Amide-containing neoepitopes: the key factor in the preparation of hapten-specific antibodies and a strategy to overcome.

For a long time, people have suffered from uncertainty, complexity, and a low success rate in generating and screening antibodies against small molecules, which have become the core bottlenecks of immunochemistry. Here, the influence of antigen preparation on antibody generation was investigated at both molecular and submolecular levels. Neoepitopes (amide-containing neoepitopes) formed in the preparation of complete antigens are one of the most important factors limiting the efficiency of generating hapten-specific antibodies, which was verified by different haptens, carrier proteins, and conjugation conditions. Amide-containing neoepitopes present electron-dense structural components on the surface of prepared complete antigens and, therefore, induce the generation of the corresponding antibody with much higher efficiency than target hapten. Crosslinkers should be carefully selected and not overdosed. According to these results, some misconceptions in the conventional anti-hapten antibody production were clarified and corrected. By controlling the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) during the synthesis of immunogen to limit the formation of amide-containing neoepitopes, the efficiency of hapten-specific antibody generation could be significantly improved, which verified the correctness of the conclusion and provided an efficient strategy for antibody preparation. The result of the work is of scientific significance in the preparation of high-quality antibodies against small molecules.

The effect of chlorophyll on the enzyme-linked immunosorbent assay (ELISA) of procymidone in vegetables and the way to overcome the matrix interference.

BACKGROUND: There is now an increasing demand for the immunoassay of procymidone residue in foodstuffs. However, the matrix interference could significantly affect the analysis. Till now there is no detailed information on the source of the interference and the mechanism involved, which greatly limits the real application of these techniques. RESULTS: Significant matrix effect was observed in the enzyme-linked immunosorbent assay (ELISA) of procymidone in negative vegetable samples (leek, broccoli and cucumber). By the investigation with both vegetable extracts and standard solutions, the chlorophyll was confirmed as an important source of the matrix effect. Therefore, a new strategy was proposed for the pretreatment based on the exploitation of 5-sulfosalicylic acid. It was demonstrated to effectively eliminate chlorophyll and exhibited little effect on procymidone and the competitive indirect ELISA (ci-ELISA) performance. The established technique was validated with different vegetables. With the spiking concentration of procymidone investigated, the recovery rate of ci-ELISA was 71.52-120.37%, and the relative standard deviation was 4.05-17.61%. CONCLUSION: Chlorophyll was for the first time illuminated as an important source of matrix interference to the immunoassay of procymidone in vegetables. A new pretreatment based on 5-sulfosalicylic acid was established to remove chlorophyll and therefore eliminate the matrix effect. Validated with different vegetable samples, the new technique was demonstrated much better efficiency in comparison to conventional methods, which indicated its promising application for the development of immunoassays of herb-origin samples. © 2021 Society of Chemical Industry.

Hapten-Branched Polyethylenimine as a New Antigen Affinity Ligand to Purify Antibodies with High Efficiency and Specificity.

Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR-PEI) was conjugated to CNBr-Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR-PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL-18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.