Specific and Generic Immunorecognition of Glycopeptide Antibiotics Promoted by Unique and Multiple Orientations of Hapten

Abstract

Conjugation chemistry does not always provide adequate spatial orientation of hapten in immunogens for the best presentation of generic or individual epitopes. In the present study, the influence of unique and multiple orientations of immunizing hapten on the immune response repertoire was compared to select generic recognition system. The glycopeptides, teicoplanin (TPL) and ristomycin (RSM), were conjugated to BSA to produce immunogens with unique and multiple orientations of haptens. Polyclonal antibodies generated against TPL conjugated through a single site were of uniform specificity and demonstrated selective TPL recognition, regardless of the coating conjugates design. The sensitivity (IC50) of 4 enzyme-linked immunosorbent assays (ELISAs) for TPL varied little within the 3.5–7.4 ng/mL, with a dynamic range of 0.2–100 ng/mL. RSM was coupled to BSA through several glycoside sites that evoked a wider repertoire of response. This first described anti-RSM antibody was selective for RSM in homologous hapten-coated ELISAs with IC50 values in the range 4.2–35 ng/mL. Among the heterologous antigens, periodate-oxidized TPL conjugated to gelatine was selected as the best binder of generic anti-RSM fraction. The developed ELISA showed group recognition of glycopeptides RSM, TPL, eremomycin, and vancomycin with cross-reactivity of 37–100% and a 10–10,000 ng/mL dynamic range. Thus, multiple presentations of immunizing hapten help expand the repertoire of immune responses and opportunities for the selection of the required fine-specificity agent.

Immunotechniques for the Group Determination of Macrolide Antibiotics Traces in the Environment Using a Volume-Mediated Sensitivity Enhancement Strategy

“…The literature search on the competitive immunoassay of small analytes has shown that an effective way to shift the cross-reactivity profile towards the desired cross-reactant is to use the latter as a coating or a labeled hapten. The phenomenon of immunorecognition enhancement was clearly observed with such analytes as spiramycin, vancomycin [27], and teicoplanin [28]. As can be seen from Figure 2, five coating conjugates were prepared.…”

Changing Cross-Reactivity for Different Immunoassays Using the Same Antibodies: Theoretical Description and Experimental Confirmation

“…They are often aggravated by the low immunogenicity or non-uniqueness of the selected structures and, moreover, require complex preliminary chemical synthetic work. An alternative approach to competitive immunoassays is to use different antigen derivatives in immunization and analysis [19][20][21][22][23][24][25][26][27][28]. This so-called "heterologous" immunoassay ensures that not all antibodies produced are involved in the competitive analytical interactions, and the spectrum of selectivity is thereby narrowed in comparison with the traditional "homologous" assay.…”