This project aims at the development of immunoelectrode probes for fast analysis of toxins in sea food.
The first objective of this project was the production of polyclonal and/or monoclonal antibodies against select toxins as okadaic-acid, saxitoxin, domoic acid.
The second objective was the production of electrochemical biosensors for enzyme activity determination.
Okadaic acid was used as toxin guide for polyclonal and monoclonal antibody production, meanwhile an electrochemical procedure to measure the activity of the enzymes which could be labelled to the toxins or to the antibodies for the electrochemical detection of toxins was carried out successfully.
Okadaic acid was the first toxin with which we started the production of polyclonal and monoclonal antibodies.
Also preparation of saxitoxin, domoic and okadaic acid antigens and the relative immunisation schedule for antibody production was carried out.
Since okadaic acid was the first toxin obtained, it was processed with production and purification of its polyclonal antibodies and then with the production of monoclonal Ab. Purification of polyclonal antibodies has been performed by protein A chromatography. Purity was tested by SDS PAGE (Polyacrilammide Gel Electrophoresis) and resulted to be 95 %. A direct ELISA test was used to calculate the average dissociation constant of the polyclonal antibodies.
The purified polyclonal antibodies obtained from immunised mice, after only 3 rounds of immunisation, were used to set up anti okadaic acid ELISA. The mouse with the higher specific antibody titre was sacrificed and splenocytes were used for somatic hybridisation. Among growing clones just one was found to produce specific anti okadaic acid antibodies. The monoclonal antibody containing supernatant was used in further experiments.
Spectrophotometric ELISA tests were performed binding curves were obtained.
For comparison a MAb anti okadaic acid purchased from Calbiochem (cat. 495605) was used in spectrophotometric ELISA system. Data obtained are reported in the graph of Figure 1.
(For Figure 1 contact the Coordinator)
In this system blanks are quite high showing that the blocking solution is critical.
Using the data showed in the Fig. 1 a competitive ELISA was set up following Friguet. Results obtained are shown in Figure 2.
Antibodies produced and commercially available were characterised and a one step ELISA with spectrophotometric detection for okadaic acid was developed.
Results of the ELISA for okadaic acid obtained with two systems are reported in the table.
Production of electrochemical biosensors for enzyme activity determination was successfully carried out by all the partners.
We used the enzymes Horseradish Peroxidase (HPR), Alkaline Phosphatase (AP) and Glucose Oxidase (GOD) coupled with platinum, platinised and rutinised carbon and glassy carbon electrodes.
The electrochemical detection of alkaline phosphatase activity has been evaluated using both platinum and glassy carbon electrodes.
Nature of buffer and its concentration has been optimised.
The best sensitivity was obtained using phenyl phosphate as substrate. Phosphate was determined using 30 mMol/L veronal buffer at pH 10.
(For Figure 2 contact the Coordinator)
Mode Linear range Limit of detection
Displacement 11.11 ?g to 1.69 ng/ml 0.560 ng/ml
Competition 3.70 ?g to 5.08 ng/ml 0.188 ng/ml
Mode Linear range Limit of detection
Displacement 1.56 ?g to 0.38 ng/ml 0.38 ng/ml
Competition As above As above
The sensitivity of the platinum sensor has been then evaluated by measuring the activity of different amounts of alkaline phosphatase in the reaction medium. A linear response of the sensor was obtained in the range: 0.0001 - 0.1 International Units/ml (I.U./ml).
The same experiment has been performed using a commercially available AP-labelled model antibody (AP-anti FITC) and the same linear range obtained i.e. 0.0001 - 0.1 I.U./ml.
In a second part of the work, we tested a glassy carbon electrode as working electrode (from Bioanalytical Systems), and the sensitivity of the sensor has been studied by determining the useful activity range for enzyme monitoring.
Then, with the enzyme tested with the model AP-labelled antibody, the linear range found was 0.00005 - 0.1 I.U./ml. As previously seen in the case of the platinum sensor, the range is in agreement with what was required in our objectives.
The activity of Horseradish Peroxidase (HPR) enzyme as label for immunosensors with electrochemical detection has been tested using several substrates, some of them never used for electrochemical detection in ELISA. From cyclic voltammetry results at glassy carbon electrode, two substrates were selected for further experiments, the tetramethylbenzidine (TMB) and the hydroquinone. All conditions for their use were studied, i.e. potential to be applied, concentration of substrates and their ratio to the second substrate for the enzymatic reaction H2O2, flow rate, volume loop, etc.
Using the optimised conditions, calibration curves for the enzyme in solution have been performed, and a linearity range between 10-4 and 2 ? 10-3 U/ml using hydroquinone (Hq), and 5?10-6 and 5?10-4 U/ml using TMB as substrate for the HPR was obtained.
ELISA competitive and not-competitive tests with electrochemical detection have subsequently been performed, using IgG and antiIgG labelled with HPR as model of our antigen and antibody. After optimisation of IgG and antiIgG concentration, type of blocking solution and time for detection, we obtained, using TMB as electrochemical substrate, a linearity range of 50 ng/ml ? 5 ?g/ml .
Similarly, the electrochemical detection of glucose oxidase (GOD) activity has been evaluated using a platinum electrode. The enzyme oxidised at the platinum electrode. The enzyme oxidised glucose as substrate and the hydrogen peroxide produced is oxidised at the platinum electrode polarised at + 650 mV in a reaction medium composed of 0.1 M acetate buffer, 0.1 M KCl at pH 6.
Preliminary results were obtained with glucose oxidase free in solution. The optimum glucose concentration was 50 mM and in these conditions, by measuring the activity of different enzyme amounts, the determination of the sensitivity of the platinum sensor gave an activity range linear from 0.0008 to 0.1 I.U./ml.
The production of polyclonal and monoclonal antibody for selected toxins will lead to the assembling of novel electrochemical immunosensors with improved characteristics of sensitivity and selectivity. Also the construction of a small, portable and cost effective instrument will lead to the measurement of sea-food toxins in the field.
1. J. Carlson, M.L. Lever, B.W. Lee, and P.E. Guire, "Development of Immunoassays for Paralytic Shellfish Poisoning: A Radioimmunoassay for Saxitoxin", E.P. Ragelis, Ed., Seafood Toxins, American Chemical Society, Washington, 1984, p. 181, and references therein
2. J.H. Gentile, in Microbial Toxins, Vol. VII, Algal and Fungal Toxins. S. Kadis, A. Ciegler and S.J. Ajl Eds., Academy Press, New York, 1971.
3. "Official Methods of analysis of the Association of Official Analytical Chemists", Twelfth Edition, W. Horowitz, Ed., AOAC, Washington, 1975, sections 18.070-18.076.
4. E.J. Schantz "Historical Perspective on Paralytic Shellfish Poison", E.P. Ragelis. Ed., Seafood toxins, American Chemical Society, Washington, 1984, p.99.
5. The Merck Index, 10th edition, M. Windholz, S. Budavari, R.F. Blumetti and E.S. Otterbein Eds., Merck and Co., Inc., Rathway, N.J. 1983.
6. T. Yasumoto, in Toxic Dinoflagellates, Proc. 3rd Int. Conf. on Toxic Dinoflagellates, St. Andrews, New Brunwick, Canada, 1985, Elsevier, New York, 1985.
The development of electrode probes for the rapid assay sea-food toxins is a goal which is of crucial importance for food analysis and food industry mainly in the area of fisheries.
To reach this goal a strategic point is the production of poly and/or monoclonal antibodies against selected toxins.
At present analysis of sea food toxins is carried out with separation procedure or expensive mouse time to death bioassays to estimate the toxicity of fish and shellfish. Improved analysis and screening techniques are required to minimise the risk associated with landed seafood and identify and monitor safe commercial harvesting areas.
Our objective is to couple the biosensor technology using an electrochemical detection coupled with conjugation of enzymes with toxins or their produced antibodies. This approach will make a toxin analysis rapid and cost effective. There is also the possibility to build a small instrument for toxin analysis "in loco".
Our new technology can be applied to several industries involved in the use of seafood for further processing such as domestic get foods or commercial canneries of oysters, crabs and tuna.
The quick turn-around time of the electroimmunochemical biosensor testing will decrease the chance that humans could become infected with toxin-containing material and will shorten the time needed from catch to market.
This will improve the overall freshness of the seafood available to the consumer and will ensure the safety of recreational use of ocean waters in areas where toxins are common.
Funding SchemeCSC - Cost-sharing contracts