3R-Project 52-96

Development of an interleukin-1 (IL-1) assay with rabbit blood as an alternative to the rabbit pyrogen test

Thomas Hartung
Biochemical Pharmacology, University of Konstanz, 78434 Konstanz, Germany
thomas.hartung@uni-konstanz.de, thomas, hartung@cec.eu.int

Keywords: rabbit; whole blood; cytokines, growth factors; fever; replacement; diagnostic approaches: pyrogens

Duration: 3 years Project Completion: 2000

Background and Aim
In the 1940’s drug testing for pyrogen contamination(fever-inducing impurities) was standardized using the rabbit pyrogen assay. Recently we proposed an alternative method using human whole blood (PyroCheck; the kit version was developed in conjunction with DPC Biermann, Bad Nauheim, Germany). This test is not restricted to endotoxins from gram-negative bacteria.

The goal of this project was to establish a rabbit IL-1-ELISA to validate the PyroCheck assay by comparing IL-1beta production in rabbit and human whole blood. This assay will potentially eliminate the need to test in rabbits.

Method and Results
The samples to be tested for contamination with pyrogens are incubated with human blood. The generation of the endogenous fever mediator interleukin 1beta (IL-1beta), indicative of a primary fever reaction in humans, is measured. This method detects the reaction of the targeted species. However, pronounced differences were reported in the potency of several bacterial pyrogens in different species. As part of the validation of the new method using human blood, discrepancies between this whole blood in vitro system and the rabbit in vivo test might occur. In these cases the rabbit blood assay will help to distinguish between an in vitro artefact and genuine species’ differences.
The rabbit IL-1beta protein (produced by Dr. Reto Crameri, Schweizerisches Institut für Allergie- und Asthmaforschung Davos, Switzerland; SIAF) is currently being used at the University of Constance to immunize chickens and sheep into producing polyclonal antibodies. Likewise, murine monoclonal antibodies are being prepared.
After the production of murine monoclonal antibodies, they were tested in combination with a second antibody to detect the rabbit IL-1ß. In order to establish a new ELISA to detect rabbit IL-1beta, it is necessary to find the optimal combination of two antibodies. The first, a monoclonal mouse anti-rabbit IL-1beta antibody covers the surface of the microtiterplate, on which the antibody can bind selectively with the protein IL-1. The second, a polyclonal goat-anti rabbit IL-1 antibody, binds to this complex and can initiate a colour reaction necessary for the detection of the amount of the protein by a photometric method (ELISA). At this stage, we should be able to optimize this system to detect Interleukin in rabbit blood. With this method, we would have an instrument available, with which to compare the human and the rabbit reaction to pyrogen contamination. Therefore in the prevalidation phase we could differentiate between a species-specific reaction to exogenous pyrogens (rabbit-man).
Using a rabbit TNF-ELISA (another fever signal transmitter like IL-1) we could show that in principle special cytokines are produced and detectable in rabbit blood.

Conclusions and Relevance for 3R
After successful validation of the rabbit whole blood assay in combination with the human assay PyroCheck, it will be possible to distinghuish between in vitro artefacts and true species differences. With this information, the PyroCheck Test will replace the rabbit in vivo pyrogen test completely.

References
(1) Fennrich, S., Wendel, A., Hartung, T. (1999): New applications of the Human Whole Blood Pyrogen Assay (PyroCheck). ALTEX 16, 146-149.

(2 )Hartung, T., Wendel, A. (1996): Detection of Pyrogens using human whole blood.In Vitro Toxicology, 9, 353-359.

(3) Eperon, S. and Jungi, T.W. (1996) The use of human monocytoid lines as indicator of endotoxin, J. Immunol. Methods. 194, 121-129.

(4) Fennrich, S., Fischer, M., Hartung,T., Lexa, P., Montag-Lessing,T., Sonntag, H.-G., Weigandt, M., Wendel,A. (1999): Detection of Endotoxins and other pyrogens using human whole blood. In: Brown F., Hendriksen, C., Sesardic D. (eds): Alternatives to animals in the development and control of biological products for human and veterinary use. Dev Biol Stand. Basel, Karger, 1999, Vol 101, 131-139.

(5) Bonenberger, J., Diekmann, D., Fennrich, S., Fischer, M., Friedrich, A., Hansper, M., Hartung, T., Jahnke, J., Löwer, J., Montag, T., Petri, E., Sonntag, H.-G., Weigand, M., Wendel, A., Zucker, B. (2000): Pyrogentestung mit Vollblut. Bundesgesundheitsblatt-Gesundheitsforschung-Gesundheitsschutz. Springer-Verlag 2000, 43, 525-533.

(6) Hartung, T., Crameri, R., Wendel, A. (1998): Entwicklung eines Pyrogentests mit Kaninchenblut. ALTEX 15, 17-18.

(7) Jahnke, M., Weigand, M., Sonntag, H.-G. (2000): Comparative testing for pyrogens in parenteral drugs using the human whole blood pyrogen test, the rabbit in vivo pyrogen test and the LAL test. European Journal of Parenteral Sciences 2000; 5(2): 39-44.

(8) Schindler S., Bristow A., Cartmell T., Hartung T. and Fennrich S. (2003) Comparison of the reactivity of human and rabbit blood towards pyrogenic stimuli. ALTEX 20, 59-63.