Batch immunoextraction method for efficient purification of aromatic cytokinins.
A range of benzylaminopurines naturally occur in plants and exhibit high biological activity. Others have been synthesized, such as 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (2OH3MeOBAPR), which has shown interesting anti-cancer activity under in vitro conditions. In order to study the biological activity of this interesting compound in more detail, a rapid and highly efficient method for its purification from complex samples (e.g. blood and plant extracts) is needed. Therefore, we prepared monoclonal antibodies against 2OH3MeOBAPR. The antibody had undetectable cross-reactivity with all natural isoprenoid cytokinins, but relatively high cross-reactivity with aromatic cytokinins as well as some synthetic di- and tri-substituted 6-benzylaminopurines and the corresponding ribosides. The antibody also showed strong responses and specificity in enzyme-linked immunoassays (ELISAs). In addition, it was used to prepare, for the first time, an immunoaffinity sorbent with high specificity and capacity for aromatic cytokinins. A batch immunoextraction method was then developed and optimized for the purification of 2OH3MeOBAPR from murine blood samples. The high efficacy and simplicity of this method (in off-line combination with HPLC-MS) for the isolation of target analytes from biological material is demonstrated in this study.
Hauserová
Eva
E
Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Olomouc, Czech Republic.
Swaczynová
Jana
J
Dolezal
Karel
K
Lenobel
René
R
Popa
Igor
I
Hajdúch
Marián
M
Vydra
David
D
Fuksová
Kvetoslava
K
Strnad
Miroslav
M
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003583:Cytokinins / Q000097:blood / Q000302:isolation & purification*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C
D003583:Cytokinins