Abstract
Antigen Used for Immunization
Recombinant human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) protein expressed in Escherichia coli.
Method of Immunization
Mouse immunization was performed as described previously (Park et al., 2016) with minor modifications. Female BALB/c mice (8 weeks of age) were immunized with 50 μg of purified recombinant human GAPDH protein supplemented with 50 μg poly (I:C) (InvivoGen, San Diego, CA) and 50 μg of anti-mouse CD40 monoclonal antibody (BioXcell, West Lebanon, NH) as adjuvants. The mice were injected twice with a 2 week interval and sera were tested by Western blot. Three days before fusion with myeloma cells, the final boost was injected through the tail vein.
Parental Cell Line Used for Fusion
Sp2/0-Ag14 myeloma cell line.
Selection and Cloning Procedure
Monoclonal antibodies were produced by minor modification of the methods previously described (Park et al., 2016; Sanchez et al., 2014). Hybridomas were selected in HAT (hypoxanthine–aminopterin–thymidine) medium and cloned by flow cytometry. Positive clones were identified by Western blot using a HeLa cell lysate.
Heavy and Light Chains of Immunoglobulin
DSHB-hGAPDH-2G7 is an IgG1 and DSHB-hGAPDH-4B7 is an IgG2a. Both antibodies have kappa light chains.
Specificity
Specificity was checked by Western blot analysis. The antibodies bound to human GAPDH, but not yeast, Caenorhabditis elegans, mouse, or rat GAPDH. GAPDH is an enzyme important in carbohydrate metabolism and expressed in all cell types (Barber et al., 2005; Schmittgen and Zakrajsek, 2000). Its ubiquitous expression is classified as a “housekeeping protein” and is often used as a loading control for Western blot analyses.
Specific Antigen Identified
Human GAPDH protein.
Availability
Monoclonal antibodies DSHB-hGAPDH-2G7 and DSHB-hGAPDH-4B7 are available from the nonprofit Developmental Studies Hybridoma Bank (