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Gastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models. MKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 μg) response curves were performed in subcutaneous models. Orthotopic models received 50 μg of M5A-IR800 or 50 μg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. In subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors. Humanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection. [ABSTRACT FROM AUTHOR]

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery. [ABSTRACT FROM AUTHOR]

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry. [ABSTRACT FROM AUTHOR]

Background/Aim: Fluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models. Materials and Methods: Humanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated. Results: Intra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76). Conclusion: Anti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic. [ABSTRACT FROM AUTHOR]

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex. [ABSTRACT FROM AUTHOR]