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960 Identification of snow-crab antigens in air sampling of a production plant
422
957
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY 1996
Fel d I Levels in Wellington Primary Schools and on Children's Clothing, KL Patchett. S Lewis. K Wickens BA. l
959
M Wissenbach PhD, S KIysner PhD, M Svangfort PhD, H Ipsen Msc. Hersholm, Denmark. The major grass pollen allergens of the Poaceae, the group 1 and group 5 allergens, appear to exist as various isoallergens in each grass species. Recombinant allergens however represent only one isoallergen. If recombinant grass pollen allergens should be used as diagnostic and therapeutic agents, the significance of this variation for sensitization and treatment of grass pollen allergic subjects has to be elucidated. For this purpose group 5 allergens were cloned from Phleum pratense. Six unique eDNA clones encoding Phi p 5 isoallergens were obtained applying the polymerase chain reaction (PCR). These were expressed as maltose binding protein-Phi p 5fusion proteins in Escherichia coli, using the pMAL-c vector. A proteolytic cleavage site for Factor Xa had been introduced between the two protein parts by genetic engineering. All six fusion proteins bound to IgE from a serum pool of allergic patients and a Phi p 5specific monoclonal antibody on immunoblots, indicating structural similarity to natural Phi p 5. Fusion proteins were isolated from bacterial lysates by affinity chromatography, cleaved with Factor Xa and further purified by ion exchange chromatography. The resulting proteins were more than 90% pure. Quantitative assays will be used to investigate the antigenic structures of the isoallergens. The high degree of overexpression of Phi p 5 (ca 30 % of the coomassie staining activity on SDS-Polyacrylamide gels, estimated by digital scanning) and the rather simple purification procedure makes this system attractive for large scale production of recombinant Phi p 5 isoallergens.
Crane FRACP. P Fitzharris MD. Wellington, New Zealand. No information is available on allergen levels in New Zealand classrooms. We collected data on buildings, cleaning schedules and cat ownership by 7-9 year old pupils in 9 schools (11 classrooms, 202 children) in July 1995. Dust waS collected using standardised collection procodmes and Fel d I levels were measured by ELISA. The table below summuris~ the findings (*dlffexeace (p<0.05))
I Fei d I
noors
| geon3_ean(95% ci)
~g............. 1.15 (.0.61-2.14).
range 0.03-9.36
(n= 26)
, l.tg/m2 "{)~68(0:21 -'~2:'24) ............. 0:~'I--2"()~52" carpet I . ~ . g ............ 2.21 {1.27-3...84.~* 0.2-9.36 (n=lT) | ~tg/m2 '3:62"(1::/'3'-'7:~ ) ............"0.'(~:~'-~:52"no carpet [..IJ'~.Ig........... 0..3..,3,(0:..09=.1.:.1..4.).*....... 0:03-3:45...... (n=9) | Bg/m2 10.03 (0.005-0.19) 0.01-3.93 shirts I tt~/e i 33.35 1.24-4092 (n=202) | l.tg/shirt i 4.54 , 0.056-1937 At least I cat was owned by 55% of children. Fei d I on floors was positively cortdated with pupil cat owncrsMp (p=0.005) and mean c ~ m shirt allergen (p=0.04) Cats are rarely allowed in classrooms yet all classrooms had at least 1 carpeted tom" sample above the proposed sensitization level of 2;xg/g, 2 samples being >8gg/g. These results suggest that clothing is an important source of classroom cat allergen and that carpet acts as a major rese~oir.
958
Cloning and analysis of different isogenes encoding Bet v 1 isolated f r o m pollen of North A m e r i c a n birch. Y Boutin PbD. R Labourdette BSc. J Boulanger MSc and J H£nert MD. Ste.Foy Canada. Bet v I, the major allergen o f birch pollen, displays a high degree o f heterogeneity. Recent studies demonstrated that this heterogeneity could he attributed to isogenes encoding the Bet v 1 rather than post-translational modifications o f the protein. However, these studies were conducted on birch pollen from Europe. The aim of this study is to analyze and compare the isogenes encoding Bet v 1 in birch o f North America and Europe. Total RNA was extracted from North American birch pollen and first strand o f eDNA was synthesized with reverse transcriptase, eDNA was then amplified by PCR using appropriate primers designed so that the.re were available 3' Xba I and 5' EcoR V sites on amplified product. The Bet v 1encoding genes obtained from this reaction were cloned into the BlueScript vector. Clones coding for Bet v 1 isoforms were isolated, and both strands were sequenced by the dideoxy chain termination method. We isolated eighteen clones and compared their amino acid sequence and we found that they could be grouped in 5 different families. The sequences o f clones in the same family differ no more than 2 amino acids and may represent all¢lic variations. We also compared the deduced amino acid sequence o f our clones with those previously described in other studies. As previously reported by otber studies, we observed a high variability in amino acid positions 7-9, 30, 57, 62, 76 to 91, 109 to 118, and 132 to 142. These data indicate that the isogenes encoding Bet v 1 from North American birch arc similar to those from Europe.
O v e r e x p r e s s i o n o f r e c o m b i n a n t Phi p 5 i s o a l l e r g e n s in
Escherichia coli
960
Identification
of
snow-crab
antigens
in
air
s a m p l i n g of a p r o d u c t i o n p l a n t . P. Chr~tien. M.D.. R. Brassard. R.T.. C. Chiauette. R.T.. J.-L. Malo. M.D.. A. Cartier. M,D-. M. McCants. R.T.. S. Lehrer. Ph.D. Baie-Comeau and Montreal, Canada, New Orleans, LA. Introduction and aim: We previously assessed the prevalence of occupational asthma (OA) due to snow-crab in a production plant (Cattier A. et al., JACI, 1984). We also showed that this type of OA is related to immediate immunological reactivity as demonstrated by skin reactivity and increased specific IgE antibodies (Cartier A., e.t al., JACI, 1986). However, we did not show that snow-crab antigens could be found in the air sampling, causing immunological reactivity and OA. This was the purpose of the current work. Material and mfth0ds: Personal air sampling at 4 different worksitcs ofa soow-crab producing plant was carried out on PVC filters with an SKC pump run at 1.5 L/rain for 2 hours. Snow-crab was being boiled and processed during the air sampling periods. Filters were anlaysed by RAST inhibition in a blind manner (i.e. without knowledge of the worksite where the filter originated). Results: Eluate from one of the four filters (#1) had the highest protein concentration and yielded the highest percent inhibition of RAST - 13% inhibition with the snow-crab meat, 23% and 28% inhibition with the snow crab water RAST in two separate assays. A second cluate taken from another filter(#2) showed borderline reactivity (10% and 13% inhibition in two assays) whereas the two other ones and a control filter were negative. The two filters that contained snowcrab proteins were the one nearest the boiling process, filter #I being the nearest followed by filter #2. It was estimated that a %inhibition of 28% corresponded to -8.6 v-g of proteins and to 1.5 Bg of allergens on the filter. Conclusion: This study shows that airborne snow-crabderived proteins, released during the boiling process, are the cause of immunological reactivity and of OA to snow-crab.
957
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY 1996
Fel d I Levels in Wellington Primary Schools and on Children's Clothing, KL Patchett. S Lewis. K Wickens BA. l
959
M Wissenbach PhD, S KIysner PhD, M Svangfort PhD, H Ipsen Msc. Hersholm, Denmark. The major grass pollen allergens of the Poaceae, the group 1 and group 5 allergens, appear to exist as various isoallergens in each grass species. Recombinant allergens however represent only one isoallergen. If recombinant grass pollen allergens should be used as diagnostic and therapeutic agents, the significance of this variation for sensitization and treatment of grass pollen allergic subjects has to be elucidated. For this purpose group 5 allergens were cloned from Phleum pratense. Six unique eDNA clones encoding Phi p 5 isoallergens were obtained applying the polymerase chain reaction (PCR). These were expressed as maltose binding protein-Phi p 5fusion proteins in Escherichia coli, using the pMAL-c vector. A proteolytic cleavage site for Factor Xa had been introduced between the two protein parts by genetic engineering. All six fusion proteins bound to IgE from a serum pool of allergic patients and a Phi p 5specific monoclonal antibody on immunoblots, indicating structural similarity to natural Phi p 5. Fusion proteins were isolated from bacterial lysates by affinity chromatography, cleaved with Factor Xa and further purified by ion exchange chromatography. The resulting proteins were more than 90% pure. Quantitative assays will be used to investigate the antigenic structures of the isoallergens. The high degree of overexpression of Phi p 5 (ca 30 % of the coomassie staining activity on SDS-Polyacrylamide gels, estimated by digital scanning) and the rather simple purification procedure makes this system attractive for large scale production of recombinant Phi p 5 isoallergens.
Crane FRACP. P Fitzharris MD. Wellington, New Zealand. No information is available on allergen levels in New Zealand classrooms. We collected data on buildings, cleaning schedules and cat ownership by 7-9 year old pupils in 9 schools (11 classrooms, 202 children) in July 1995. Dust waS collected using standardised collection procodmes and Fel d I levels were measured by ELISA. The table below summuris~ the findings (*dlffexeace (p<0.05))
I Fei d I
noors
| geon3_ean(95% ci)
~g............. 1.15 (.0.61-2.14).
range 0.03-9.36
(n= 26)
, l.tg/m2 "{)~68(0:21 -'~2:'24) ............. 0:~'I--2"()~52" carpet I . ~ . g ............ 2.21 {1.27-3...84.~* 0.2-9.36 (n=lT) | ~tg/m2 '3:62"(1::/'3'-'7:~ ) ............"0.'(~:~'-~:52"no carpet [..IJ'~.Ig........... 0..3..,3,(0:..09=.1.:.1..4.).*....... 0:03-3:45...... (n=9) | Bg/m2 10.03 (0.005-0.19) 0.01-3.93 shirts I tt~/e i 33.35 1.24-4092 (n=202) | l.tg/shirt i 4.54 , 0.056-1937 At least I cat was owned by 55% of children. Fei d I on floors was positively cortdated with pupil cat owncrsMp (p=0.005) and mean c ~ m shirt allergen (p=0.04) Cats are rarely allowed in classrooms yet all classrooms had at least 1 carpeted tom" sample above the proposed sensitization level of 2;xg/g, 2 samples being >8gg/g. These results suggest that clothing is an important source of classroom cat allergen and that carpet acts as a major rese~oir.
958
Cloning and analysis of different isogenes encoding Bet v 1 isolated f r o m pollen of North A m e r i c a n birch. Y Boutin PbD. R Labourdette BSc. J Boulanger MSc and J H£nert MD. Ste.Foy Canada. Bet v I, the major allergen o f birch pollen, displays a high degree o f heterogeneity. Recent studies demonstrated that this heterogeneity could he attributed to isogenes encoding the Bet v 1 rather than post-translational modifications o f the protein. However, these studies were conducted on birch pollen from Europe. The aim of this study is to analyze and compare the isogenes encoding Bet v 1 in birch o f North America and Europe. Total RNA was extracted from North American birch pollen and first strand o f eDNA was synthesized with reverse transcriptase, eDNA was then amplified by PCR using appropriate primers designed so that the.re were available 3' Xba I and 5' EcoR V sites on amplified product. The Bet v 1encoding genes obtained from this reaction were cloned into the BlueScript vector. Clones coding for Bet v 1 isoforms were isolated, and both strands were sequenced by the dideoxy chain termination method. We isolated eighteen clones and compared their amino acid sequence and we found that they could be grouped in 5 different families. The sequences o f clones in the same family differ no more than 2 amino acids and may represent all¢lic variations. We also compared the deduced amino acid sequence o f our clones with those previously described in other studies. As previously reported by otber studies, we observed a high variability in amino acid positions 7-9, 30, 57, 62, 76 to 91, 109 to 118, and 132 to 142. These data indicate that the isogenes encoding Bet v 1 from North American birch arc similar to those from Europe.
O v e r e x p r e s s i o n o f r e c o m b i n a n t Phi p 5 i s o a l l e r g e n s in
Escherichia coli
960
Identification
of
snow-crab
antigens
in
air
s a m p l i n g of a p r o d u c t i o n p l a n t . P. Chr~tien. M.D.. R. Brassard. R.T.. C. Chiauette. R.T.. J.-L. Malo. M.D.. A. Cartier. M,D-. M. McCants. R.T.. S. Lehrer. Ph.D. Baie-Comeau and Montreal, Canada, New Orleans, LA. Introduction and aim: We previously assessed the prevalence of occupational asthma (OA) due to snow-crab in a production plant (Cattier A. et al., JACI, 1984). We also showed that this type of OA is related to immediate immunological reactivity as demonstrated by skin reactivity and increased specific IgE antibodies (Cartier A., e.t al., JACI, 1986). However, we did not show that snow-crab antigens could be found in the air sampling, causing immunological reactivity and OA. This was the purpose of the current work. Material and mfth0ds: Personal air sampling at 4 different worksitcs ofa soow-crab producing plant was carried out on PVC filters with an SKC pump run at 1.5 L/rain for 2 hours. Snow-crab was being boiled and processed during the air sampling periods. Filters were anlaysed by RAST inhibition in a blind manner (i.e. without knowledge of the worksite where the filter originated). Results: Eluate from one of the four filters (#1) had the highest protein concentration and yielded the highest percent inhibition of RAST - 13% inhibition with the snow-crab meat, 23% and 28% inhibition with the snow crab water RAST in two separate assays. A second cluate taken from another filter(#2) showed borderline reactivity (10% and 13% inhibition in two assays) whereas the two other ones and a control filter were negative. The two filters that contained snowcrab proteins were the one nearest the boiling process, filter #I being the nearest followed by filter #2. It was estimated that a %inhibition of 28% corresponded to -8.6 v-g of proteins and to 1.5 Bg of allergens on the filter. Conclusion: This study shows that airborne snow-crabderived proteins, released during the boiling process, are the cause of immunological reactivity and of OA to snow-crab.