Untersuchung zum Risikoprofil hinsichtlich einer Sensibilisierung mit Rinderallergen unter besonderer Berücksichtigung der verschiedenen Rinderrassen

Abstract

Aim of the study: In some patients commercial test preparations of cow allergen do not confirm obvious cow related symptoms. The aim of this study was to investigate four different commercial cow allergen extracts and to compare them with self prepared extracts of different cattle breeds by immunoblotting. We used the sera of 42 German farmers with asthma and rhino conjunctivitis caused by cattle. The cow related symptoms could be confirmed by specific IgE antibodies in the sera of 68 % of the patients.Method: The detection of specific IgE antibodies was performed by Pharmacia CAP Rast . For every patient immunoblot experiments were performed with extracts from the lyophilised raw material used for the commercial extracts and from the hair of the cattle which are present on their farm. The hair of their own cattle was extracted by a 24 hour incubation at 6oC in a 0.1 M NH4HCO3 solution. After lyophilisation and dissolution in small volumes of solvent the total protein content was about 1 mg per ml. Proteins were separated on 15% gels by SDS-Page, and the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Biorad). Subsequently the membranes were incubated with sera from patients in dilution 1:5 and 1:20. The detection of IgE binding was performed with an anti-IgE-antibody (Sigma), which was coupled with alkaline phosphatase. After incubation with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP/NBT) a violet colour showed immunoreactivity.Results: The cow related symptoms could be confirmed by specific IgE antibodies in the sera of 68 % of the patients. After immunoblotting we found distinct bands in all symptomatic farmers, even in twelve farmers with a negative RAST result. Bands with molecular weights in the range between < 14 and about 67 kDa were observed; reactivity with the major allergen Bos d 2 at 20 kDa was detected in all farmers, although not as the strongest band in every case. Independent of the different raw materials the 42 farmers with clear cow related symptoms showed allergen reaction. Results of the four commercial cow allergen extracts: In addition to the previosly described major allergen at 20 and 22 kDa, reactivity against proteins at MW 14, 30, 32, 40/42, 55, 67 and higher were found in more than 50 % of the patients. Results of the extracts from hair of different breeds of cattle: There were no striking differences in the sensitisation pattern from the exposed breeds of the cattle detectable. Comparison between commercial and self prepared cow allergen extracts: In both commercial and self prepared cow allergen extracts we observed a reaction at MW 20 and 22 in all races. In some races we could see small additional reactions at MW 24/25, 38/40, 60 and greater than 97. In contrast to most of the commercial extracts investigated, the self prepared extracts frequently showed reactivity at certain molecular weights; at MW 14, 55 and between 66 and 97 kDa reactivity was seen in more than 50 % of the farmers. All patients showed high reactivity at MW 20 and 22 in all four commercial cow allergen extracts. Only in a few cases reactivity was seen at MW 18, 28, 35, 44 and about 97 with the four commercial extracts. Differences were seen in IgE binding capacity especially at MW 14, 30, 32, 40/42, 55, 67 and more than 67. At these molecular weights the reactivity differs from one commercial extract to the other between 4.8 and 85.7 %, contrasting to staining in SDS-Page as described previously. An association between the sensitization pattern and the breeds of the cattle was not detectable. In contrast to our self prepared extracts some proteins of the same molecular weight in some commercial extracts did not show a distinct band.Conclusion: Our results of the IgE binding are in agreement with previous studies; additionally we showed reactivity in immunoblotting at MW 32, 44 and more than 67 kDa in up to 33 % of the farmers. In many farmers with negative RAST results we found distinct reactions with cow allergen in immunoblotting experiments with the high concentrations used. Therefore we suggest that skin tests with native cattle hair should be performed in cases with clearly cow related symptoms. The most striking result of our investigation lay in the altered capacities of proteins in some commercial extracts to bind with IgE antibodies. We presume that some proteins had lost their ability to react with IgE antibodies as a consequence of methods of production. Another reason might be the low concentration of allergens in the commercial extracts. The lack of the allergen IgE-binding capacity of some commercial cow allergen extracts may be a possible explanation for the differences between clinical symptoms and results obtained with a commercial test in some patients.