Development of determination of di-n-octyl phthalate (DOP) residue by an indirect enzyme-linked immunosorbent assay

Figures & data

Figure 1.  Synthetic route for hapten DOAP: (a) di-n-octyl 4-nitrophthalate (DONP) and (b) di-n-octyl 4-aminephthalate (DOAP).

Figure 2.  LC-MS analysis of DOAP: (a) the positive ions LC spectrum of DONP and (b) the mass spectrum of DONP.

Figure 3.  LC-MS analysis of DOAP: (a) the positive ions LC spectrum of DOAP and (b) the mass spectrum of DOAP.

Table 1. Effects of the factors on the performance of ELISA.

Factor	Amax	IC50	Amax/IC50
Coating method
4°C, overnight	1.58±0.06	33.88±1.0	0.047±0.003
37°C, 2 h	1.54±0.08	36.42±1.4	0.042±0.004
37°C, 2 h then 4°C, overnight	1.37±0.07	34.64±1.9	0.039±0.002
Ionic strength (mM)
5	1.54±0.08	35.49±1.3	0.043±0.003
10	1.76±0.05	34.66±0.8	0.051±0.002
20	1.78±0.11	35.72±1.5	0.050±0.002
40	1.82±0.12	36.94±3.5	0.049±0.002
pH
6.0	1.46±0.11	39.51±1.2	0.037±0.002
7.4	1.48±0.08	34.10±0.8	0.043±0.001
8.5	1.36±0.08	34.24±0.9	0.039±0.003
9.5	1.31±0.10	35.69±1.3	0.037±0.002
Methanol content (%)
5	1.52±0.14	32.21±1.0	0.047±0.006
10	1.45±0.13	28.23±0.9	0.051±0.006
20	1.44±0.11	24.12±0.9	0.060±0.004
30	1.46±0.13	26.35±1.6	0.055±0.004
Competitive time (h)
0.5	1.43±0.13	20.38±0.6	0.070±0.006
1	1.49±0.10	19.83±0.7	0.075±0.006
2	1.54±0.14	24.75±1.3	0.062±0.005
Colouration time (min)
10	1.05±0.17	21.28±1.8	0.049±0.001
20	1.54±0.11	19.92±1.1	0.077±0.004
30	1.57±0.15	21.07±1.6	0.074±0.002

Figure 4.  ELISA standard inhibition curves for DOP: ‘B/B₀’ is binding ratio of antibody/coating antigen in wells.

Table 2. Cross-reactivity of structure related compounds to DOP determined by competitive indirect ELISA.

Analytes	IC50/ng ml^−1	CR^b (%)
Di-n-octyl phthalate (DOP)	19.2	100
Di-n-octyl 4-nitrophthalate (DONP)	35.4	54.2
Di-n-octyl 4-aminophthalate (DOAP)	29.3	65.5
Di-iso-octyl phthalate (DIOP)	84.9	22.6
Di-hexyl phthalate (DHP)	110	17.6
Di-n-butyl phthalate (DBP)	90.3	21.2
Phthalic acid	>2000	<0.1
Phthalic anhydride	>2000	<0.1
^aELISA conditions were the same as that described in Figure 4.
^bCross-reactivity (%)=(IC50 of DOP/IC50 of other compounds)×100.

Figure 5.  Comparison between ELISA and HPLC-ESI-MS/MS analysis.