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-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-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-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-o-hemisuccinate with a carboxylic spacer arm attached to the 3ß-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.

[Enzyme immunoassay of (24R)-brassinosteroids].

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.

Potentiation of the tolerogenicity of benzylpenicilloylated eicosa-L-lysine by conjugation with 4-(hydroxymethyl)benzyl 3 beta-cholestanyl succinate.

It was previously found that amino acid polymers such as oligolysines bearing haptenic groups in high densities efficiently suppress anti-hapten IgE antibody formation. Such conjugates are strong elicitors of anaphylaxis and therefore may not be used for desensitization of drug allergic patients. Here we report on the synthesis and immunological evaluation of benzylpenicilloylated (BPO) eicosa-L-lysines containing none, one, or two lipophilic p-(hydroxymethyl)benzyl cholestan-3 beta-yl succinate (OSuco) groups. The lipophilic derivatives suppress primary as well as ongoing anti-BPO IgE antibody formation in mice much more efficiently than their hydrophilic counterpart. The lipophilic but not the hydrophilic derivatives form stable micelles in water and suppress the antibody formation according to different cellular mechanisms. The relationship between structure, hydrophobicity, and mode of action is discussed.

Induction of immunological tolerance to the penicilloyl antigenic determinant--IV. The effect of BPO-oligolysines and cholestanol-bearing BPO-oligolysines on murine IgE responses.

Penta-, deca- and eicosalysine carriers were synthesized in solution and conjugated with benzylpenicillin to give BPO-conjugates of high haptenic density. Each oligolysine conjugate was prepared in two forms--with a free C-terminus and with an esterified C-terminus carrying via a benzylester bridge in essence a lipophilic cholestanol moiety [p-oxymethylbenzylcholestan-3 beta-yl succinate (OSuco group)]. Decalysines that carried a single haptenic BPO group and succinyl groups on the other amino functions were also prepared. Suppression of IgE responses was studied in BALB/c mice. It was found that BPO-specific suppression could be induced by injecting OSuco-bearing deca- or eicosalysine conjugates before immunization with BPO-Asc in A1(OH)3. The pentalysine conjugate was only slightly effective as were all OSuco-deficient conjugates. Ongoing IgE responses were only slightly suppressed and OSuco-bearing conjugates were not more effective than OSuco-deficient derivatives. When the monohaptenic OSuco-bearing decalysine, which exhibited weak tolerogenic effects on primary as well as on ongoing responses, was applied under conditions that favour suppressor T-cell induction, a pronounced unresponsiveness resulted. Direct evidence for suppressor T-cell involvement in the abrogation of anti-BPO responses by OSuco-bearing BPO-conjugates was obtained from cell transfer experiments. The study shows that relatively small haptenic conjugates, the lower limit of effectiveness being approximately represented by decalysine conjugates, may be effective tolerogens depending on the immune status.