The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10–1000？μg/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule. 1. Introduction Lymphomas (primary malignant neoplasms of lymphoreticular origin) represent one of the major health problems worldwide [1]. Despite the availability of several options to the treatment of lymphomas [2], the identification of novel tumor-associated antigens that are universally expressed in these malignancies is necessary for the development of newer immunotherapeutic strategies [3]. On the other hand, as it is well known, the main cause of cancer-related death is due to metastasis of primary tumors to secondary sites within the body [4]. Usually, different primary tumors tend to spread to preferred metastatic sites, although both regional and distant lymph nodes are the most common metastatic targets for a variety of primary malignancies such as skin, breast, colon, stomach, and lung [5]. Gangliosides are sialic acid-containing glycosphingolipids engaged in many biological events that take place at vertebrate’s cell membrane [6]. Unusual glycolylated gangliosides have been identified by immunohistochemical methods in some malignant tumors and their metastasis becoming attractive targets for immunotherapy [7–10]. The tissue reactivity of 14F7 Mab, a highly specific IgG1 against the NeuGcGM3
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