Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.
We have recently discovered the existence of 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-o-hemisuccinate with a carboxylic spacer arm attached to the 3β-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.
Cancer Research Center of Toulouse, UMR 1037 INSERM-University Toulouse III, Toulouse, France. phildemedina@yahoo.fr
Philippe
P
de Medina
Michael R
MR
Paillasse
Gregory
G
Segala
Talal
T
Al Saati
Jeannine
J
Boyes
Georges
G
Delsol
Cuider
C
Allal
Sabrina
S
Marsili
Sandrine
S
Silvente-Poirot
Marc
M
Poirot
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D001688:Biological Products / Q000032:analysis* / Q000276:immunology; D060326:Chemistry Techniques, Synthetic; D004083:Cholestanol / Q000032:analysis* / Q000276:immunology; D002777:Cholestanols / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D006825:Hybridomas / Q000166:cytology; D007093:Imidazoles / Q000032:analysis* / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013095:Spermidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology
C582666:5-hydroxy-6-(2-(1H-imidazol-4-yl)ethylamino)cholestan-3-ol; C582667:5-hydroxy-6-(3-(4-aminobutylamino)propylamino)cholest-7-en-3-ol; D000906:Antibodies; D001688:Biological Products; D002777:Cholestanols; D006241:Haptens; D007093:Imidazoles; D007106:Immune Sera; D004083:Cholestanol; D013095:Spermidine