ABSTRACT
BACKGROUND: The transcription-reverse transcription concerted reaction (TRC) test is a novel molecular-based procedure, which can assess nodal metastasis accurately and quickly. We examined the usefulness of the TRC test with a double marker, cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) mRNA, to detect sentinel lymph nodes (SLN) metastasis in breast cancer patients. METHODS: A total of 264 SLNs from 131 breast cancer patients were assigned to a training set (109 SLNs from 50 patients) and validation set (155 SLNs from 81 patients). Cytokeratin 19 and CEA mRNA were detected by TRC tests, and the sensitivity and specificity of the SLN metastasis between the TRC and histology cohorts were compared. RESULTS: Mean copy numbers of CK19 and CEA by TRC tests were increased according to the metastatic size. In the training set, TRC test showed 100% sensitivity, specificity and concordance rates against the permanent histopathology test. In the validation set, sensitivity was 97.1%, specificity was 99.2% and the concordance rate was 99.4%. CONCLUSION: Our results showed that the detection of CK19 and CEA mRNA using the TRC test is, an accurate and rapid method for detection of SLN metastasis and can be applied as an intraoperative molecular diagnosis in breast cancer patients.
Subject(s)
Breast Neoplasms/pathology , Lymphatic Metastasis/diagnosis , Molecular Diagnostic Techniques , Axilla/pathology , Biomarkers, Tumor/genetics , Carcinoembryonic Antigen/genetics , DNA Copy Number Variations , Female , Humans , Intraoperative Period , Keratin-19/genetics , Middle Aged , RNA, Messenger , Reverse Transcription , Sensitivity and SpecificityABSTRACT
In the preparation of F(ab')(2) fragments of monoclonal antibodies (mAbs) of IgG class, heavy (H) chains are truncated by pepsin and light (L) chains are remained intact. However, F(ab')(2) fragments formed by pepsin-digestion of a mouse mAb PM373, which was of the IgG1 class and raised against human prostate specific antigen (PSA), indicated that the L chains of 31 kDa were cleaved into 23-kDa fragments as well as the cleavage of H chains of 50 kDa into 28-kDa fragments. On the other hand, F(ab')(2) fragments formed by digesting the mAb by cathepsin D showed that the L chains were intact and the H chains were truncated. The immunoreactivities against PSA of the F(ab')(2) fragments containing the intact L chains and those containing the truncated L chains were almost the same as that of the parental mAb, suggesting that the truncation of the L chains does not affect the interaction of the mAb with its specific antigen.
Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Light Chains/metabolism , Pepsin A/metabolism , Animals , Antibodies, Monoclonal/chemistry , Cathepsin D/chemistry , Cathepsin D/metabolism , Chromatography, High Pressure Liquid , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Immunoglobulin Light Chains/chemistry , Mice , Pepsin A/chemistry , Prostate-Specific Antigen/immunologyABSTRACT
Monoclonal antibodies (mAbs) of the IgG class produced by mouse hybridomas raised with NS-1 myelomas have been shown to contain two types of immunoglobulin light (κ) chains derived from the myelomas and antigen-stimulated spleen lymphocytes, and the hybridomas produce three mAb species with light chain heterogeneity (Abe and Inouye, 1993). In the present study, 9 hybridoma lines secreting homogeneous mAbs have been isolated from 63 lines cloned from an established hybridoma line producing three mAbs. They secrete homogeneous mAbs containing light chains derived from either myeloma or spleen cells. They contain either κ gene derived from the respective cells, and the other gene was deleted during the cultivation. The deletion frequency of the κ gene of myelomas is 3 times higher than that of spleen cells, although 80-85% of hybridomas reach the stable state containing both κ genes.
ABSTRACT
We have clearly shown by primary culture of hepatocytes that a promoting factor for proliferation of parenchymal hepatocytes appeared in the portal blood of rats which received 70% partial hepatectomy, and that such a liver-regenerating factor exists not only in murine small-intestinal mucosa, but also in bovine intestinal mucosa at all times. We also observed that in hepatopathy induced in rats by chemical agents such as CCl4 and D-galactosamine, intraperitoneal injection of the bovine small-intestine mucosal extract containing this factor suppressed the increases of GOT, GPT and other enzyme activities in the rats' serum and modified the hepatopathy. These results indicate that the in-vitro effects of these factors on hepatocyte cultures reflect those found in vivo on hepatopathy.