383/448 nm) was controlled. After 20 min of substrate incuba- tion fluorescence intensity was such high that raw-data were taken for a four parametric calculation. Fig. 1 demonstrates a typical terbuthylazine standard curve.

Compared to already published results [1], substrate incuba- tion time was reduced from 20 h to 20 min so that fluorescence immunoassay becomes more interesting.

Acknowledgements. We would like to thank T. Giersch (Techni- cal University of Munich-Weihenstephan) for making available monoclonal antibodies and the Deutsche Forschungsgemein- schaft (DFG) for financial support.

51

References

1. Ulrich P, Niessner R (1992) Fresenius Environ Bull 1 : 2 2 - 27

2. Giersch T, Hock B (1990) Food Agric Immunol 2 : 8 5 - 97

3. Schlaeppi JM, F6ry W, Ramsteiner K (1989) J Agric Food Chem 37:1532-1538

4. Ulrich P, Weller MG, Weil L, Niessner R (1991) Vom Wasser 76: 251 -- 266

5. Weller MG, Weil L, Niessner R (1991) Mikrochim Acta (in press)

Fresenius J Anal Chem (1992) 3 4 3 : 5 1 - 52 - © Springer-Verlag 1992

002

Determination of triazine herbicides by ELISA - Optimization of enzyme tracer synthesis

M. G. Weller, L. Weil, and R. Niessner

Institut fiir Wasserchemie und Chemische Balneologie, Technische Universit/it Miinchen, Marchioninistrasse 17, W-8000 M/inchen 70, Federal Republic of Germany

Introduction

Several immunoassays have been developed to detect atrazine [1] or similar compounds [2]. Some standard methods for the preparation of tracers based on horseradish peroxidase (HRP) were tested [3]. Irreproducible results or laborious procedures forced to several improvements. The hapten needs to be a carboxylic acid, which can be transformed into a reactive NHS- ester [4, 5].

o.3

0.2-

0.1~

[]

0.001 0.01 0.1 1 10 100 1000

Terbuthylazine [t.t g/ l l

Fig. 1. Standard curve of terbuthylazine, error bars." range. (Antibody: KI F4 [2] l : 20 000, tracer 1 : 40 000, tracer-incuba- tion time: 7 min, development: 5 min)

Materials and methods

Tracer synthesis'. 16.2 mg of (5.0' 10 -5 mol) 2-(tert-butyl- amino)-4-[(1-carboxypent-5-yl)amino]-6-(ethylamino)-s-triazine [6] and l l . 5 m g of (1 .0-10-4mol) N-hydroxysuccinimide (NHS) are dissolved in 1 ml of 1,2-dimethoxyethane (glyme). 20 mg of di-(N-succinimidyl)-carbonate (DSC) [7] is added in small portions. Development of CO2 indicates, that traces of water have been present. 40mg (2 .1 .10-4mol) of N- (dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is added to the chilled solution (0°C) and stirred for 10 h (25 ° C). Activation may be controlled by TLC using silica gel sheets (Macherey-Nagel, A L U G R A M SIL G/UV254). The plates should be prewashed with solvent mixture. As mobile phase t-butylmethylether (98%)/ethanol (99%)/formic acid (85%), 90: 9:1 is suitable.

5 mg of horseradish peroxidase is dissolved in sodium tetraborate-buffer (500 gl, 0.1 mol/1). 120 gl of the NHS-ester solution is added at room temperature in four aliquots in time increments of 10 rain to the well-stirred enzyme solution. The solution is stored at 4 ° C for about one day. If any precipitates are visible, the solution should be centrifuged before the chro- matographic step.

Purification. Gel permeation chromatography is performed on a Sephadex PD-10 column, equilibrated with PBS-buffer. An empty microtiter plate can be taken as manual fraction collector.

The enzyme content of the fractions can be measured photometrically with a microplate reader at 405 nm. The tracer (undiluted) can be stored at 4°C without any stabilization for several months. For long time storage 1% bovine serum albumine (BSA) and 0.05% Thimerosal (ethylmercury thiosali- cylic acid, sodium salt) should be added.

Characterization. The tracer quality can be checked by per- forming an ELISA (enzyme-linked immunosorbent assay). The ELISA procedure was performed as previously described [6, 8].

Results

Several triazine-peroxidase conjugates were prepared and tested for their suitability as tracers in ELISAs for the determination of triazine herbicides. In Fig. 1 a standard curve of an immunoassay for terbuthylazine is examplarily shown. Very high dilutions of enzyme tracer can be used, which are favorable in respect to sensitivity and to avoid unspecific binding.

Conclusions

This method of preparing enzyme conjugates has several ad- vantages:

52

- glyme facilitates the control by TLC, as the solvent evaporates easily

- dryness of reagents is not critical - DSC does not produce any problematic hydrolysis prod-

ucts - EDC and the corresponding urea compound separate in

a favorable form and are water soluble: purification of NHS- ester usually is not necessary

- reproducible results are obtained. Carboxylic acids with a very low solubility in glyme are

difficult to handle. In this case one should switch to dimethylsulfoxide (DMSO) as solvent or to a mixture of glyme/ DMSO (4:1).

Acknowledgements. We want to thank T. Giersch for providing monoclonal antibodies. This work was supported by a grant of the Technical University of Munich.

References

1. Schlaeppi JM, F6ry W, Ramsteiner K (1989) J Agric Food Chem 37:1532-1538

2. Giersch T, Hock B (1990) Food Agric Immunol 2 : 8 5 - 9 7 3. Tijssen P (1985) Practice and theory of enzyme immuno-

assays, Elsevier, Amsterdam, p 285 4. Anderson GW, Zimmermann JE, Callahan FM (1964) J Am

Chem Soc 86:1839-1842 5. Bodanszky M, Bodanszky A (1984) The practice of peptide

synthesis, Springer, Berlin Heidelberg New York, p 125 6. Weller MG, Weil L, Niessner R (1992) Mikrochim Acta (in

press) 7. Sauer MJ, Morris BA (1987) J Steroid Biochem 26 :165-

167 8. Ulrich P, Weller M, Weil L, NieBner R (1991) Vom Wasser

76: 251 - 266

Fresenius J Anal Chem (1992) 343:52 - © Springer-Verlag 1992

003

Specific quantification of proteins by zone immunoelectrophoresis assay, ZIA

O l o f Vesterberg

National Institute of Occupational Health, S-171 84 Solna, Sweden

Introduct ion

Zone immunoelectrophoresis assay, ZIA, is an improved form of rocket immunoelectrophoresis. ZIA can advantageously be used for quantification of several proteins in blood, cere- brospinal fluid (CSF) and urine, e.g. albumin [1] and lipoproteins [2]. For ZIA reviews see ref. [3] and [4]. With ZIA it is possible to measure also very low protein concentrations [5]. This is of special interest for quantification of proteins in urine and CSF. ZIA does not require expensive equipment or special procedures nor expensive kits as used for radioimmuno- assay (RIA) and ELISA.

Among the proteins studied the following should be mentioned: albumin, ~-fetoprotein (AFP), ~l-microglobulin, ]~2-microglobulin, apo-lipoproteins, ferritin, orosomucoid, re- tinol binding protein (RBP) and transferrin.

M e t h o d s and results

Working procedures for ZIA have been described in detail [1 - 5]. The concentrations of many proteins in urine samples from persons with and without kidney malfunction were studied as well as the stability of the proteins upon storage. Results obtained with reference material and in interlaboratory comparisons indicate high accuracy and reliability of ZIA. For ZIA the imprecision (within and between run) is often better than 5%.

Discuss i on and conclus ions

ZIA has many advantages: 1. simple apparatus and procedures (no gel punching nor cooling); 2. minimal antiserum consump- tion (1 ml may allow > 1000 assays); 3. electrophoresis may be

performed in a few hours or overnight; 4. low coefficient of variation; 5. calibration curves are usually linear, requiring only a few standard points (a new computerprogram facilitates calculations of concentrations in samples); 6. low detection limit <10 ng/ml; 7. wide concentration measuring ranges; 8. the specificity of antisera must not be absolute because unspecificity can be seen easily (> 1 precipitate in a gel rod); 9. no kits nor unique preparations of antisera such as IgG fractions are required as with RIA and ELISA; 10. good agreement with the results of other methods.

RIA has the following drawbacks: complicated procedures to produce and check the reagents, short halflife of isotopes and assay kits, extra precautions with radioactive isotopes and expensive apparatus. ZIA uses cheap equipment, is simple to perform, straightforward, easy adaptable to different demands and thus has many advantages over other methods.

A new possibility for diagnosis is to quantify some proteins in serum or plasma and the erythrocyte sedimentation rate (ESR) and to calculate coefficients specific for certain diseases [6]. Also isoproteins, i.e. multiple molecular forms of proteins, can be quantified with ZIA after separation by electrophoresis, e.g. to detect effects of alcohol on the liver [7]. ZIA can improve diagnosis.

To conclude ZIA has a good potential for specific quantification of several proteins including antibodies. A more detailed description of ZIA will be published in a special issue of Environ. Geochem. Health, 1992. A short history of electrophoretic methods has been published [8].

References

1. Vesterberg O (1981) Clin Chim Acta 113:305-310 2. Holmquist L, Vesterberg O (1986) Clin Chim Acta 156:131 -

144 3. Vesterberg O (1980) Hoppe-Seyler's Z Physiol Chem

361:617-624 4. Vesterberg O (1983) Protein quantification with zone immu-

noelectrophoresis assay (ZIA) In: Tscbescbe H (ed) Modern methods in protein chemistry-review articles. De Gruyter, Berlin New York, pp 187- 206

5. Vesterberg O (1983) Clin Chim Acta 135:99-101 6. Ruhenstroth-Bauer G, Schedler K-G, Scherer R, Vesterberg

O (1990) J Clin Chem Clin Biochem 28:845--850 7. Petrrn S, Vesterberg O (1989) Electrophoresis 10: 6 0 0 - 604 8. Vesterberg O (1989) J Chromatogr 4 8 0 : 3 - 1 9