Chemical strategies for triggering the immune response to the mycotoxin patulin

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.

Hapten Design and Monoclonal Antibody to Fluoroacetamide, a Small and Highly Toxic Chemical

Fluoroacetamide (FAM) is a small (77 Da) and highly toxic chemical, formerly used as a rodenticide and potentially as a poison by terrorists. Poisoning with FAM has occurred in humans, but few reliably rapid detection methods and antidotes have been reported. Therefore, producing a specific antibody to FAM is not only critical for the development of a fast diagnostic but also a potential treatment. However, achieving this goal is a great challenge, mainly due to the very low molecular weight of FAM. Here, we design two groups of FAM haptens for the first time, maximally exposing the fluorine or amino groups, with the aid of linear aliphatic or phenyl-contained spacer arms. Interestingly, whereas the hapten with fluorine at the far end of the hapten did not induce an antibody response to FAM, the hapten with an amino group at the far end and phenyl-contained spacer arm triggered a significantly specific antibody response. Finally, a monoclonal antibody (mAb) named 5D11 was successfully obtained with an IC50 value of 97 μg mL−1 and negligible cross-reactivities to the other nine functional and structural analogs.

Simple Synthesis of 17-β-O-hemisuccinate of Stanozolol for Immunoanalytical Methods

The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17β-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

Competitive ELISA for N-terminal pro-brain natriuretic peptide (NT-proBNP) determination in human plasma

A competitive ELISA has been developed for the determination of NT-proBNP using a rational hapten design for its antibody production and also, for the development of the ELISA.

Vaccines against Drug Abuse: Morphine-Hapten Design and Synthesis

Drugs of abuse are small molecules that typically do not induce an antibody response following the administration. To induce antibodies against these kind of molecules, structural changes have to be made to obtain so called “haptens”. The hapten must be coupled to immunogenic proteins, called “carriers”. These connected derivatives are typically drug-linker adducts, in which the linker has a terminal functional group (i.e., carboxylic acid or aliphatic amine) that forms a covalent bond with the carrier. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Six nor-normorphine compounds were reacted with ethyl acrylate and ethyl bromoacetate. After the synthesis of the specific esters we hydrolyzed them to receive the N-carboxymethyl- and N-carboxyethyl-normorphine derivatives. The next step was the coupling phase with glycine ethyl ester, but the reactions didn’t work or the work-up process was not accomplishable. As an alternative route the normorphine-compounds were reacted with N-chloroacetyl glycine ethyl ester. These products were hydrolyzed in alkaline media and after the work-up process all of the derivatives contained the free carboxylic group of the glycine sidechain. All of the glycine ester and the glycine carboxylic acid derivatives are under biological tests.

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.