Production and Characterization of Monoclonal Antibodies against Sarafloxacin and Cross-Reactivity Studies of Related Fluoroquinolones

Abstract

Monoclonal antibodies were developed that bind sarafloxacin, a fluoroquinolone approved by the Food and Drug Administration for use against Escherichia coli in poultry. Splenocytes from mice immunized with a bovine serum albumin−sarafloxacin conjugate were fused with SP2/0 myeloma cells, and hybridomas secreting antibodies against sarafloxacin were selected and cloned. An enzyme-linked immunoassay was developed, and 50% inhibition of control values ranged from 7.3 to 48.3 ppb using sarafloxacin as the competitor. Tissue samples were spiked with sarafloxacin, and the average percent recoveries at 10, 50, and 100 ppb were 132, 78, and 81%, respectively. Monoclonal antibodies exhibiting high relative affinity for sarafloxacin were also characterized for their ability to detect five structurally related quinolones. The specificity and cross-reactivities of these antibodies are discussed in relation to three-dimensional, computer-generated molecular models of the fluoroquinolones. Keywords: Sarafloxacin; fluoroquinolone; ELISA; immunoassay

An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody

“…The substituent at position 8 in the structure of gatifloxacin was also believed to be a contribution to the high specificity of anti-gatifloxacin antibody [28]. The importance of substitution at position 1 in fluoroquinolone structures for antibody binding has also been stressed previously [27,30]. The poor affinity (<0.1%) of the produced anti-norfloxacin antibody toward ofloxacin, enrofloxacin and marbofloxacin seems reasonable due to different substituents at positions 1 and 7 in the structures of these compounds from those of norfloxacin.…”

“…Previous research has revealed that the substituents located in quinolone rings may influence the cross-reactivity of antibody [27,30]. All fluoroquinolones possess a nitrogen-containing, six-membered heterocyclic aromatic ring, a carboxylic group at position 3, a ketone group at position 4 and a fluorine substituent at position 6 (Table 1).…”

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review

“…When studying cross-reactivity and antibody recognition, the three-dimensional conformations and electrostatic potential isosurfaces are most often generated for the analyte of interest, hapten, and other competitors, and then compared to determine what structural features contribute to antibody recognition [33][34][35]. The electrostatic potential isosurface results from coloring the electronic density surface according to the calculated electrostatic potential values.…”

“…However, AMBER is a better force field than MM2 for proteins and nucleotides [66]. CAChe [34,35,39,50,51], Chemoffice [33], Hyperchem [30,36,47,48,53], Insight II [37], and Sybyl [32] can all be used for modeling the three-dimensional conformations of molecular surfaces or for computational docking experiments. However, the specific description of these computational programs is constantly changing due to the rapid development of the computational industry.…”

“…Many immunoassays used for detection of low molecular weight food contaminants such as an algaecide [30], antibiotics [6,16,[31][32][33][34][35], cork taint [36], herbicides [37][38][39][40], mycotoxins [41,42], pesticides [43][44][45][46][47][48], veterinary drugs [49][50][51] and other chemicals [52][53][54][55][56] have been developed with the use of CAMM methods (Table 1). CAMM can be used to assist in hapten design, study antibody-antigen recognition in cross-reactivity studies and model antibody binding sites (Table 1).…”