MCPA, dichlorprop and 2,4-D do not affect the analytical method of determining 2,4,5-T. At 50% displacement 2,4-DB display affinity for the antiserum. (I50 value ...
BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 13: 177–178 (1999)
EXTENDED ABSTRACTS
Quanti®cation of 2,4,5-trichlorophenoxy acetic acid by ¯uorescence enzyme-linked immunosorbent assay with secondary antibody F. GarcõÂa SaÂnchez,1* A. Navas DõÂaz,1 A. F. GonzaÂlez DõÂaz1 and S. A. Eremin2 1 2
Department of Analytical Chemistry, Faculty of Sciences, University of Malaga, Malaga 29071, Spain Moscow State University, Moscow, 119899 Russia
Received 24 June 1998; accepted 29 June 1998
INTRODUCTION For detection and quantification at ng mLÿ1 levels of a specific pesticide in a sample, an immunoassay is adequate because it is an analytical technique based on the specific combination of antigens and antibodies. Assay sensitivities have also been greatly enhanced by methods for attaching easily detected enzyme labels to antigens or antibodies such as, for example, in enzymelinked immunosorbent assay (ELISA) procedures (Clark et al., 1986). Fluorometry based on the use of fluorogenic substrates in ELISA should, in principle, be superior to spectrophotometric detection based on the use of chromogenic substrates. This method has the advantage that one label can be used for measuring any antigen for which a suitable first antiserum is available.
EXPERIMENTAL
15 min at 4 °C. One column received no 2,4,5-T to determine the maximum fluorescent reading. The immobilized antibodies were detected by labelled anti-Ig antibodies. Goat anti-rabbit–horseradish peroxidase conjugate (1:300) was added to the plates and incubated for 1 h at 4 °C. Substrate solution containing 3 mg mLÿ1 of phydroxyphenyl acetic acid, H2O2 (7.35 10ÿ5 M) and Tris-HCl (0.1 M) pH 8.5 buffer was prepared. 200 mL of this solution was added to each well. Fluorescence was allowed to develop for1 hand wasmeasured asa 320 nm wavelength excitation (slit excit. 5 nm) and 420 nm wavelength emission (slit em. 10 nm).
RESULTS AND DISCUSSION Standard curves To construct a calibration curve aliquots of 2,4,5-T
Instrumentation Fluorescent readings were accomplished in a PerkinElmer plate reader accessory. A bifurcated high-grade fused silica fibre optic with a high transmission range of 240–2200 nm was used to transfer the excitation and emission energies between the well plate and the spectrometer. ELISA procedure The immunoassay used was double antibody sandwich (DAS)-ELISA. Polystyrene microtiter plates were coated [200 mL of HGG-245-T conjugate (Fleeker 1987) (Habeed 1966) to each well] and incubated for 24 h at 4 °C. Diluted antiserum (1:1250) in phosphate buffer, pH 7 was preincubated for 1 h with 2,4,5-T at different concentrations; 200 mL of these preincubated mixtures was transferred to the wells of the microtiter plate and incubated for *Correspondence to: F. Garcı´a Sa´nchez, Department of Analytical Chemistry, Faculty of Sciences, University of Malaga, Malaga 29071, Spain. Copyright 1999 John Wiley & Sons, Ltd.
Figure 1. ELISA standard calibration curve for 2,4,5-T. CCC 0269–3879/99/020177–02 $17.50
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F. Garcı´a Sa´nchez et al.
EXTENDED ABSTRACTS
Table 1. Cross-reactivity of the 2,4,5-T and related compounds Formula
Compounds 2,4-D
2,4-DB
*% RC50%a
*% RC50%b
0
1.4
0.0374
3.3
2,4,5-T
100
100
MCPA
0
0
Dichlorprop
0.6
* Cross-reactivity estimated at 50% displacement of signal. a Fluorescence ELISA. b Polarization fluoroimmunoassay.
covering the range 0.01–5000 ng mLÿ1 were preincubated with antiserum, and then incubated for 15 min with previously coated HGG-2,4,5-T. Free 2,4,5-T was then quantified by its competition with the coated 2,4,5-T for binding to anti-2,4,5-T (antibody). In this method, HRP is conjugated to a secondary antibody and its concentration on the wells is inversely proportional to 2,4,5-T concentration. A plot of fluorescent readings against concentration (ng mLÿ1) of the standard 2,4,5-T (Fig. 1) in logarithmic scale, displays a typical calibration graph fitted to an IC50 four parameter logistic model. A dynamic range covering standard concentration of 2,4,5-T between 0.023 and 5000 ng mLÿ1 is deduced. The minimum detectable concentration (MDC) (O’Connell et al., 1993) was 0.023 ng mLÿ1 and the relative standard deviation was 6.9% (P = 0.05).
displacement were determined and the cross-reactivity (CR) of a compound was determined as (Miller et al., 1992): 50% CR = (2,4,5-T50%)/(CR50%) 100. [2,4,5T50%: concentration of 245-T at the assay midpoint (F/ Fo = 0.652). CR50%: concentration of cross-reactant at the assay midpoint (F/Fo = 0.652)]. MCPA, dichlorprop and 2,4-D do not affect the analytical method of determining 2,4,5-T. At 50% displacement 2,4-DB display affinity for the antiserum (I50 value = 13 mg mLÿ1 and I50 value for 2,4,5-T is 5 ng mLÿ1). A comparison of the antibody specificity in two different methods [fluorescence ELISA (*%CR50%a) and the automated polarization fluoroimmunoassay (*%CR50%b) enabled us to make a conclusion: a fluorescence ELISA shows more specificity than polarization fluoroimmunoassay (Table 1).
Speci®city of the assay Analytical interference due to the limited selectivity of antibodies is commonly referred to as cross-reactivity (the ratio of competitive binding between two or more structurally similar ligands with the antibody binding sites). The cross-reactivity was calculated by using the 50% displacement method. Displacement curves were prepared by incubation of various doses of cross-reactant with a constant amount of antibody. After fitting the curves, the doses of cross-reactant that give 50%
Copyright 1999 John Wiley & Sons, Ltd.
REFERENCES Clark, M. F., Lister, R. M. and Bar-Joseph, M. 1986. Methods Enzymol. 118: 742. Eremin, S. A., Egorov, A. M., Melnichenko, O. A. and Tumanov, A. A. 1995. Journal of Analytical Chemistry. 50: 198. Fleeker, J. J. 1987. Assoc. Off. Anal. Chem. 70: 874. Habeed, A. F. S. A. 1966. Anal. Biochem. 14: 328. Miller, J. T., Valdes, R. 1992. J. Clin. Immunoassay, 15: 97. O’Connell, M. A., Balanger, B. A. and Haaland, P. D. 1993. Chemom. Intell. Lab. Syst. 20: 97.
Biomed. Chromatogr. 13: 177–178 (1999)
