The hapten rigidity improves antibody performances in immunoassay for rifamycins: Immunovalidation and molecular mechanism

The immunogenicity of haptens determines the performance of the resultant antibody for small molecules. Rigidity is one of the basic physicochemical properties of haptens. However, few studies have investigated the effect of hapten rigidity on the strength of an immune response and overall antibody performance. Herein, we introduce three molecular descriptors that quantify hapten rigidity. By using of these descriptors, four rifamycin haptens with varied rigidity were designed. The structural and physicochemical feasibility of the designed haptens was then assessed by computational chemistry. Immunization demonstrated that the strength of induced immune responses, i.e., the titer and affinity of antiserum, was significantly increased with increased rigidity of haptens. Furthermore, molecular dynamic simulations demonstrated conformation constraint of rigid haptens contributed to the initial binding and activation of naïve B cells. Finally, a highly sensitive indirect competitive enzyme-linked immunosorbent assay was developed for detection of rifaximin, with an IC50 of 1.1μg/L in buffer and a limit of detection of 0.2–11.3μg/L in in raw milk, river water, and soil samples. This work provides new insights into the effect of hapten rigidity on immunogenicity and offers new hapten design strategies for antibody discovery and vaccine development of small molecules. Environmental implication Rifamycins (RFs) exhibit broad-spectrum antimicrobial properties and are widely used in human and veterinary medicine. However, overuse and illegal use of RFs in humans, livestock, and aquatic organisms has resulted in their release into the environment. Due to their residue in water and sediments, RFs may induce chronic toxicity and endocrine disruption in aquatic life and in humans. Further, excessive use or abuse of RFs can lead to antibiotic-resistance genes in pathogenic microorganisms. Therefore, it is necessary to identify high-affinity, broad-specific antibodies as a means by which to establish rapid, sensitive, and high-throughput immunoassays for the measurement of RFs. Such measurement will reduce the risk of exposure to RFs.