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 variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) 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-phage sera as detector. With this ELISA (sensitivity 0.05 microgram/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart ( approximately 3 mg/g of protein), while liver tissue and endothelial 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 mg/g of protein on the day before birth to 3.6+/-0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically 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 contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins 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 proteins in their natural context.
Pelsers
M M
MM
Cardiovascular Research Institute Maastricht (CARIM), Department of Physiology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Lutgerink
J T
JT
Nieuwenhoven
F A
FA
Tandon
N N
NN
van der Vusse
G J
GJ
Arends
J W
JW
Hoogenboom
H R
HR
Glatz
J F
JF
D000818:Animals; D000914:Antibodies, Viral; D000918:Antibody Specificity; D001435:Bacteriophages / Q000276:immunology*; D018955:CD36 Antigens / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D016172:DNA Fingerprinting; D003920:Diabetes Mellitus / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D027361:Organic Anion Transporters; D019151:Peptide Library; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
D000914:Antibodies, Viral; D018955:CD36 Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D027361:Organic Anion Transporters; D019151:Peptide Library