A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase CD36 in cardiac and red skeletal muscle and up-regulation in diabetes

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a vari ety of membrane processes), is implicated in the transport of long-chain fa tty acids across cellular membranes. To set up an immunoassay for quantific ation of FAT in different tissues, we isolated a series of anti-FAT antibod ies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted speci fically with FAT, and most likely recognize the same or closely located imm unodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-I V7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-ph age sera as detector. With this ELISA (sensitivity 0.05 mu g/ml), the FAT c ontent in isolated cardiomyocytes was found to be comparable with that of t otal heart (approximate to 3 mg/g of protein), while liver tissue and endot helial cells were below the detection limit (<0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7 +/- 0.7 m g/g of protein on the day before birth to 3.6 +/- 0.4 mg/g of protein on da y 70, Comparing control with streptozotocin-induced diabetic rats, a statis tically significant (P < 0.05) 2-4-fold increase of FAT was seen in heart ( from 4.2 +/- 2.3 to 11.0 +/- 5.7 mg/g of protein), soleus (from 0.6 +/- 0.1 to 1.4 +/- 0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3 +/- 0.1 to 1.2 +/- 0.8 mg/g of protein). In addition, the FAT co ntents of each of these muscles were found to be of similar magnitude to th e contents of cytoplasmic heart-type fatty-acid-binding protein in both dia betic rats and controls, supporting the suggested roles of these two protei ns in cellular fatty acid metabolism, This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane prot eins in their natural context.