Isoallergen Analysis of Pectate Lyases (Amb a 1 and Amb a 2) from Commercial Short Ragweed Pollen

Abstract

RATIONALE: To provide the basis for the decision if a single recombinant isoallergen of Amb a 1/2 is sufficient or if a mixture of isoallergens should necessarily be included in a recombinant immunotherapeutic vaccine for the treatment of ragweed allergy.METHODS: Different oligonucleotide primer were designed based on the 5'- and 3'-UTRs of known Amb a 1 and Amba 2 isoallergen genes and were used to amplify Amb a 1 and Amb a 2 genes from cDNA generated from commercial short (common) ragweed pollen (Allergon, Sweden) originated from the United States. Additionally, spots of Amb a 1/2 proteins separated by 2D-gel electrophoresis of ragweed pollen extract were analyzed by MSMS-sequencing.RESULTS: With the PCR approach we analyzed 78 sequences. The majority (49%) were Amb a 1.03 sequences, followed by Amb a 1.01, Amb a 1.02, Amb a 2.01 and Amb a 1.04. Ten new isoallergen variants were found, but additional isoallergens could not be detected. The MSMS-analysis of protein spots confirmed the existence of the 5 isoallergens, however the most abundant isoallergen was Amb a 1.01.CONCLUSIONS: On a first sight it seems that Amb a 1.01 is the most important isoallergen of short ragweed Amb a 1/2. This is in line with published data showing a high IgE-binding capacity of Amb a 1.01. However, this needs to be confirmed with a large number of sera from ragweed allergic subjects and a set of natively folded recombinant isoallergens of Amb a 1. RATIONALE: To provide the basis for the decision if a single recombinant isoallergen of Amb a 1/2 is sufficient or if a mixture of isoallergens should necessarily be included in a recombinant immunotherapeutic vaccine for the treatment of ragweed allergy. METHODS: Different oligonucleotide primer were designed based on the 5'- and 3'-UTRs of known Amb a 1 and Amba 2 isoallergen genes and were used to amplify Amb a 1 and Amb a 2 genes from cDNA generated from commercial short (common) ragweed pollen (Allergon, Sweden) originated from the United States. Additionally, spots of Amb a 1/2 proteins separated by 2D-gel electrophoresis of ragweed pollen extract were analyzed by MSMS-sequencing. RESULTS: With the PCR approach we analyzed 78 sequences. The majority (49%) were Amb a 1.03 sequences, followed by Amb a 1.01, Amb a 1.02, Amb a 2.01 and Amb a 1.04. Ten new isoallergen variants were found, but additional isoallergens could not be detected. The MSMS-analysis of protein spots confirmed the existence of the 5 isoallergens, however the most abundant isoallergen was Amb a 1.01. CONCLUSIONS: On a first sight it seems that Amb a 1.01 is the most important isoallergen of short ragweed Amb a 1/2. This is in line with published data showing a high IgE-binding capacity of Amb a 1.01. However, this needs to be confirmed with a large number of sera from ragweed allergic subjects and a set of natively folded recombinant isoallergens of Amb a 1.