[Preparation and characterization of monoclonal antibodies against AR protein].
OBJECTIVE
To prepare monoclonal antibodies (mAbs) against aldose reductase (AR) and compare with anti-aldose reductase-like protein (ARL-1) mAb.


METHODS
The AR gene was gained by RT-PCR and inserted into pGEX-4T-1 (His)(6)C. Recombinant protein GST-AR was used to immunize BALB/c mouse. MAb was prepared by hybridoma technique and detected by ELISA and Western blot. Simultaneously, according to the analysis of AR by software Clustalx and Antheprot, GST-dAR(80-142 aa), GST-dA1(1-79 aa), GST-dA2(80-99 aa), GST-dA3(111-142 aa) and GST-dA4(143-316 aa) were expressed in E. coli Rosetta. All the proteins were used to analyze the binding sites of the mAb and AR protein by Western blot.


RESULTS
Three clones secreting anti-AR mAb were obtained. They were all of IgG1. And the titer of mAb in ascites was 1:400,000 while in cell culture was 1:10,000. All of the three anti-AR mAbs reacted to GST-AR and proteins of placenta tissues and had no cross-reaction to GST-ARL-1 and GST protein. And the three anti-AR mAb could recognize GST-dA1, GST-dA3 and GST-dA4, respectively.


CONCLUSIONS
Three specific mAbs against AR are obtained and recognize the 1-79, 111-142, 143-316 amino acid sites of AR, respectively. The anti-AR mAb, together with the anti-ARL-1 mAb, may be a useful tool for further study of the function of AR and ARL-1 and the relationship between the two proteins and relevant diseases as well as for the epidemiological investigation.
Ma
Da
D
Genetics Research Center, Southeast University, Nanjing 210009, China.
Jin
Jun-fei
JF
Sun
Ming-kuan
MK
Shan
Jun
J
Zhang
Jian-qiong
JQ
Xie
Wei
W
D000449:Aldehyde Reductase / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000449:Aldehyde Reductase