using Swiss rolls of 15 entire colorectal cancer resections, inappropriate expression of SIMA epitopes was detected in all cancers, in adjacent transitional mucosa and remote morphologically normal mucosa, extending as far as resection margins (73%). SIMA expression, whether preexisting or reactive to the tumor, may predispose to malignant change and tumor recurrence.