Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk
ABSTRACT As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal
antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several
heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used
as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different
dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic
in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive
assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%.
We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases
(11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml).
The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good
sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.