Neurite adhesion molecule L1 has been implicated in neuron-neuron and neuron-Schwann cell adhesion in vertebrates. L1-like molecules, found in mouse, rat, chicken, and human, promote axonal elongation and may also play a role in regeneration of axons after injury. Molecular cloning data suggest 87% amino acid identity between mouse and human L1 molecules. 5G3 antigen (Ag), originally defined by monoclonal antibody 5G3, is considered to be the human homologue of mouse L1. The 5G3 antibody was developed against a human neuroblastoma cell line to use as a probe for the elucidating the biological characteristics of neuroblastoma. 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa and its 200 kDa precursor. The 215 kDa molecule is expressed on the cell surface; whereas the 200 kDa precursor is shed from the cell surface. The 215 and 200 kDa species also differ in their posttranslational modification patterns. The 5G3 antibody has been used as a marker for neuroblastoma, and to purify 5G3 Ag from normal adult human brain.
The antibody recognizes human L1 on human neuroblastoma cell lines and tissues. Reactivity has been tested on a variety of malignant and normal tissues. Squamous lung, squamous skin, and osteogenic sarcoma cell lines were positive, as were two out of eight melanoma cell lines tested. A variety of other cell lines and tumor tissues tested negative. 5G3 did not react with either T or B lymphoblastoid cell lines or a fibroblast cell line. Among all the normal tissues tested, mAb 5G3 reacted only with cerebellum.
The molecular masses observed using mAb 5G3 may vary among immunoprecipitation isolates. In normal human cerebellum, 5G3 Ag migrated as a 190/200 kDa doublet, 140 kDa band with minor bands at 80 and 65 kDa. 5G3 Ag isolated from SK-N-AS cells migrates as 200 to 215 kDa bands, or as a diffuse band ranging from 200 to 215 kDa. Additional bands have been described at 140 to 150 kDa in SK-N-AS cells. Only the 200 kDa band has been detected in culture media from SK-N-AS cells.
The antibody was conjugated to BD Horizon™ BV650 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 650-nm. BD Horizon BV650 can be excited by the violet laser and detected in a filter used to detect APC-like dyes (eg, 660/20-nm filter). Due to the excitation and emission characteristics of the acceptor dye, there will be spillover into the APC and Alexa Fluor® 700 detectors. However, the spillover can be corrected through compensation as with any other dye combination.