Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

Abstract

A protein conjugate of streptomycin (streptomycin-bovine serum albumin (BSA) conjugate) was prepared and used as immunogen to produce monoclonal antibodies (MAb). One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentration (IC(50)) value of 4.65 ng/ml and the IC(20) value of 0.21 ng/ml in phosphate buffered saline (PBS). At optimum conditions, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic assay (CGIA) were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/ml were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum detection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

Fig. 1

Ultraviolet scan analyses of streptomycin-BSA (a) and streptomycin-OVA (b) conjugates Str: streptomycin; Str-BSA: streptomycin-BSA conjugate; Str-OVA: streptomycin-OVA conjugate

Fig. 1

Ultraviolet scan analyses of streptomycin-BSA (a) and streptomycin-OVA (b) conjugates Str: streptomycin; Str-BSA: streptomycin-BSA conjugate; Str-OVA: streptomycin-OVA conjugate

Fig. 2

Mean calibration curves of the ELISA in the two matrices Data represent the means of four determinations

Fig. 3

Detection of streptomycin with the CGIA A series of dilutions (0, 20, 30, 40, 50, 60 and 70 ng/ml) of streptomycin were prepared in streptomycin-free milk (a) and swine urine (b)

Fig. 3

Detection of streptomycin with the CGIA A series of dilutions (0, 20, 30, 40, 50, 60 and 70 ng/ml) of streptomycin were prepared in streptomycin-free milk (a) and swine urine (b)

Fig. 4

Specificity analysis of the CGIA 1, 2, 3, 4, 5, 6 and 7 were streptomycin, neomycin, gentamycin, kanamycin, sulfamethazine, penicillin G and chlortetracycline, respectively