The Wuerzburg Hybridoma Library againstDrosophilaBrain

Abstract

This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

Transcellular spreading of huntingtin aggregates in theDrosophilabrain

“…2A and Movie S1). These neurons appear identical to the large cells labeled by the monoclonal antibody nb169 from the Würzburg hybridoma library (25,26) (Fig. 2B).…”

“…Primary antibodies include chicken anti-GFP (1:500) (Life Technologies), rabbit anti-DsRed (1:500) (Clontech), mouse anti-Bruchpilot (Brp) (1:50) (Developmental Studies Hybridoma Bank, University of Iowa), rat anti-HA (1:50) (Sigma-Aldrich). The mouse monoclonal antibody nb169 (1:20) (Würzburg Hybridoma Library) (25,26) was provided by Erich Buchner (University of Würzburg, Würzburg, Germany). Species-specific Alexa Fluor conjugated secondary antibodies were used at 1:500 (Invitrogen), including Alexa Fluor 488, 568, and 633.…”

Bruchpilot, A Synaptic Active Zone Protein for Anesthesia-Resistant Memory

“…Mouse monoclonal anti-Brp, nc82 (Hofbauer et al, 2009), and Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen) were applied as the primary and secondary antibodies, respectively. Brains for Brp quantification were counterstained with Phalloidin (catalog #R415; Invitrogen).…”

Behavioral and Synaptic Plasticity Are Impaired upon Lack of the Synaptic Protein SAP47

“…In this sense, studying associative learning and memory, rather than being particularly difficult, may be tackling the relation between brain and behavior in a particularly accessible form. Within this context, we focus on the role of a specific synaptic protein [the synapse-associated protein of 47 kDa (SAP47) (Reichmuth et al, 1995;Funk et al, 2004;Hofbauer et al, 2009)] for associative function and synaptic physiology in larval Drosophila. This seems timely, given the importance of synaptic processes for learning in general [e.g., Lechner and Byrne (1998) and Pittenger and Kandel (2003)] and in larval Drosophila in particular (e.g., Michels et al, 2005).…”

“…1A) was identified by a monoclonal antibody from a hybridoma library raised against Drosophila brain (Hofbauer et al, 2009). It codes for a synaptic protein with a BSD domain and is highly conserved between nematode, fly, fish, and human (Reichmuth et al, 1995;Funk et al, 2004).…”