Fluorescence polarisation for immunoreagent characterisation

Journal of Immunological Methods 213 Ž1998. 31–39

Fluorescence polarisation for immunoreagent characterisation ¨ P. Onnerfjord

a,)

, S. Eremin b, J. Emneus ´ a, G. Marko-Varga

c

a

b

Department of Analytical Chemistry, Lund UniÕersity, P.O. Box 124, 221 00 Lund, Sweden Department of Chemistry, DiÕision of Chemical Enzymology, M.V. LomonosoÕ Moscow State UniÕersity, Moscow 119899, Russian Federation c Astra Draco, Bioanalytical Chemistry, Preclinical Research and DeÕelopment, P.O. Box 34, 221 00 Lund, Sweden Received 21 August 1997; revised 15 December 1997; accepted 14 January 1998

Abstract Antibodies were characterised using fluorescence polarisation, a homogeneous assay technique in which all reagents are in solution. Kinetic studies on the association and dissociation of the immunocomplex were performed. A competitive assay was used and the sensitivities, operational linearities, as well as the specificities of the immunoassays were experimentally determined for various antibody preparations with specificity for triazines. Detection limits for atrazine in water samples were determined to be within the range of 0.08–0.4 ng mly1 using a 5-min incubation time and a 0.5-ml sample volume. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antibody characterisation; Fluorescence polarisation; Homogenous immunoassay; Triazines; Atrazine

1. Introduction The quality and characteristics of immunoreagents, as well as the kinetics of immunocomplex formation are of the utmost importance to any immunological method. Antibodies are the key reagents for the development of an immunoassay and their quality generally needs to be high when used for analytical purposes. The potential and improvements of tailor-made antibodies have accelerated the development of recombinant technologies ŽKramer and Hock, 1996., antibody engineering ŽHock et al., 1995; Marco et al., 1995., and recently molecular imprinting techniques using polymer chemistry ŽPiletsky et al., 1995; Siemann et al., 1996.. Enzyme )

Corresponding author.

linked immunosorbent assays ŽELISAs. commonly performed on microtiter plates are among the most sensitive and widely used assay techniques. However, these techniques can present certain drawbacks such as complex sample preparation before the ELISA, multiple pipetting steps Žboth washing and reagent addition. and incubation steps which are relatively time-consuming and error-prone Žif manual.. In addition to the above well established assay techniques, immunosensors have gained in interest, and are now increasingly being recognised as alternatives. Notably, optical immunosensors such as surface plasmon resonance, SPR ŽBrecht et al., 1995., and evanescent wave sensors, EWS ŽOrozlan et al., 1993. are making steady progress. In order to make predictions about antibody characteristics, multi-array

0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 1 9 - 2

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¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

approaches utilising statistical calculation programs ŽWortberg et al., 1996, 1995., as well as identification using mass spectrometry linked to chromatographic techniques ŽPichon et al., 1995. have recently been developed. In this paper, a fast, simple and sensitive method, based on polarisation fluoroimmunoassay ŽPFIA. is described for the characterisation of five different anti-s-triazine antibodies using a fluorescein-labelled tracer ŽAg).. The main goal of this work was to characterise the various Ab-preparations in order to develop other assays Že.g., flow immunoassay systems. on the basis of the information obtained.

2. Materials and methods 2.1. Chemicals and immunoreagents Desisopropyl-atrazine, E-103320 Lot 30128; desethyl-atrazine, E-103310 Lot 30505; 2-hydroxyatrazine, E-103330 CH 21123; atrazine, IPO 005, simazine, IPO 692; terbuthylazine, E-173000 were all purchased from Dr. Ehrenstorfer ŽAugsburg, Germany.. Propazine 139-40-2 was purchased from Polyscience ŽNiles, IL, USA.. Stock solutions of the different s-triazines were prepared in acetonitrile and kept in the dark at q48C. The stock solutions were further diluted with 0.01 M PBS buffer ŽpH 7.4. when used for PFIA measurements. All other chemicals were of analytical reagent grade. Fluorescein thiocarbamyl ethylene diamine ŽEDF. was synthesised as previously described ŽPourfarzaneh et al., 1980. from fluorescein isothiocyanate isomer I ŽFITC. ŽCat. No. F-4274, Lot 105H2611, Sigma, St. Louis, MO, USA.. The derivative 4,6-dichloro-N-Žisopropyl.-1,3,5-triazin-2-amine was synthesised from cyanuric chloride ŽSigma. and isopropylamine ŽMerck, Darmstadt, Germany. as described by Goodrow et al. Ž1990.. The tracer ŽiPrrClrEDF., see Table 1, was synthesised by reacting 2 mg Ž10 m mol. of 4,6-dichloroN-Žisopropyl.-1,3,5-triazin-2-amine dissolved in 0.2 ml dimethylformamide ŽDMF. ŽFluka, Buchs, Switzerland. with 4 mg Ž8 m mol. of EDF. The reaction mixture was stirred for 1 h at room temperature and kept overnight at q48C. The 50-m l portions of the reaction mixture were then separated by thin-

Table 1 Compound

Structural information R1rR2rR3

Tracer ŽI. Tracer ŽII. Immunogen ŽI. Immunogen ŽII. Immunogen ŽIII. Immunogen ŽIV. Immunogen ŽV. Atrazine ŽATR. Simazine ŽSIM. Desisopropylatrazine ŽDIA. Desethylatrazine ŽDEA. Propazine ŽPROP. Hydroxyatrazine ŽHYA. Terbuthylazine ŽTBA.

NHiPrrClrEDF NHiPrrSŽCH 2 . 2 CO-EDFrNHEt NHiPrrClrNHŽCH 2 .5 CONH-KLH NHiPrrClrNHŽCH 2 .5 CONH-KLH NHŽCH 2 .5 CONH-KLHrClrNHEt NHiPrrSŽCH 2 . 2 CONH-KLHrNHEt NHiPrrSŽCH 2 . 2 CONH-BSArNHEt NHiPrrClrNHEt NHEtrClrNHEt NH 2 rClrNHEt NHiPrrClrNH 2 NHiPrrClrNHiPr NHiPrrOHrNHEt NhterbrClrNHEt

The structures of fluorescein labelled tracer I–II ŽEDF s fluorescein thiocarbamyl ethylene diamine., immunogen structures ŽI–V., atrazine and other s-triazines structures. The different R-groups that are substituted at the symmetrical 2,4,6-positions of the 1,3,5-triazine are denoted R1rR2rR3. Abbreviations: iPr s isopropyl; Et sethyl; terbs terbuthyl.

layer chromatography, TLC ŽKieselgel 60, Merck. using methylene chloridermethanol Ž5:1 vrv. as the eluent. The major yellow band at R f 0.5, containing the tracer, was isolated and stored in methanol at y208C. The molecular weight of the tracer product was confirmed using MALDI-TOF MS ŽVoyager DE, Perseptive Biosystems, Framingham, MA, USA. and a-cyano-hydroxycinnamic acid ŽFluka. as the sample matrix. Another tracer ŽiPrrSŽCH 2 . 2 COEDFrEt. was synthesised using a derivative of atrazine, obtained from a reaction with thiopropionic acid ŽGoodrow et al., 1990.. An amount of 8 mg Ž80 m mol. N-hydroxysuccinimide and 8 mg Ž40 m mol. of N, N X-dicyclohexyl-carbodiimide was added to the solution of 5.7 mg Ž20 m mol. s-triazine COOH-derivative in 1 ml of N, N X-DMF. The reaction mixture was stirred for 4 h at room temperature. A total of 250 m l Ž5 m mol. of this solution was added to 2.5 mg Ž5 m mol. of EDF. The reaction mixture was stirred for 3 h at room temperature. Small portions of the reaction mixture Ž20 m l. were separated by TLC using methylene chloridermethanol Ž4:1 vrv. as the eluent. The main yellow band at R f s 0.7 was isolated and stored in methanol at y208C. The concentrations of the two tracers were estimated spectrophotometrically at 492 nm, assuming

¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

an adsorptivity in sodium bicarbonate buffer Ž50 mM, pH 9.0. to be the same as for fluorescein Ž ´ s 8.78 P 10 4 My1 cmy1 .. The tracer solution was further diluted in 0.01 M PBS buffer ŽpH 7.4. and used for PFIA measurements. 2.2. Antibodies The different antibodies were obtained and designated according to the following: Antibody I Žpolyclonal, from rabbit: 12r2d. was kindly provided by Dr. M.-P. Marco ŽCID-CSIC, Barcelona, Spain.; Antibody II Žmonoclonal: K4E7. was kindly provided by Dr. T. Giersch ŽTechnical University, Munich, Germany.; Antibody III Žpolyclonal, from rabbit: Sima. and Antibody IV, Žpolyclonal, from rabbit: Arina., were kindly provided by Immunotech ŽMoscow, Russia.; Antibody V Žpolyclonal, from rabbit: BSA-D. was kindly provided by Dr. M. ŽVeterinary Research Institute, Brno, Czech Franek ´ Republic.. All antibody preparations were purified using ŽNH 4 . 2 SO4 precipitation Žat 50% saturation. and centrifugation for 10 min Ž4000 = g .. The pellets were resuspended in 0.01 M PBS buffer ŽpH 7.4. and precipitated a second time before further clean-up was performed using dialysis for approximately 24 h in 0.01 M PBS Žthree times change of buffer.. Dialysis tubing from Spectrum ŽLos Angeles, CA, USA. with MW cut-off of 12–14 kDa was used. The protein concentrations were determined by calculating the difference in absorbance at 280 nm against a PBS blank. Assuming that the IgG concentration of 1 mg mly1 corresponds to 1.35 absorbance units ŽHarlow and Lane, 1988., IgG concentrations for Abs I–V were found to be 1.9, 2.0, 3.2, 5.2 and 3.3 mg mly1 , respectively. 2.3. Instrumentation and method A FPM-1 ŽJolley Consulting, Round Lake, IL, USA. instrument was used for all experiments and measurements were performed in borosilicate glass cuvettes 12 = 75 mm, ŽSchott Gerate, ¨ Hofheim, Germany.. The FPM-1 was connected to a recorder and a PC-computer with data acquisition software for the FPM ŽFPM-1 Data Management. was used for the collection of data. The total time for one measure-

33

ment was 13 s including lamp control. Readings could be made in both static Žone measurement. and kinetic Žmeasurements every 13 s. mode. PFIA standard curves were obtained by sequentially adding to the cuvette 0.5 ml of a standard or sample, 0.5 ml of tracer, and finally 0.5 ml of antibody at the dilution, which gave approximately 70% of maximum binding of tracer Ži.e., 200–250 mP. from the antibody dilution curve. The incubation time before readout was 5 min.

3. Result and discussion PFIA is a technique which utilises polarised light to detect the speed of molecular rotation in aqueous solutions ŽDandliker et al., 1981.. It is a homogenous assay, which relies on the increase in fluorescence polarisation of a fluorescent tracer when bound to a corresponding antibody, compared with the fluorescence polarisation of unbound tracer. The difference in the polarised fluorescence can be expressed according to Eq. Ž1.. P s Ž I V y IH . Ž I V q IH .

Ž 1.

P equals the polarisation factor and I V and I H are the vertical and horizontal components of the emitted fluorescence intensity. The measured polarisation will be in a stoichiometric relationship to the amount of free and bound tracer present in the sample. 3.1. Immunossay optimisation Due to the demands of high sensitivity for antigen determinations, an immunoassay should normally be in the nanogram per milliliter–picogram per milliliter range, whereas, the specificity of the assay depends on the purpose of the assay. In cases where the metabolites and transformation products are of special interest due to, e.g., toxicity and biological activity, a broad specificity of the antibody is desirable. If, on the other hand, the determination of a specific analyte is of interest, antibodies with a narrow specificity are preferably used. The specific characteristic of a given antibody preparation will ultimately be determined by the synthesised carrier protein conjugate, the immunisation, and the purifications undertaken with the serum.

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¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

Hence, conjugation position, structure and chain length of the anchoring group between target molecule and the carrier protein will have dramatic effects on the Ab properties. The sensitivity and selectivity of the immunoassay ŽGoodrow et al., 1990; Marco et al., 1995. is affected by these factors. The different antibody preparations ŽAb I–V. used in this work, were obtained from various corresponding immunogen structures ŽI–V., illustrated in Table 1. Several carrier proteins, as well as a variety of structures and positions of the linker arm were used. As seen in Table 1, the immunogen for Ab IV and V consisted of a sulphur-containing spacer –SŽCH 2 . 2 CONH– substituted at the R2 position. Immunogens I–III have the same spacer arm –NHŽCH 2 .5 CONH–, whereas immunogens I–II have the protein conjugated at the R3 position and immunogen III at the R1 position. The conjugated protein was keyhole limpet hemocyanin ŽKLH. for immunogens I–IV and bovine serum albumin ŽBSA. for immunogen V. In a similar fashion, the chemical structure of the tracer, i.e., labelling position, structure and chain length of the anchoring group influence the sensitivity, as well as the selectivity of the assay ŽEremin and Samsonova, 1994; Eremin et al., 1994.. Theoretically, a sensitive competitive immunoassay can be developed when the antibody affinity constant Ž K aff . for the tracer is of the same order of magnitude as for the analyte. However, for a small target antigen, the affinity for the tracer is normally higher than for the analyte as the antibody can recognise parts of the anchoring chain used for the hapten-carrier protein binding. To overcome this problem, antigens can be labelled at different positions to the one used for the immunogen protein conjugation. In addition or alternatively, a linker with different length and structure to that employed for the immunogen, can be used. Previously, it was found that the fluorescein-labelled tracer iPrrClrEDF with a short two carbon bridge between fluorescein and the antigen resulted in the highest sensitivity for Ab III and IV ŽEremin and Samsonova, 1994; Eremin et al., 1994., and, therefore, this tracer was selected to be used in this work.

means that the concentrations of analyte, tracer and antibodies must be within the same order of magnitude. As the tracer signal Ži.e., tracer concentration. sets the sensitivity of an assay, the lowest possible tracer concentration should be used for highly sensitive assays. For the iPrrClrEDF tracer, the lowest concentration was determined to be 0.9 nM, giving a signal approximately 10 times higher than the background signal from buffer Žtotal fluorescence intensity.. The optimum antibody concentrations were determined by recording dilution curves Žsee Fig. 1. for the five different antibody preparations ŽAb I–V., using the optimal tracer concentration Ž0.9 nM.. A tracer volume of 1 ml was added to 0.1 ml of 100, 33, 11, 3.7, and 1.2 m g mly1 of Ab I–V, respectively. The solutions were mixed and incubated at room temperature for 5–7 min, as this ensured that ) 90% of the mixtures reached equilibrium. As seen in the dilution curves in Fig. 1, sufficient binding of tracer and antibody could be obtained with all antibodies except with Ab V and Ab 2,4,5-T. However, by using a newly synthesised fluorescent tracer iPrrEtrSŽCH 2 . 2 CO-EDF Žsee details in Section 2., Ab V could also be used in an atrazine immunoassay Žsee Fig. 2.. The anti-2,4,5-T antibody was used as a control to investigate non-specific binding of the tracer to IgGs in general, although the non-specific binding of the iPrrClrEDF tracer was negligible as seen in Fig. 1. The optimum antibody concentration was chosen when approximately 70% of the tracer

3.1.1. Tracer and antibody conditions For small molecules, e.g., triazine pesticides, the competitive assay format is commonly used. This

Fig. 1. The influence of print antibody I–V dilutions, Ab I Žv ., Ab II Ž,., Ab III Žl., Ab IV Žn., Ab V Ž'., and Ab 2,4,5-T Žq. on the fluorescence polarisation ŽmP.. The 1-ml Ž0.9 nM. iPrrClrEDF tracer and 100 m l Ab were incubated for 5 min.

¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

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Table 2 Selectivity of Ab I–IV measured with various cross-reactants and expressed as cross-reactivity values Ž%. vs. the atrazine response

Fig. 2. Dilution curves of Ab V using two different tracers. Tracer I iPrrClrEDF Ž`. and tracer II iPrrEtrSŽCH 2 . 2 CO-EDF Žv .. wtracer Ix s 0.9 nM and wtracer IIx s 0.4 nM

was bound to antibodies, which for Abs I–V were used at IgG-concentrations of 4, 1.1, 10, 24, and 15 m g mly1 , respectively. 3.2. SelectiÕity As previously discussed, an important aspect to consider when choosing an appropriate antibody for a particular assay is selectivity in terms of cross-reactivity ŽCR.. Competitive PFIA procedures were performed, using the optimised Ab and tracer concentrations with analyte Žatrazine or cross-reactant. concentrations between 0.01 to 10 000 ng mly1 . CR data obtained from these calibration curves was calculated according to Eq. Ž2., cross-reactivity Ž % .

Analyte

Ab I

Ab II

Ab III

Ab IV

ATR SIM DIA DEA PROP HYA TBA

100 1 - 0.1 0.4 33 - 0.1 1

100 48 3 4 500 0.7 20

100 69 6 8 450 0.1 4.5

100 6.4 2.3 51 250 3.9 2.6

body can be selected. In fact, using Ab IV makes it possible to monitor all the selected triazines at acceptable sensitivities. The signal response will be related to the sum of the cross-reactants and, therefore, can be used as a group-selective screening method. However, to obtain the individual concentrations, a separation step must be included. This can be useful in following the transformation rate, allowing investigation of the mechanisms which increase or decrease the rate of transformation. On the other hand, when using Ab I it is possible to follow the variation in concentration of the parent compound and one transformation product without chromatographic separation. This analytical approach is of interest when the transformation products are non-toxic or of less importance. 3.3. Kinetics

s 100 = IC 50 Ž atrazine. rIC 50 Ž cross-reactant.

Ž 2. where IC 50 is the concentration at which 50% of the antibodies are bound to the analyte. The CR-values using Ab I–IV for seven s-triazines, i.e., the parent compound atrazine, simazine, and five well known transformation products of these, are shown in Table 2. Antibody preparations II–IV showed high cross-reactivity values for ATR, SIM, PROP and DEA but lower values for the other analytes. Ab I had the most narrow CR-profile, showing significant affinity Ž) 1%. only for atrazine Ž100%. and propazine Ž33%.. Thus, depending on the purpose of an assay, i.e., the measurement of a single or a group of analytes, the appropriate anti-

In order to develop immunoassays with a high sample throughput, it is of the utmost importance to investigate the kinetics of the immunoreactions. The time required to reach equilibrium in the PFIA system was evaluated by mixing equivalent volumes of sample, tracer and antibody at concentrations of 4, 1, 10, and 24 m g mly1 for Ab I–IV, respectively. The concentration of tracer was kept at 0.3 nM and measurements were made in the kinetic mode every 20 s. The results, shown in Fig. 3, indicate that incubation times of 4.5, 2.0, 7.0 and 2.5 min, respectively, were sufficient to reach ) 90% of the equilibrium at ambient temperature. The replicability of this experiment is illustrated in Fig. 3a, where two consecutive runs are overlaid. As shown in Fig. 3,

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Fig. 3. Association kinetics of AbAg) complex using Ža. Ab I, Žb. Ab II, Žc. Ab III, Žd. Ab IV with iPrrClrEDF tracer Ž0.9 nM.. At the marked arrow, displacement of Ag) with excess of atrazine occurs Ž100 m l at 10 m g mly1 .. For Ab I, two consecutive injections were made, showing good repeatability. The wAbx were 4, 1.1, 10, and 15 m g mly1 , respectively.

Ab II has the slowest dissociation ŽFig. 3b. while Ab IV has the fastest ŽFig. 3d.. Ab II is a monoclonal antibody with a rather long displacement time but an almost constant dissociation slope. This can be explained by the fact that monoclonal Abs, which all have the same structure, only have one type of affinity mechanism resulting in a constant slope. However, the polyclonals ŽI, III and IV. have various affinities Žhigh affinity and low affinity populations. observed as different slopes at the beginning and end of the dissociation curve. A drawback when using polyclonal serum is that it contains a population of several different antibodies in the same preparation, thus making it difficult to derive kinetic constants. However a rough estimation of the affinity constant Žmean value for the polyclonal antibodies. can be made at the midpoint of the calibration curve, where K aff s 1rIC 50 . The estimated constants for Ab I–IV were found to be 0.8–2.4 = 10 8 My1 , which is considered to be normal for most immunoassays. 3.4. Displacement or association assay The kinetics during the association of immune complexes are much faster than the dissociation ki-

netics and, therefore, non-equilibrium conditions are present when using the dissociation mode. If the antibody affinity for the tracer and analyte differ considerably, the order of mixing the reagents can drastically change the sensitivity. Three modes of mixing are possible, as shown in the following reactions.

AgqAg)qAb| AbAg)qAbAg

Ž 3.

AgqAb| AbAg ™qAg) | AbAgqAbAg)

Ž 4.

Ag)qAb| AbAg) ™qAg | AbAgqAbAg)

Ž 5.

Reaction Ž3., the association mode, pre-incubation of Ag with Ag) and the following addition of Ab will give Ab equal possibilities to bind either Ag or Ag). The assay sensitivity will be determined by the different affinities for the analyte and the tracer. Reaction Ž4., the Ag-displacement mode, pre-incubation of Ag with Ab to form the Ab–Ag complex and then the following addition of the tracer. This assay will be less sensitive if the affinity for the tracer is lower than the affinity for the Ag, as the

¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

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Fig. 4. Influence on sensitivity using association or displacement immunoassays with Ab I Žunfilled symbols. and Ab IV Žfilled symbols.. The association experiment Ž`,v ., Ag-displacement experiment Žn,'., and Ag)-displacement experiment Že,l. were carried out according to the text. The tracer and Ab-concentrations were the same as in Fig. 3.

tendency for the equilibrium to shift from Ab–Ag to Ab–Ag) is low. Reaction Ž5., the Ag)-displacement mode, pre-incubation of Ag) with Ab to form the Ab–Ag) complex followed by the addition of analyte. For reasons mentioned earlier, this assay format will be less sensitive if the affinity for the Ag is lower than for the tracer Ag). One advantage of the last method is that a single-reagent mixture, containing tracer and antibody ŽAg)–Ab. can be used. The storage stability of such mixtures are usually better than when the two solutions are stored separately ŽColbert et al., 1984; Meltnichenko et al., 1996.. A calibration experiment was carried out with continuous measurements using the three mixing modes described above. For each value on the time axis a separate calibration curve can be obtained. However, in Fig. 4, the calibration curves using Ab I and Ab IV were obtained after a reaction time of 5 min. The sensitivity of the association mode and the Ag-displacement mode are almost equal while the Ag)-displacement mode is considerably less sensitive. Consequently, following the previous discussion, it can be concluded that the affinity for the tracer is greater than the affinity for atrazine using

either Ab I or Ab IV. However, it should be mentioned that if the displacement calibration curves are plotted for samples at equilibrium, there should not be any significant difference in sensitivity. 3.5. Calibration and sensitiÕities Calibration graphs, using atrazine as the analyte and Ab I–IV at 70% of binding, are presented in Fig. 5. The detection limit was defined as the sample

Fig. 5. PFIA calibration curves of atrazine, at 70% of binding, using Ab I Ž'., Ab II Ž,., Ab III Žv ., and Ab IV Ž`. at the same concentrations as in Fig. 3, and the fluorescein labelled tracer iPrrClrEDF at 0.9 nM.

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¨ P. Onnerfjord et al.r Journal of Immunological Methods 213 (1998) 31–39

concentration corresponding to a signal equal to the blank sample minus 3 = RSD for a blank sample Ž3 = 2.7%, n s 10.. The obtained LOD-values were found to be 0.2, 0.4, 0.08 and 0.35 ng mly1 for Ab I–IV, respectively. The relative standard deviation within a sample Žreplicate measurements, n s 10. at 1 ng mly1 where 2.2% at a 95% confidence level. The linear range of detection for atrazine lies within the concentration interval of 0.3–20 ng mly1 . The sensitivity using the FPM-1 was about one order of magnitude higher compared to the Abbot TDX instrument ŽEremin, 1994..

The information that can be received from fast assay experiments employing this technique can be used in order to select the proper combination of immunoreagents for developing other fluorescence based immunoassays. The informations from this study have been used to develop a flow immunoassay based on the separation of bound and free tracer from the equilibrium conditions in Eq. Ž3. using a ¨ small reversed-phase column ŽOnnerfjord et al., 1997.. Acknowledgements

4. Conclusions Fluorescence polarisation has been demonstrated to be a highly efficient and sensitive technique for the characterisation of immunoreagents. Fluorescence polarisation is a homogeneous assay technique, fully applicable as an optical immunoassay for studying binding of labelled ligands in the low nanogram per milliliter-range. The current experiments were carried out in a single sample mode, whereby sample throughput is rather low. However, by utilising the FPM-2 instrument ŽJolley Consulting, Round Lake, IL, USA., a 96-well Žor 384-well. plate format assay, the technique can be utilised for screening purposes obtaining sample throughputs fully compatible with conventional automated ELISAs. A disadvantage is a significant loss in sensitivity due to a different optical construction of this instrument as compared to FPM-1. The advantages of PFIA as compared to other techniques such as ELISA and SPR, are mainly speed, simplicity and avoidance of immobilisation steps. However, several disadvantages such as manual sample handling steps and matrix dependence should be mentioned. Samples containing more than 2% Žvrv. organic solvent could not be run using the FPM-1, as there was a huge decrease in polarisation. The errors in manual sample handling could have been avoided by using automated sample preparation systems, but this was outside the scope of this study. Furthermore, as the polarisation technique is based on the difference in molecular weight between bound and free-labelled antigen, it is of limited applicability when there is a smaller mass difference between receptor and target molecules.

Financial support from the European Community EC No. EV5V-CT92-0109, Swedish National Board for Industrial and Technical Development ŽNUTEK. are gratefully acknowledged. The Swedish Institute is also acknowledged for financially supporting the research stay of Sergei Eremin in Lund. References Brecht, A., Piehler, J., Lang, G., Gauglitz, G., 1995. A direct optical immunosensor for atrazine detection. Anal. Chim. Acta 311, 289. Colbert, D.L., Smith, D.S., Landon, J., Sidki, A.M., 1984. Single-reagent polarisation fluoroimmunoassay for barbiturates in urine. Clin. Chem. 30, 1765. Dandliker, W.B., Hsu, M., Levin, J., Rao, B.R., 1981. Equilibrium and kinetic inhibition assays based upon fluorescence polarisation. Methods Enzymol. 74, 3. Eremin, S.A., 1994. Polarisation fluoroimmunoassay for rapid, specific detection of pesticides. In: Nelson, J.O., Karu, A.E., Wong, R.B., ŽEds.., 207th National Meeting of the American Chemical Society. ACS, San Diego, CA, 223. Eremin, S.A., Samsonova, J.V., 1994. Development of polarisation fluoroimmunoassay for the detection of s-triazine herbicides. Anal. Lett. 27, 3013. Eremin, S.A., Samsonova, Z.V., Egorov, A.M., 1994. Immunochemical methods for the assay of herbicides of the 1,3,5-triazine group. Russ. Chem. Rev. 63, 611. Goodrow, M.H., Harrison, R.O., Hammock, B.D., 1990. Hapten synthesis, antibody development, and competitive inhibition enzyme immunoassays for s-triazine herbicides. J. Agric. Food Chem. 38, 990. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 673. Hock, B., Dankwardt, A., Kramer, K., Marx, A., 1995. Immunochemical techniques—antibody production for pesticide analysis: a review. Anal. Chim. Acta 311, 393. Kramer, K., Hock, B., 1996. Recombinant single-chain antibodies against s-triazines. Food Agric. Immunol. 8, 97.

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