ATLA::Alternatives to Laboratory Animals

Volume 29, Number 6

Human haemoclassification by use of specific yolk antibodies obtained after immunisation of chickens against human blood group antigens.

ATLA 29, 717-726, November/December 2001

Esteban Gutiérrez Calzado,¹ Rosa Mireya Gracía Garrido,² and Rüdiger Schade³

¹Laboratory of Antibodies and Experimental Biomodels, Santiago de Cuba Medical University, Calle 23 y Carretera del Caney S/N Reparto Vista Alegre, P.O. Box 4032, Santiago de Cuba, Cuba; ²Department of Biology, Faculty of Mathematics and Natural Sciences, University of Oriente, Ave. Patricio Lumumba, Santiago de Cuba, Cuba; ³Institute of Pharmacology and Toxicology, Medical Faculty (Chariti), Humboldt University, Dorotheenstrasse 94, 10117 Berlin, Germany

SUMMARY

Polyclonal antibodies, widely used in research and diagnostics, are conventionally isolated from the blood of immunised mammals, especially rabbits. The fact that antibodies can also be detected in the yolk of eggs laid by immunised hens, led to the development of yolk antibody technology (IgY-technology) as an alternative method that is less stressful to animals. This technology has become a worthwhile alternative to the blood-dependent techniques. Furthermore, because of the phylogenetic distance between birds and mammals, avian antigens have a very specific immune response to highly conserved antigens of mammals, such as human erythrocyte antigens. To evaluate the humoral immune response of hens immunised with human red erythrocyte antigens, 22 White Leghorn hens were kept in cages and immunised with total red blood cells or stroma of the human rhesus positive (Rh+) system (D antigen) by weekly intramuscular and intravenous injections, without the use of an adjuvant. The haemagglutination assay was used to evaluate the dynamics of the production of IgY antibodies against human erythrocyte antigens, and single radial immunodiffusion was used to evaluate the amount of total IgY in de-lipidated supernatants from egg yolk. The highest titres were observed four weeks after the first immunisation, and these remained stable for up to seven weeks for the intravenous route. Positive reactivity against human erythrocyte antigens A, B and 0 was demonstrated in de-lipidated supernatants from the egg yolks of immunised hens. The strongest reaction was observed against blood group O Rh+ (0+). Furthermore, the antibodies obtained recognised erythrocyte antigens that belong to neither the Rh system nor the Landsteiner-Weiner antigen. This finding opens the possibility of using hens instead of rabbits to produce polyclonal antibodies for human haemoclassification.

Keywords: IgY egg yolk antibody, haemoclassification, erythrocytes, red blood cell antigen