Enzyme-linked immunosorbent assay for the detection of neomycin in milk: effect of hapten heterology on assay sensitivity

Figures & data

Figure 1.  Chemical structure of neomycin.

Figure 2.  Titer (upper panel) and IC₅₀ (lower panel) change curves of antisera from various bleeds of rabbits. Neomycin-OVA_E was used as coating antigen at the concentration of 1 µg/ml. The titer of antiserum was defined as the dilution factor which gave 2.1 times OD values than negative serum. Each point represents the average of three well replicates.

Table I. Titers and IC₅₀ values of final antisera to neomycin-OVA_E and neomycin-G-OVA.

		Coating antigen^a (titer^b/IC50 ^c)
Immunogen	Antiserum	Neomycin-OVAE	Neomycin-G-OVA
Neomycin-BSA	Serum 1	3686 400/4.21	921 600/0.89
	Serum 2	921 600/1.56	230 400/0.45
Neomycin-HAS	Serum 3	230 400/12.5	576 00/1.01
Neomycin-G-BSA	Serum 4	115 200/8.93	230 400/ > 200
^aThe amount of neomycin-OVAE and neomycin-G-OVA was 100 ng/well. ^bThe titer of antiserum was defined as the dilution factor which gave 2.1 times OD values than negative serum. ^cThe unit of IC50 value is ng/ml.

Figure 3.  Representative standard curves of the neomycin ELISA using neomycin-OVA_E (▪), neomycin-F-OVA (•) and neomycin-G-OVA (▴) as coating antigen, respectively. B and B₀ are the absorbances of the sample with/without neomycin, respectively. Each point represents the average of three well replicates, and error bars represent standard deviations.

Table II. B₀ and IC₅₀ values of ciELISA under optimum concentration of antiserum and coating antigens.

				Heterology^e
Coating antigen^a (ng/well)	Antiserum dilution^b	B0 ^c	IC50 ^d (ng/ml)	Spacer arm	Structure	Site
Neomycin-OVAE (40)	1:250 000	1.88	1.21	HO	HO	HO
Neomycin-F-OVA (10)	1:50 000	1.64	0.35	HE	HO	HO
Neomycin-G-OVA (10)	1:50 000	1.71	0.33	HE	HO	HO
Neamine-OVAE (100)	1:100 000	1.53	>100	HO	HE	HO
Neamine-G-OVA (50)	1:20 000	1.86	0.15	HE	HE	HO
Neomycin-OVAP (50)	1:20 000	1.78	0.098	HE	HE	HE
^aThe concentration of each coating antigens has been optimized to give the best sensitivity. ^bSerum 2, which was generated against neomycin-BSA, was used. ^cB0 is the absorbances of the sample without neomycin. ^dThe unit of IC50 value is ng/ml, and the values are the average of triplicates. ^eHO, homologous; HE, heterologous.

Figure 4.  Influence of matrix in milk on the neomycin ELISA standard curve. The dilution times of milk in assay buffer (PBS, pH 7.0) were as follows: control (▪), 50-fold (•), 20-fold (▴), and 10-fold (▾). Each point represents the average of three well replicates, and error bars represent standard deviations.

Table III. Recoveries of neomycin from spiked milk (n=4).

Level added (ng/ml)	Level found ±SD (ng/ml)	Recovery (%)	CV (%)
2.5	2.74±0.24	109	8.8
5	5.19±0.40	104	7.7
10	9.70±0.46	97	4.7
20	17.30±1.21	87	7.0